US20040170607A1

US20040170607A1 - Oncolytic virus

Info

Publication number: US20040170607A1
Application number: US10/743,649
Authority: US
Inventors: John Bell; Nahum Sonenberg; David Stojdl; Earl Brown; Harold Atkins; Ricardo Marius; Brian Lichty; Shane Knowles
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-09-17
Filing date: 2003-12-23
Publication date: 2004-09-02
Also published as: US8147822B1; US20070166287A1; US20040208849A1

Abstract

The present invention is directed to a method of reducing the viability of a tumor cell involving administering a virus that is not a common human pathogen to the tumor cell. Preferably, the virus exhibits differential susceptibility, in that normal cells are not affected by the virus. This differential susceptibility is more pronounced in the presence of interferon. The tumor cell is characterized by having low levels, or no, PKR activity, or as being PKR−/−, STAT1−/− or both PKR−/− and STAT1−/−. The virus is selected from the group consisting of Rhabdovirus and picornavirus, and preferably is vesicular stomatitis virus (VSV) or a derivative thereof.

Description

This is a divisional of U.S. patent application Ser. No. 09/664,444, filed Sep. 18, 2000, the contents of which are incorporated herein by reference. This application claims the benefit of U.S. Provisional Patent Application No. 60/287,590, having an effective filing date of Sep. 17, 1999, the contents of which are incorporated herein by reference.[0001]

The present invention relates to a novel cancer therapeutic. More specifically, this invention relates to viruses that selectively infect and inhibit tumour cell growth.

BACKGROUND OF THE INVENTION

The use of oncolytic bacteria, or compositions of oncolytic bacterias, for combatting neoplasms in humans and animals is known. For example EP 564 121, GB 1,587,244 and U.S. Pat. No. 3,192,116 disclose the use of non-pathogenic bacteria that result in the liquification and lysis of tumours in vertebrates. However in many instances, for example with the use of Clostridium, the tumours are only partially destroyed, and tumour regrowth may still occur. To ensure control of tumour growth the administration of bacteria, followed by chemotherapeutic drugs, for example 5-fluorodeoxyuridine or alkylating agents, has been suggested (e.g. GB 1,069,144).

Several viruses have also been shown to exhibit tumoricidal properties, for example parvovirus H-1 (Dupressoir et al., 1996. Cancer Res, 49:3203-3208), Newcastle disease virus (Reichand et al., 1992. J. Surg. Res, 52:448-453) or retroviral vectors containing drug susceptibility genes (Takamiya et al., 1993. J. Neurosurg, 79:104-110). WO97/26904 and WO96/03997 disclose a mutant herpes simplex virus (HSV-1761) that inhibits tumour cell growth. Administration of HSV-1716 comprising a 759 base pair deletion in each copy of γ34.5 of the long repeat region (R _L) to tumour cells kills these cells. However, this virus is specific for neuronal cells as HSV is known to selectively inhabit the neuronal system. Furthermore, the use of common human pathogens as an oncolytic virus is limited as it is likely that the general population has been infected and acquired an immune response to such viruses. A preexisting immune response to a viral strain similar to the one used as a therapeutic agent in the treatment of a cancer may attenuate the effectiveness of the virus as therapeutic agent.

Other virus strains have reported oncolytic activity. The ONYX-015 human adenovirus (produced by ONYX pharmaceuticals) is believed to replicate preferentially in p53 negative tumour cells. This virus shows promise in clinical trials with head and neck cancer patients (Kim, D., T. et al., Nat Med, 1998. 4:1341-1342). Reovirus type 3 is being developed by Oncolytic Biotech as a cancer therapeutic, which preferentially grows in PKR−/− cells (Yin, H. S., J Virol Methods, 1997. 67:93-101; Strong, J. E. and P. W. Lee., J Virol, 1996. 70:612-616; Strong, J. E., et al., Virology, 1993. 197:405-411; Minuk, G. Y., et al., J Hepatol, 1987. 5:8-13; Rozee, K. R., et al., Appl Environ Microbiol, 1978. 35:297-300). Reovirus, type III exhibited enhanced replication properties in cells which expressed the mutatnt ras oncogene (Coffey, M. C., et al., Science, 1998. 282:1332-1334; Strong, J. E., et al., Embo J, 1998. 17:3351-1362). Mundschau and Faller (Mundschau, L. J. and D. V. Faller, J Biol Chem, 1992. 267:23092-23098) have shown that the ras oncogene product activated an inhibitor of PKR, and this coupled with the observation that the PKR chemical inhibitor 2-aminopurine increased the growth of Reo type III in normal cells implicates PKR is a critical regulator of the growth of reovirus.

WO 99/04026 teaches the use of VSV as a vector in gene therapy for the expression of a wide range of products including antibodies, immunogens, toxins, etc. for the treatment of a variety of disease disorders.

Interferons are circulating factors which bind to cell surface receptors activating a signalling cascade ultimately leading to a number of biological responses.

Two of the outcomes of interferon signalling are tightly linked: (1) an antiviral response and (2) induction of growth inhibitory and/or apoptotic signals.

U.S. Pat. No. 4,806,347 discloses the use of y Interferon and a fragment of INF-γ(known as Δ4α2) against human tumour cells.

WO 99/18799 reports the cytotoxic activity of Newcastle Disease Virus (NDV) and Sindbis virus towards several human cancer cells. However, both viruses demonstrated selectivity in their cytotoxic activity towards tumor cells.

WO 99/18799 discloses that interferon addition to normal cells renders these cells resistant to NDV, yet, this effect was not observed with interferon-treated tumor cells which continued to exhibit NDV-induced sensitivity. WO 99/18799 also discloses the cytotoxic activity of VSV cells against KB cells (head and neck carcinoma) and HT 1080 (Fibrosarcoma), and alleviation of cytotoxicity in normal and tumor cells, by VSV, in the presence of interferon. No other cell types were tested against VSV cytotoxic activity.

Certain mutant strains of VSV have been reported. Stanners, et al., Virology (1987) 160(1):255-8. Francoeur, et al., Virology (1987) 160(1):236-45. Stanners, et al., J. Gen. Virol. (1975) 29(3):281-96. Stanners, et al., Cell (1977) 11(2):273-81.

The present invention relates to viral formulations that are useful in the treatment of diseases and cancers, preferably leukaemia. Such formulations may also comprise an oncolytic VSV strain and a chemical agent, for example a cytokine which confers to normal cells, resistance to viral infection, but leaves diseased or cancerous cells susceptible to viral infection and lysis.

It is an object of the invention to overcome disadvantages of the prior art.

The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method of reducing the viability of a tumour cell comprising administering a virus to the tumour cell, wherein the virus is characterized as not being a common human pathogen. Preferably the tumour cell lacks PKR activity, and the virus is selected from the group consisting of rhabdovirus. More preferably the virus is VSV.

This invention is also directed to a method of reducing the viability of a tumour cell comprising administering a virus to the tumour cell, wherein the virus is characterized as being unable to inactivate PKR activity within a host cell. Preferably the virus is selected from the group consisting of vesicular stomatitis virus, picornavirus, influenza virus, and adenovirus.

The present invention also pertains to a method of reducing the viability a tumour cell within a population of cells comprising administering a virus to the population of cells, wherein the virus is characterized as being able to selectively infect and kill the tumour cell. Preferably the virus is further characterized by being unable to inactivate PKR activity in a host cell.

This invention also relates to the method as defined above, wherein the population of cells is treated with interferon prior to administering the virus.

This invention provides a method for identifying a tumor susceptible to treatment with a virus, comprising: (a) dividing a sample containing cells of the tumor into a first portion and a second portion; (b) treating the first portion with the virus; and (c) determining whether the percentage of dead cells in the first portion is higher than in the second portion, wherein the tumor is susceptible to treatment with the virus if the percentage of dead cells in the first portion is higher than in the second portion.

This invention provides a method for identifying a tumor susceptible to treatment with a virus, comprising: (a) dividing a sample containing cells of the tumor into a first portion and a second portion; (b) treating the first portion with the virus and an amount of interferon sufficient to improve survival of interferon-responsive cells in the presence of the virus, and treating the second portion with the virus in the absence of interferon; and (c) determining whether the percentage of dead cells in the first portion is higher than in the second portion, wherein the tumor is susceptible to treatment with the virus if the percentage of dead cells in the first portion is higher than in the second portion.

The present invention is directed to a mutant VSV, characterized in that the mutant VSV grows poorly in interferon-responsive cells. Such strains are also referred to herein as attenuated strains of VSV, or VSV strains that grow poorly in interferon-responsive cells. They can be identified by their producing smaller plaques in monolayers of interferon-responsive cells than in interferon-nonresponsive cells, as described below. Attenuated VSV strains can also be identified by their having a higher LD50 when administered intranasally to PKR+/− mice as compared to WT Indiana, in the assay described below.

The present invention also pertains to a method for isolating VSV using an affinity matrix, comprising adding the VSV to the affinity matrix to produce bound VSV, washing the bound VSV, and eluting the VSV from the affinity matrix. Also included in the present invention is a modified VSV that comprises a non-native fusion protein on the outer surface of the virus. The non-native protein may be fusion protein comprising an affinity tag and a viral envelope protein, or it may be derived from a producer cell.

The present invention is also directed to isolated nucleic acid molecules (DNA or RNA) having a sequence coding for mutant VSV proteins and sequences complementary thereto. Such nucleic acid molecules can be used in the preparation of a recombinant VSV or as a DNA vaccine.

There are several advantages for the use of a virus as described herein as a therapeutic virus over other viruses:

Rhabdoviruses are not common human pathogens. For example, VSV is found mostly in insects, rodents and domestic farm animals, and therefore a large proportion of individuals will not have been infected or immunized to VSV infection. On the other hand, Adenovirus or Reovirus are human pathogens and most of the general population have been infected and acquired an immune response to both of these viruses. A preexisting immune response to a viral strain similar to the one used as a therapeutic agent in the treatment of a cancer may attenuate the effectiveness of the virus as therapeutic agent;

VSV replicates much more quickly than either Adenovirus or Reovirus, and can be readily concentrated to high titres. Production of high titre virus preparations is a significant limitation of other potential viral therapeutic strains;

VSV is simple virus comprising only five genes, easily amenable to genetic manipulation. No such system is currently available for Reovirus;

Cellular infection by VSV is highly responsive to additional chemical agents such as interferon, a feature which enhances its therapeutic value.

VSV has a broad host range and is capable of infecting most types of human cells, whereas other viruses are more limited in regard to the types of cells they may infect.

VSV is a RNA virus and spends its entire lifecyle in the cytoplasm. Therefore it involves less danger of unwanted integration into the genome of a patient.

Collectively, these VSV attributes provide significant advantages over the use of the other viruses known to exhibit oncolytic activity.

This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: [0035]
FIG. 1 shows a general schematic of the interferon cascade. [0036]
FIG. 2 shows the effect of VSV on normal human fibroblasts, human melanoma cell line SK-MEL3, LNCaP a prostate cancer cell line, and the ovarian carcinoma cell A2780 in the presence and absence of interferon as determined by a modified cpe assay. Monolayers of cells were infected at an moi of 0.1 pfu in a 12 well plate. At [0037] time 0 and every 12 hours subsequent up to 48 hours, one well of infected cells was fixed with 0.5 ml Leukostat fixative for 2 minutes. At the end of the experiment monolayers were stained with Leukostat stains.
FIG. 3 shows the cytopathic effect of VSV in normal fibroblasts cells (FIG. 3 ([0038] a)), and tumour cell lines, including ovarian tumour cells (FIG. 3(b)) and KB tumour cells (FIG. 3(c)).
FIG. 4 shows the effect of VSV on normal human fibroblasts co-cultured with 293T tumour cells over a period of 24 hours. Co-cultures were infected at an moi of 0.1 pfu/cell and the infection allowed to proceed in the presence (IFN+) or absence (IFN−) of interferon. Cultures were stained with antibodies to large T antigen (red nuclei) to detect the 293T cells and with DAPI (blue nuclei) which stains all cell types. [0039]
FIG. 5 shows the effect of VSV in vivo on tumors implanted within nude mice. Human melanoma cells were implanted within nude mice and either mock injected (VSV(−)), injected with wild type VSV (data not presented), or injected with additional melanoma cells infected in vitro with VSV for one hour prior to injection into the tumour site VSV(+)). Size of the tumors were determined over a 7 day period. [0040]
FIG. 6 shows ulcers formed on a tumor produced within a nude mouse as described in FIG. 5. [0041]
FIG. 7: VSV and VSV infected cells inhibit growth of human melanoma xenografts in nude mice. [0042]
FIGS. [0043] 8A and 8B: PKR−/− mice are acutely sensitive to intranasal VSV infection and demonstrate a deficiency in IFN mediated resistance.
FIG. 9: Interferon can protect xenograft bearing nude mice during VSV treatment. [0044]
FIGS. [0045] 10A and 10B: Virus production from tumour cells and normal cells infected with wild type Indiana and various mutant VSV strains.
FIG. 11: Malignant cells are rapidly killed following VSV (WT Indiana) infection and are not protected by IFN-α. [0046]
FIG. 12: VSV induced cytopathic effect visible in human melanoma cells but not in primary human cells with or without IFN-α. [0047]
FIG. 13: Efficacy of a single intravenous dose of mutant VSV in treating human melanoma xenografts in nude mice. [0048]
FIG. 14: N Protein cDNA sequence of wild type and mutant VSVs. [0049]
FIG. 15: N Protein amino acid sequence of wild type and mutant VSVs. [0050]
FIG. 16: P Protein cDNA sequence of wild type and mutant VSVs. [0051]
FIG. 17: P Protein amino acid sequence of wild type and mutant VSVs. [0052]
FIG. 18: M Protein cDNA sequence of wild type and mutant VSVs. [0053]
FIG. 19: M Protein amino acid sequence of wild type and mutant VSVs. [0054]
FIG. 20: G Protein cDNA sequence of wild type and mutant VSVs. [0055]
FIG. 21: G Protein amino acid sequence of wild type and mutant VSVs. [0056]
FIG. 22: L Protein cDNA sequence of wild type and mutant VSVs. [0057]
FIG. 23: L Protein amino acid sequence of wild type and mutant VSVs.[0058]

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a novel cancer therapeutic. More specifically, this invention relates to viruses that selectively infect and inhibit tumour cell growth. [0059]
The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect. [0060]
Cancer cells gain a survival advantage over their normal counterparts by acquiring mutations in growth inhibitory or apoptotic pathways and, in the case of interferons, would do so at the expense of critical antiviral defence mechanisms. As tumour cells gain a significant growth advantage by mutating interferon response genes, they will be more susceptible to virus infection. [0061]
By “reducing the viability” of a tumour cell it is meant either killing the tumour cell or limiting its growth for a period of time. [0062]
By “not a common human pathogen” it is meant a virus that is found mostly in non-human hosts, for example, but not limited to insects, rodents, and farm animals. Such viruses are not typically found within the general human population. [0063]
As used herein Mutant I, [0064] Mutant 1, Mut 1 and M1 refer to attenuated mutant strain T1026. Mutants II, I, IV and V (and variant nomenclature analogous to Mutant I) refer to attenuated mutants T1026R, TP3, TP6 and G31, respectively.
The novel cancer therapeutic of the present invention incorporates the use of at least one oncolytic virus that selectively targets tumour cells and leads to their destruction. Preferably the oncolytic virus is a Vesicular stomatitis virus (VSV), for example the Indiana strain, or other strains, or a derivative thereof. By a derivative of VSV, it is meant a VSV virus obtained by either selecting the virus under different growth conditions, or one that has been subjected to a range of selection pressures, or one that has been genetically modified using recombinant techniques known within the art. For example, which are not to be considered limiting in any manner, a derivative of VSV may include a mutant VSV selected following infection on a human cell that has been treated with interferon as described herein, or a VSV that displays an affinity tag useful for affinity purification. [0065]
The effectiveness of oncolytic virus suppression of tumour cell growth in part resides in the differential susceptibility of tumour cells, compared to normal cells, to viral infection. Without wishing to be bound by theory, the differential susceptibility may in part be due to the down regulation or inactivation of factors within a cell that otherwise function to protect the cell from tumorous growth and virus infection. Examples of factors that when inactivated result in tumorous cell growth, and that are also involved in mediating virus infection include but are not limited to PKR (double stranded RNA dependent kinase) and PML (Promyelocytic Leukemia gene), however, it is to be understood that other factors may also play a role. [0066]
The down regulation or inactivation of PKR, through a variety of mechanisms including but not limited to PKR-related mediators, is known to be associated with tumour cell growth, while normal cells exhibit active PKR. Furthermore, wild type cells exposed to viral infection exhibit elevated PKR expression which results in the suppression of viral replication, while cells that exhibit reduced, or no, PKR activity are susceptible to viral attack and exhibit cancerous growth. Similarly, the PML gene product functions as a tumour suppressor and it is also known to suppress viral replication. [0067]
By “differential susceptibility”, it is meant a property associated with a cell that results in both tumour cell growth and the inability of the cell to suppress viral replication. Cells exhibiting differential susceptibility are preferred candidates for treatment of tumorous cell growth using the cancer therapeutic of the present invention. This differential susceptibility may be accentuated through the addition of one or more chemical agents prior to or during treatment of the tumour cell. Preferably, this chemical agent increases the resistance of a wild-type cell to viral infection, but has little or no effect on the response of a tumour cell to viral infection. An example, which is not to be considered limiting in any manner, of such a chemical agent is interferon. [0068]
By “PKR” it is meant a serine/threonine kinase that exhibits multiple functions including roles in the control of mRNA translation and gene transcription (1,2). The kinase harbors two double-stranded RNA dsRNA binding motifs in its amino terminal regulatory half and catalytic kinase domain in its carboxyl tail. Binding of dsRNA to the amino terminus induces a conformational change in the enzyme revealing and activating the catalytic kinase domain. The expression of PKR is induced by several PKR-mediators, including but not limited to, interferon. [0069]
By “PKR-mediator” it is meant proteins or compounds that directly, or indirectly affect PKR activity either at the gene or protein level and include both PKR-activators and PKR-inhibitors. Examples of PKR-activators include, but are not limited to STAT1 (see FIG. 1), Interferon regulatory factor (IRF-1), and interferon. Examples of PKR-inhibitors, include, but are not limited to, VA RNAs, p58(IPK), factors associated with the Ras pathway, the ribosomal protein L18, or proteases that degrade PKR protein. PKR activity may also be mediated through mutations to the gene encoding PKR, or to the regulatory region that drives the expression of PKR. These mutations may either increase or decrease PKR activity. Mutations to PKR that reduce PKR activity include, but are not limited to, the loss of dsRNA binding ability of PKR, or mutations that result in negative catalytic mutants. Mutations that increase PKR activity include, but are not limited to over-expression of PKR, or mutations that resulted in a more active PKR protein. [0070]
PKR regulates translation through the phosphorylation of eIF-2α, a factor involved in the initiation of protein translation. Once phosphorylated, eIF-2α-GDP, forms an inactive complex with eIF-2B resulting in a rapid inhibition of protein synthesis. PKR impinges on gene transcription indirectly via activation of NFκB. This activation appears to be carried out by PKR phosphorylation of an IκB kinase (3) which in turn phosphorylates IκB leading to its targeted destruction. [0071]
PKR Antiviral Activity [0072]
Infection of a cell by many distinct virus types leads to the formation of dsRNA (e.g. as replicative intermediates) resulting in the activation of PKR and its subsequent downstream effectors (see FIG. 1). In particular, protein synthesis is rapidly terminated and an apoptotic cascade is initiated (4,5). As a result of the activation of PKR, the production of new virions is curtailed and the spread of virus through the organism is limited. Der et al (12) report a requirement for PKR in the induction of cellular apoptosis in response to a variety of stress inducers. [0073]
Without being bound by theory, it is possible that malignancies arise as a result of multiple mutations in genes that control cell proliferation and apoptosis. PKR's role in regulating protein synthesis coupled with its antiproliferative and pro-apoptotic properties make it a target for oncogenic mutations, which directly or indirectly affect its activity. [0074]
As described in more detail in the examples an initial screen of several viruses using PKR−/− animals indicated that PKR null animals are susceptible to infection by Vesicular stomatitis virus (VSV). Similar results were obtained in vitro, where VSV infection proceeded more rapidly in PKR−/− fibroblasts, when compared to infection in PKR+/+ fibroblasts. These results demonstrate that PKR is required by mammalian cells to resist infections by VSV. Furthermore, certain cell lines, for example, but not limited to primary human bone marrow, were resistant to VSV infection, while leukemia cell lines were susceptible to VSV infection. [0075]
It is contemplated that viruses related to VSV, or other viruses that exhibit similar mechanisms of viral infection can be identified that exhibit the property of selectively infecting cells with reduced or no PKR activity. One of skill in the art can readily screen other viruses using the methods as described herein, for their ability to reduce the viability of cells, or kill cells lacking PKR activity, or PKR−/− cells, PKR−/− animals, or both PKR−/− cells and animals. [0076]
As indicated in the examples below, pretreatment of cells with interferon reduces virus infectivity by several orders of magnitude. Without wishing to be bound by theory, the addition of interferon may upregulate PKR expression resulting in this increased resistance to viral infection. [0077]
Because of its potent antiviral activity, viruses have evolved strategies to circumvent PKR. For example, HIV and Hepatitis C encode proteins dedicated to the binding and inactivation of PKR (6,7). Adenovirus encodes small RNA molecules (VA RNAs) which bind to but do not activate PKR (8). Influenza virus usurps a cellular protein p58(IPK) to inhibit PKR while polio virus initiates the proteolytic degradation of PKR (9,10). Large T antigen of SV-40 appears to function downstream of eIF-2α to promote protein translation even in the presence of activated PKR (10). [0078]
PKR and Tumour Suppression [0079]
Expression of dominant negative PKR catalytic mutants in NIH 3T3 cells leads to their malignant transformation and facilitates their growth as tumours in nude mouse models (13,14). A similar phenomena has been observed using PKR mutants which have lost dsRNA binding activity. Induced expression of PKR in [0080] S. cerevisiae leads to growth arrest in the yeast—a phenomena that can be reversed by co-expression of a non-phosphorylatable version of eIF-2α. Therefore, PKR has anti-proliferative activity and functions as a tumour suppressor.
There are several lines of evidence that PKR is inactivated, absent or reduced in expression in a broad spectrum of human malignancies: [0081]
Oncogenic Ras mutations occur in about 30% of all human tumours while mutations in upstream Ras activators (ie EGF receptor, Neu receptor, PDGF receptor) are even more common. Mundschau and Faller (15,16) have described an oncogenic Ras induced PKR inhibitor. Furthermore, Strong et al (17) demonstrated that activation of the Ras pathway results in down regulation of PKR activity. [0082]
The ribosomal protein L18 is overexpressed in primary colorectal cancer tissues and has recently been shown to bind to and inactivate PKR (18). [0083]
Patients with 5q translocations exhibit diminished PKR expression (19-21). Interferon regulatory factor 1 (IRF-1) is a transcription factor with tumour suppressor activity, which maps to the human chromosomal region 5q. PKR gene transcription is regulated in part by IRF-1. [0084]
Human PKR maps to 2p21-22 and has been recently identified as the site of translocation in a case of acute myelogenous leukemia. [0085]
In biopsies from poorly differentiated, highly malignant tumours, PKR protein was present at very low levels or was undetectable (23-25). [0086]
STAT1 [0087]
STAT1 is an essential mediator of the interferon pathway and its activation results in an upregulation of PKR mRNA and protein (see FIG. 1). [0088]
There is marked deficiency in the level/activity of STAT1 protein in interferon resistant melanoma cell lines and primary melanoma biopsy material (26), in a variety of human tumour cell lines including a myeloid leukemia, cervical carcinomas, ovarian cancer, and a lung carcinoma (27), and in a gastric adenocarcinoma (28,29). Furthermore, cutaneous T cell lymphoma (CTCL) is a malignancy which in general is responsive to interferon (however frequently clinical resistance arises in a substantial portion of cases). Sun et al (30) have reported that STAT1 protein is absent in a CTCL cell line suggesting that development of clinical resistance to interferon may arise due to STAT1 mutations. [0089]
PML: Promyelocytic Leukemia Gene [0090]
PML is an interferon induced gene that normally functions as a tumour suppressor and a key regulator of Fas, TNFα and interferon induced apoptosis. Recently, Chelbi-Alix et al have shown that another normal function of the PML gene product is to suppress virus replication. The PML-RAR fusion protein functions as a dominant negative inhibitor of interferon induced apoptosis and we would predict will also make APL cells preferentially susceptible to virus infection. [0091]
Down regulation of PKR protein or activity occurs in a broad spectrum of human malignancies. While cancer cells have attained a growth advantage and unbridled protein translation capacity by eliminating PKR or PKR-mediators, these cells have simultaneously eliminated one of the cell's primary and potent antiviral defence mechanisms. Therefore, tumour cells with reduced PKR activity will be more susceptible to infection than their normal counterparts. As indicated above, other components (e.g. STAT1 and PML) of the interferon pathway are frequently mutated in human malignancies, and loss of their activity will render tumour cells sensitive to virus infection. This differential susceptibility forms the basis for the use of viral-based cancer therapeutics of the present invention for the treatment of tumorous cell growth. [0092]
Screening of PKR null mouse strains with several different viruses indicated that PKR null animals are capable of suppressing a number of virus infections including vaccinia, influenza and EMCV. However, Vesicular Stomatitis Virus (VSV) exhibited an ability to infect PKR−/− animals. VSV, a member of the Rhabdovirus family, was observed to kill 100% of PKR null animals following intranasal infection by as little as 50 infectious virus particles (or plaque forming units, pfu). In contrast, over 20,000 times as many VSV particles were required to kill half of infected wild type littermates. [0093]
VSV is an enveloped, negative sense RNA virus with a simple five gene genome. This is a very well characterized virus family with several serologically distinct laboratory strains and a multitude of characterized mutants. The natural hosts of VSV include insects, rodents and domestic farm animals. In general, very few North Americans have come in contact with the virus—most human infections occurring in laboratory personnel and farmers. In humans infections are either asymptomatic or manifested as mild “flu”. There are no reported cases of severe illness or death amongst infected humans. [0094]
The ability of VSV to selectively infect tumour cells over wild-type cells was also observed. Tumour cell lines, following an overnight infection exhibited a 100 to 1000 times higher rate of infection than that detected in normal primary fibroblasts. Furthermore, the cytopathic effect (cpe) was accelerated in the tumour cell cultures. [0095]
Since PKR is an interferon inducible gene product, pretreatment of cells with interferon prior to exposure to VSV was tested to determined the effect of viral infection. Wild-type cell cultures, that were pretreated with interferon, were resistant to VSV infection, while tumour cell lines, for example, but not limited to, fibrosarcoma, melanoma, prostate carcinoma, leukaemia and ovarian sarcoma, were susceptible to virus infection (see Table 1, Example 2; FIG. 2). Lung carcinoma cells (LC80) were also susceptible to VSV infection in the presence and absence of interferon (data not presented). However, several tumour cell lines were resistant to VSV infection in the presence of interferon. [0096]
Ovarian carcinoma cells, fibrosarcoma, lung carcinoma, melanoma, prostate carcinoma, lung carcinoma, and leukaemia cells are VSV sensitive, and this sensitivity was maintained in the presence of interferon, therefore, such tumor cells and cancers derived therefrom may be particularly amenable to VSV treatment. However, other cancers may also be amenable to viral treatment as described herein. Studies with respect to VSV sensitivity using primary tumour material is readily available in ascites fluid. Further, since the tumour is contained within the peritoneal cavity it may prove particularly suited to localized administration of a virally based therapeutic. In this regard, live tissue from patient's ascitic fluid can be tested for the ability of the tumour cells to support VSV infection in the presence and absence of interferon. [0097]
It is expected that VSV will have therapeutic activity in vivo, and will have the ability to kill distant (metastatic) tumour growths. To date no significant organ pathology in treated mice have been observed, however, the kinetics of VSV viremia need to be further studied. Nude mice, implanted with human melanoma cells received VSV, or additional melanoma cells infected in vitro with VSV (see Example 5), to ensure the continuous production of infective particles to the tumour over a several hour period, via injection (FIG. 5). In mock-injected animals (VSV(−); injection with vehicle alone) tumours grew continuously over the course of the experiment. Animals which received only pure virus showed initially continuous growth of tumours over the first four day period, after this time the tumours began to reduce in size and continued to do so over the course of this experiment. Tumours that were injected with infected cells stopped growing and regressed to small hard nodules resembling scar tissue. In some of the larger injected tumours, ulcers formed on the tumour within 1-2 days, (see FIG. 6). While both injection of purified virus and infected melanoma cells caused significant regressions, infected producer cells were more effective. [0098]
Studies with an immunocompetent mouse tumour model (i.e. as described by Strong et al; 17) will examine the affects of antibody response to therapeutic VSV infection, and determine if VSV infection of tumour cells increases their immunogenicity and promotes recognition of tumour antigens by the host organism. [0099]
Primary human bone marrow was also found to be resistant to VSV infection in the absence of interferon pretreatment (see Table 1, Example 2), indicating that these cells have an innate resistance to VSV infection. In contrast two leukemia cell lines (M07E and L1210) were also tested and found to be susceptible to VSV infection as evidenced by cytopathic effect, virus growth and loss of cell viability. [0100]
While the results disclosed herein relate to VSV, it is to be understood that one of skill in the art, by following the methods outlined in this document, will be readily able to screen other VSV strains, derivatives of VSV including mutants of VSV, or related viruses for the ability to selectively kill tumour cells. There are several other serologically and biologically distinct strains of VSV, which can be tested for this property. Such VSV strains include, but are not limited to New Jersey, Piry, Coccal, and Chandipura. Identification of other suitable serologically unrelated strains may be useful if sequential VSV injections are required to completely eradicate tumours. Furthermore, picornaviruses (eg rhinoviruses) are known to be relatively innocuous to normal human tissues yet grow extremely well in transformed cells in tissue culture, and these viruses may also be used. Furthermore, combinations of viruses may be used to enhance the cytopathic effect observed with VSV. [0101]
In order to determine whether the presence of either a normal or tumor cell could affect the other cell type (either normal and tumor cell) and alter the resistance or susceptibility of either of these cells to VSV infection, normal cells and fibroblasts were co-cultured in the present of VSV. The culture was infected at an moi of 0.1 pfu/cell and the infection allowed to proceed in the presence or absence of interferon. At 0, 12 and 24 hours (FIG. 4) the cultures were fixed and stained with antibodies to large T antigen (red nuclei) to detect the 293T cells and with DAPI (blue nuclei) which stains all cell types (FIG. 4). The number of 293T cells (red nuclei) steadily declined during the time course and displayed severely condensed or fragmented nuclei characteristic of a cell dying from virally induced apoptosis. This selective destruction of the transformed cells was seen both in the presence and absence of interferon. The normal fibroblasts did not develop nuclear changes nor were their numbers reduced in response to VSV infection even though 293T cells were producing copious amounts of virus within the co-culture. This indicates that mixtures of cell populations may be treated with VSV while still maintaining tumor cell sensitivity, and normal cell resistance to VSV. [0102]
In addition there are a number of mutants of VSV, for example, but not limited to mutants which are impaired in the shut down of host protein synthesis or are more or less sensitive to interferon, which may exhibit differential infection between normal and tumour cells. For example, which is not intended to be limiting in any manner, other viral mutants are known which show tropism for STAT1 or PKR negative cells include an influenza virus strain which is unable to inactivate PKR has been described (36) Adenovirus mutants which lack the PKR inactivating VA gene are known to grow better in the absence of PKR. [0103]
As described herein, VSV mutants were isolated that grew poorly on interferon responsive cells. These mutants were selected based upon their ability to form small plaques in monolayers of interferon-responsive cells. On interferon non-responsive cells (i.e. tumor cells) these mutants form large plaques. The selection of mutants by size of plaque in interferon-responsive cells allows for the isolation of virus that grows poorly in normal cells. However, other VSV mutants may be obtained under different selection criteria. Mutants isolated using interferon-responsive cells were amplified and tested for their ability to kill tumour and normal cells. The rationale here is that VSV mutants, which can induce interferon in target cells, would limit their own replication in an interferon responsive cell population. These same viruses would however have unrestricted growth in tumour cells that lack interferon responsiveness. These mutants are of value, as they have even less cytopathic effect on normal tissues while maintaining oncolytic activity than wild type VSV. [0104]
Four mutants (Mut 1-4) were obtained based on their ability to form plaques in monolayers of interferon-responsive cells. These mutants, and wild type virus (moi of 1.0 pfu/cell) were used to infect melanoma cells and normal human foreskin fibroblasts. All of the mutants were able to kill tumour cells efficiently but normal cells infected with the mutants even after long periods of infection appeared completely uninfected. At this same moi wild type VSV demonstrated a cytopathic effect on the normal cells. These results indicate that the mutant virus have a greater therapeutic effect in that they kill tumour cells efficiently while sparing normal cells, and that they also have the ability to produce more virus particles and increase virus spread throughout the tumour (see Example 4). Surprisingly these mutants grew more rapidly than wild type VSV (Indiana) in HCT 116 colon carcinoma cells but not in OSF7 cells (See Example 21 and FIGS. 10A and 10B). VSV mutants that display rapid growth in the tumour cell of interest but not in normal cells are preferred. [0105]
Earlier experiments indicated that PKR−/− mice were killed with VSV by several routes of infection, however, these mice were not affected by intravenous injections of the virus. In order to determine whether plasma components were inactivating the virus upon contact, VSV produced from several sources including within mouse L cells was incubated with human serum (from normal uninfected donor) and the virus titer after incubation determined (Example 6). The viral titer of L cell-produced VSV dropped four hundred fold, while VSV produced in human melanoma cells was unaffected by incubation in plasma. These results indicate that the choice of cell line for the production of VSV is critical. Based on this observation it is possible to screen human cell lines for those that produce optimum amounts of virus that is not sensitive to human serum. [0106]
Without wishing to be bound by theory, it may be that the difference in these two virus preparations reflects the nature of the cohort of proteins found on the surface of the virus producing cells. As part of its replicative cycle, VSV buds through the plasma membrane and acquires cellular protein on its envelope. Certain proteins found on L cells when expressed in the context of the virus particle could activate complement. Indeed, it has been shown earlier that retrovirus particles produced in certain mouse cells are inactivated by serum while the same virus produced in a subset of human cell lines was unaffected by plasma. (Pensiero, M. N., et al. Hum Gene Ther, 1996. 7:1095-1101). [0107]
Conventional techniques for VSV production are difficult to scale up for industrial production. Therefore, the purification of VSV, using an affinity matrix, for example affinity chromatography was explored. (See Example 7). However, other protocols for affinity purification may also be used as known within the art, for example, but not limited to, batch processing a solution of virus and affinity matrix, pelleting the VSV-bound matrix by centrifugation, and isolating the virus. In order to provide the virus with an affinity tag to be used for the purification of the virus, the virus may be genetically modified, using techniques well known in the art, to express one or more affinity tags on its surface, preferably as a fusion viral envelope protein, or producer cell lines may be engineered to express one or more affinity tags on their plasma membranes which would be acquired by the virus as it buds through membrane, however, endogenous viral envelope proteins may also be used. One well characterized affinity tag involve the use of Histidine residues which binds to immobilized nickel columns, however, it is to be understood that other affinity tags may also be employed. [0108]
Cell lines can be prepared that act as a universal producer of VSV, or other virus, that expresses a chimeric VSV protein with nickel binding, or other affinity tag properties. This universal producer cell may be used for the production of a chimeric protein (affinity tag) for any enveloped virus (including all enveloped RNA and DNA viruses). For the purification of virus which bud through the nuclear membrane (such as Herpes virus), a tag to be expressed on the viral envelope protein expressed in the nuclear membrane is engineered. [0109]
Other affinity tags include antibodies, preferably an antibody which recognizes a particular peptide under conditions of low salt, low temperature or in the presence of a critical cation/anion. Physiological salt concentrations, thermal elution or chelation could effect elution. Antibodies generated against di or tripeptides may also be used for purification. In this manner, two or more of these tags on the surface of a single virus particle would allow for the sequential affinity purification of the virus. [0110]
VSV may be genetically modified in order alter its properties for use in vivo. Methods for the genetic modification of VSV are well established within the art. For example a reverse genetic system has been established for VSV (Roberts A. and J. K. Rose, Virology, 1998. 247:1-6) making it possible to alter the genetic properties of the virus. Furthermore, standard techniques well known to one of skill in the art may be used to genetically modify VSV and introduce desired genes within the VSV genome to produce recombinant VSVs (e.g. Sambrook et al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press). [0111]
VSV may be targeted to a desired site in vivo to increase viral efficacy. For example, modification of VSV G protein to produce fusions that target specific sites may be used to enhance VSV efficiency in vivo. However, it is to be understood that other protein targets in addition to the VSV G protein may also be modified to produce such fusion proteins. Such fusion proteins may comprise, for example, but not limited to, Single chain Fv fragments (Lorimer, I. A., et al. Proc. Natl. Acad. Sci. U.S.A., 1996. 93:14815-20) that have specificity for tumour antigens. An example of such a single chain Fv fragment that may be used to prepared a VSV G fusion protein, is an Fv fragment that targets a mutant EGF receptor found on about 80% of human breast tumour cells. [0112]
VSV may also be modified to express one or more suicide genes capable of metabolizing a pro-drug into a toxic metabolite thereby permitting VSV infected cells to be killed by administration of a pro-drug. For example, VSV comprising the herpes virus thymidine kinase gene or the cytosine deaminase gene encodes an enzyme that can convert ganciclovir or 5-FC, respectively, into a toxic compound. However, it is to be understood that other suicide genes may also be employed. As it is well established that ganciclovir metabolites kill not only cell expressing HSV TK but also cells in the immediate vicinity, rVSV comprising these suicide genes exhibit several advantages. For example, the effective killing by the virus is increased since one infected cell kills ten or more surrounding tumour cells, furthermore rVSV comprising a suicide gene permits the elimination of virus if desired from an individual infected with the virus. This may be important in situations where it is unclear how VSV may affect an individual. For instance, an immune comprised individual may be unexpectedly susceptible to VSV. Thus the addition of a suicide gene would be an improvement on the safety of the viral therapeutic. [0113]
VSV may also be modified by the introduction of a mammalian gene product. Such a mammalian gene product would limit VSV growth in normal cells, but not the growth of VSV in tumour or diseased cells. For example, rVSV capable of expressing one or more transactivators of p53, activates apoptotic pathways in normal cells but not tumor cells. Such rVSVs therefore selectively limit virus spread in normal tissues. However, it is to be understood that other mammalian gene products may also be expressed within VSV for this purpose. Another example, which is not to be considered limiting in any manner is the PKR gene. A rVSV expressing the PKR gene limits virus replication in all normal cells, however, in cells that express PKR inhibitors, the virally encoded PKR is inactivated. An example of a cell that expresses one or more PKR inhibitors is a chronically Hepatitis C infected cell. Since Hepatitis C encodes and expresses two known inhibitors of PKR (i.e. NS5A and E2), a VSV encoded PKR gene product is be neutralized, and VSV allowed to replicate freely. [0114]
The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution. [0115]
The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner. [0116]

EXAMPLE 1

PKR Negative Cells are Susceptible to VSV Infection

In Vivo Experiments [0117]
Initial studies were directed to identifying viruses that are capable of infecting PKR−/− animals and cells. Using homologous recombination strategies, PKR null mouse strains were generated (35, which is incorporated by reference) and tested for their ability to fight virus infections. Since these mice are PKR−/−, they should be susceptible to virus infection. Several species of virus were administered to PKR null animals over a range of concentrations. [0118]
Infection of PKR Null Mice: [0119]
A PKR null mouse line was generated using conventional knockout technology (Abraham, N., et al., J Biol Chem, 1999. 274:5953-5962.). Groups of five female mice, 3 months of age or greater, were infected intranasally with varying amounts of vesicular stomatitis virus (Indiana strain). Age matched wild type animals were infected in parallel and both sets of animals were monitored on a daily basis for signs of infection. These include, hydration, piloerection, activity level, appetite, hind limb paralysis, respiratory rate, body weight and any other symptoms indicating that the animal was in distress. [0120]
Wild type animals showed few and only transient symptoms at multiplicities of infection up to 10[0121] ⁵pfu with VSV. In contrast, PKR null animals very rapidly developed dehydration, piloerection, loss of appetite, rapid respiratory rate, decreased activity and squinting crusty eyes. At high doses of VSV infection (10⁵pfu) the animals showed symptoms in less than 24 hours and usually succumbed to the infection within 48 hours. At doses of infection as low as 25 pfu, 100 percent of the PKR null animals died of VSV infection within 5 days. In separate experiments groups of five wild type and PKR null animals were sacrificed at 48 hours post infection with VSV and organs were removed to assess viral titres. In the PKR animals titres in excess of one million PFU/ml of lung homogenate were found at this time while in wild type animals virus titres ranged from 0 to 100 pfu per ml of lung homogenate. In the wild type and PKR null animals similar amounts of virus were found in the brain. The remainder of the tissues in both mouse strains had undetectable virus at this time post infection.
Vesicular stomatitis virus, a member of the Rhabdovirus family was able to kill 100% of PKR null animals following intranasal infection by as little as 50 infectious virus particles (or plaque forming units, pfu). In contrast, over 20,000 times as many VSV particles were required to kill half of infected normal littermates. [0122]
These results indicate that PKR null animals are capable of suppressing a number of virus infections including vaccinia, influenza and EMCV. However, VSV exhibited an ability to infect PKR−/− animals. These results also indicate that PKR is required by mammalian cells to resist infections by VSV (Indiana laboratory strain). [0123]

EXAMPLE 2

Selective Killing of Tumour Cells with VSV

In Vitro Experiments [0124]
Several tumour cell lines were chosen at random from the Ottawa Regional Cancer Center and tested for their susceptibility to VSV infection. Primary fibroblast cultures from healthy adult volunteers or primary bone marrow samples from healthy donors were used as control cells. [0125]
Infection of Tumour Cells with VSV: [0126]
As a first test of the oncolytic properties of VSV, virus production and cytopathic effect following an overnight incubation with VSV was assessed. Monolayers of cells were incubated with the Indiana strain of VSV at a multiplicity of infection (moi) of 0.1 plaque forming units (pfu). After allowing virus to adsorb for 30 minutes at 37 C, the cultures were rinsed thoroughly with phosphate buffered saline (PBS) and then cultured an additional 18 hours at 37 C. At this time, the cultures were examined microscopically for cytopathic effect (cpe) and photographed. The 18 hour supernatant was removed and virus titres per ml of medium determined. In some experiments, cultures were preincubated for 12 hours with human alpha interferon (100 units/ml) prior to infection. [0127]

To examine the kinetics of infection of the assorted cell types a modified cpe assay (Heise, C., et al., Nat Med, 1997. 3:639-645) was used. Essentially, monolayers of cells were infected at an moi of 0.1 pfu in a 12 well plate. At time 0 and every 12 hours subsequent up to 48 hours, one well of infected cells was fixed with 0.5 ml Leukostat fixative (Fisher Diagnostics) for 2 minutes. At the end of the experiment monolayers were stained with

Leukostat stains

1 and 2 following manufacturers instructions. Since PKR is an interferon inducible gene product, the pretreatment with interferon, 100 units/ml of human alpha interferon 12 hours prior to infection, was tested to determined if interferon could enhance protection within the assorted cell cultures. The data are presented in Table 1 and FIGS. 2-3.

TABLE 1


Cell lines tested for VSV sensitivity

			Untreated	Interferon
			Overnight Virus	Overnight
Cell line	cell type	Reference	Yield	Virus Yield

OSF 16	human normal fibroblast	ORCC¹	1 × 10⁵pfu	0 pfu
AG1522	human foreskin fibroblast	[20]		0 pfu
OSF 7	human normal fibroblast	ORCC	1 × 10⁶	0 pfu
OSF 12	human normal fibroblast	ORCC	2 × 10⁵pfu	0 pfu
MN11	mouse fibrosarcoma	[21]	1 × 10⁸	1 × 10⁴
A2780	human ovarian carcinoma	[22]	2 × 10⁸	1 × 10⁷
H-1078	normal human bone	ORCC	0 pfu (moi 10 pfu)	Not determined
	marrow
M07E	human leukemic cell line	[23]	2 × 10⁶(moi 1.0 pfu)	Not determined
L1210	mouse leukemic cell line	[24]	4 × 10⁶	2 × 10⁴
SK-MEL3	human melanoma	[25]	Not determined:	Not determined: cpe
			cpe assay	assay positive
			positive
LNCAP	human prostate	[26]	Not determined:	Not determined; cpe
	carcinoma		cpe assay	assay positive
			positive
293T	fibrosarcoma transformed	[27]	1 × 10⁸	8 × 10⁷
	with SV-40 Large T and
	Adeno E1A
OVCA 432		[28]	1 × 10⁷	0 pfu
C13	ovarian carcinoma	[29]	1 × 10⁸	1 × 10⁵
OVCA 3		[30]	5 × 10⁷	Not determined
COS	Large T transformed	[31]	2 × 10⁸	Not determined
	simian kidney cell line
HCT 116	colon carcinoma	[32]	Not determined:	Not determined
			cpe assay	cpe assay positive
			positive
OVCA 420		[28]	1 × 10⁸	3 × 10⁶

1 established at the ORCC from forearm biopsy. [0129]
From the data in Table 1 it can be seen that although normal human fibroblasts can support viral replication, the amount of virus produced and the progression to cell lysis was substantially delayed when compared to tumour cells. An even more substantial difference in virus production was observed following pretreatment with interferon. While normal human fibroblast monolayers were completely protected from the cytolytic affect of VSV by interferon, tumour cells remained sensitive, producing copious amounts of viral particles and rapidly undergoing cytolysis. [0130]
Other cells lines, inlcuding a lung carcinoma cell line (LC80) and a leukaemia cell line, AML5 (acute myelogenous leukemia 5) cells were also found to be effectively killed by VSV. In the case of AML5, at a moi of 1.0 pfu/ml cells were completely killed within 24 hours, while at 0.0001 pfu/ml the cells were killed within 72 hours, further indicating the sensitivity of leukaemia cells to VSV. [0131]
As can be seen in FIG. 2, monolayers of tumour cells were much more rapidly destroyed by VSV infection as compared to normal human fibroblasts. The human melanoma cell line SK-MEL3, the LNCaP prostate cancer cell line and the ovarian carcinoma cell A2780 all showed substantial cpe as early as 12 hours post infection. Although the normal human fibroblast cultures were infected and capable of producing virus (see Table 1), the kinetics of infection was substantially slower than in the three tumour cell lines tested in this experiment. In addition, as with the overnight virus growth assay (Table 1, FIG. 2), interferon alpha treatment completely protected the normal human fibroblasts, but was ineffective at protecting the three tumor cell lines from the cytopathic effect of VSV. [0132]
The results obtained for Table 1 demonstrate that a screening strategy for determining the types of tumours which are susceptible to killing by VSV may be employed using for example, but not limited to, the NIH/NCI standard panel of tumour cell lines available from ATCC. These cell lines are screened in order to determine the time to complete cpe and/or virus growth using various initial multiplicities of infection. These experiments are done in the presence and absence of interferon so that the number of and types of tumours that are VSV sensitive and are resistant to interferon's antiviral activity are determined. [0133]
VSV Treatment of Leukemia [0134]
VSV does not productively infect bone marrow stem cells, even at high moi of 10 pfu/cell (H-1078; Table 1). The treated cultures retained all of their stem cell characteristics. Two leukemia cell lines (M07E and L1210; Table 1) were killed following an overnight infection and produced large amounts of virus. [0135]
To determine whether VSV could kill primary leukemia cells from a cancer patient, a peripheral blood sample was obtained from an AML patient and white blood cells collected and plated in RPMI media plus 10% FBS (10[0136] ⁷/well in 6 well plate, each infection in duplicate). Cells were mock infected or infected at an moi of 10.0/cell. VSV selectively killed myeloid leukemic cells as indicated by the decrease in the percentage of blast cells (leukemic blasts), while the overall cell number was minimally affected (i.e. neutrophils flourished). The leukemic sample produced titres of VSV exceeding 10⁷pfu/ml at 16 hours post infection. The number of blast cells in the sample was dramatically reduced at 21 hours post infection while the proportion of normal neutrophils increased. Mock infected cells (−VSV) contained almost 70% blast cells in a monolayer, while in cells infected with VSV (+VSV) normal cells predominated. These results demonstrate VSV is able to preferentially kill primary leukemic blast cells while sparing normal blood cells.

EXAMPLE 3

Killing of Tumour Cells in Mixed Cultures

Normal human fibroblasts and 293T tumour cells were co-cultured in a 50:50 mixture. Since 293T cells express the large T antigen which is not found in normal cells, the two cell types can be distinguished by immunofluoresence. [0137]
In this experiment cultures were infected at an moi of 0.1 pfu/cell and the infection allowed to proceed in the presence or absence of interferon. At 0, 18 and 24 hours (FIG. 4) the cultures were fixed and stained with antibodies to large T antigen (red nuclei) to detect the 293T cells and with DAPI (blue nuclei) which stains all cell types (FIG. 4). Initially both cell types displayed a spindle-like morphology with large oval nuclei. After 18 hours the number of 293T cells (red nuclei) were reduced and many of the remaining 293T cells displayed altered nuclear morphology. By 24 hours post-infection very few 293T cells were detected and those few that remained displayed severely condensed or fragmented nuclei characteristic of a cell dying from virally induced apoptosis. [0138]
This selective destruction of the transformed cells was seen both in the presence and absence of interferon. The normal fibroblasts did not develop nuclear changes nor were their numbers reduced in response to VSV infection even though 293T cells were producing copious amounts of virus within the co-culture. [0139]

EXAMPLE 4

VSV Mutants as Oncolytic Agents

VSV mutants were isolated based upon their ability to form small plaques in monolayers of interferon-responsive cells, as compared to the size of plaques in monolayers of interferon-nonresponsive cells. Viral isolates, which form small plaques in interferon-responsive cells were picked, amplified and re-cloned. Mutants isolated in this way were amplified and tested for their ability to kill tumour and normal cells. The rationale here is that VSV mutants, which can induce interferon in target cells, would limit their own replication in an interferon responsive cell population. These same viruses would however have unrestricted growth in tumour cells that lack interferon responsiveness. These mutants would be of value, as they should have even less cytopathic effect on normal tissues while maintaining oncolytic activity. [0140]
Four mutants (Mut 1-4) were obtained based on their ability to form small plaques in monolayers of interferon-responsive cells. These mutants were initially identified by Dr. Lauren Poliquin (University of Quebec at Montreal) and provided by him. After five rounds of plaque purification, these mutants and wild type virus (moi of 1.0 pfu/cell) were used to infect melanoma cells and normal human foreskin fibroblasts and titres of released virus determined 12 and 24 hours post infection. [0141]
All of the mutants were able to kill tumour cells efficiently but normal cells infected with the mutants even after long time points appeared completely uninfected. At this same moi wild type VSV demonstrated a cytopathic effect on the normal cells. It was also observed that all of the VSV mutants produced approximately ten times more virus than the wild type VSV following an overnight infection of melanoma cells. On normal cells, while the Mutants 1-4 had significantly less cytopathic effect than wild type VSV, similar amounts of virus were produced from the infected cultures. These results indicate that the mutant virus have a greater therapeutic effect in that they kill tumour cells efficiently while sparing normal cells, and that they also have the ability to produce more virus particles and increase virus spread throughout the tumour. [0142]

EXAMPLE 5

Infection of Nude Mice Bearing Human Tumour Xenografts

Nude mice were implanted with human melanoma cells and divided into groups. One group received a mock injection (VSV(−)), and the other were injected with wild type VSV or injected with additional melanoma cells infected in vitro with VSV for one hour prior to injection into the tumour site in order to deliver cells that would continuously produce infective particles to the tumour over a several hour period (VSV(+)). The results of these experiments are seen in FIG. 5 which shows the average of the tumour area with time in treated and mock injected animals. [0143]
In the case of mock-injected animals (VSV(−); injection with vehicle alone) tumours grew continuously over the course of the experiment. Animals which received only pure virus showed initially continuous growth of tumours although at [0144] day 4 post infection the tumours began to shrink and continued to do so over the course of this experiment. Tumours that were injected with infected cells demonstrated the most dramatic regressions. Essentially most tumours stopped growing and regressed to small hard nodules resembling scar tissue.
In some of the larger injected tumours, ulcers formed on the tumour within 1-2 days, (see FIG. 6), followed by continuous shrinkage of the once rapidly growing malignancy. While both injection of purified virus and infected melanoma cells caused significant regressions, infected producer cells were more effective. [0145]

EXAMPLE 6

The Choice of Cell Line for Producing VSV Affects Sensitivity of the Virus to Plasma

Earlier experiments indicated that PKR−/− mice were killed with VSV by several routes of infection, however, these mice were not affected by intravenous injections of the virus. Without wishing to be bound by theory, this could be because the PKR−/− vascular endothelial cells provide a barrier to tissue infection or because plasma components were inactivating the virus upon contact. To test this latter idea VSV produced from several sources including within mouse L cells was incubated with human serum (from normal uninfected donor) and the virus titer after incubation determined. [0146]
Following incubation of VSV in human serum, the viral titer of L cell-produced VSV dropped four hundred fold. On the on the other hand VSV produced in human melanoma cells was unaffected by incubation in plasma. [0147]
These results indicate that the choice of cell line for the production of VSV is critical. Based on this observation it is possible to screen human cell lines for those that produce optimum amounts of virus that is not sensitive to human serum. [0148]

EXAMPLE 7

Strategy for VSV Concentration and Purification

Conventional techniques for VSV production include centrifugation steps and gradient purification—both of these approaches difficult to scale up for industrial production. Therefore, alternate protocols for the purification of VSV, for example affinity columns for the simultaneous concentration and purification of virus particles has been explored. [0149]
In order to provide the virus with an affinity tag to be used for the purification of the virus, endogenous proteins may be used or, the virus may be engineered to express one or more affinity tags on its surface, or producer cell lines may be engineered to express one or more affinity tags on their plasma membranes which would be acquired by the virus as it buds through membrane. The unique viral envelope proteins can be purified using affinity chromatography. [0150]
One such affinity tag may involve the use of Histidine residues which binds to immobilized nickel columns, however, it is to be understood that other affinity tags may also be employed. This approach has been tested using the bacterial virus M13. Using a phage peptide display system (Koivunen, E., et al., J. Nucl Med, 1999. 40:883-888), viral particles expressing Histidine containing peptides which bind to nickel columns, but that can be eluted with imidazole, were selected including: CTTHRHHTSNC (SEQ ID NO:1); CLNAHRTTHHHC (SEQ ID NO:2); CHGLHSNMRHC (SEQ ID NO:3); CHHHHRLNC (SEQ ID NO:4); CHSHHHRGC (SEQ ID NO:5); CWDHHNHHC (SEQ ID NO:6); CDNNHHHHC (SEQ ID NO:7); CHHHRISSHC (SEQ ID NO:8). The expression of these peptides on the surface of M13 phage resulted in the purification concentration of the virus on nickel resins and their elution using low concentrations of imidazole. [0151]
One or more of these sequences can be integrated into the VSV G protein to result in an increased concentration of the viral particles bearing these peptides on nickel residues. The eluted virus is expected to retain its infectivity. [0152]
In this manner a cell line that can be a universal producer of VSV, or other virus, that expresses a chimeric VSV protein with nickel binding properties is produced. This universal producer cell may be used for the production of such a chimeric protein (affinity tag) for any enveloped virus (including all enveloped RNA and DNA viruses). For the purification of virus which bud through the nuclear membrane (such as Herpes virus), a tag to be expressed on the viral envelope protein expressed in the nuclear membrane is engineered. [0153]
Other affinity tags include antibodies, preferably an antibody which recognizes a particular peptide under conditions of low salt, low temperature or in the presence of a critical cation/anion. Physiological salt concentrations, thermal elution or chelation could effect elution. Antibodies generated against di or tripeptides may also be used for for purification. In this manner, two or more of these tags on the surface of a single virus particle would allow for the sequential affinity purification of the virus. [0154]

EXAMPLE 8

Use of VSV to Treat Chronic Infections

Some human disorders arise as a result of chronic viral infections including latent herpes infection, hepatitis, AIDS and cervical cancer. In each of these cases, the causative viral agent has evolved mechanisms to inactivate components of the interferon response pathway including PKR (e.g. Chelbi-Alix, M. K. and H. de The, Oncogene, 1999. 18:935-941; Gale, M. J., Jr., et al., Virology, 1997. 230:217-227; [0155]
Gale, M. J., et al., Clin Diagn Virol, 1998. 10:157-162; Gale, M., Jr. and M. G. Katze, Methods, 1997.11:383-401; Barnard, P. and N. A. McMillan, Virology, 1999. 259:305-313). Therefore, the administration of VSV, or interferon inducing VSV mutants, or a combination thereof, to individuals suffering from these disorders, selectively ablates the chronically infected cells. Further therapeutic efficacy could be found by targeting through cell or viral receptors only the chronically infected cells. [0156]

EXAMPLE 9

Genetic Modification of VSV

A reverse genetic system has been established for VSV (Roberts A. and J. K. Rose, [0157] Virology, 1998. 247:1-6) making it possible to alter the genetic properties of the virus.
Targeting VSV to Desired Sites In Vivo [0158]
Presently VSV can bind to most mammalian cell types although its replication once inside the cell can be restricted (i.e. by interferon responsive gene products including PKR). Thus the effective dose of virus that can actually find target cells (i.e. tumour cells) for productive infection can be greatly limited simply by the “sink” that other normal tissues provide. Therefore, VSV may be genetically modified in order to bind and infect only tumour cells. [0159]
Recombinant DNA techniques well known in the art (e.g. Sambrook et al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press) are used to modify VSV G protein. Single chain Fv fragments (Lorimer, I. A., et al. Proc. Natl. Acad. Sci. U.S.A., 1996. 93:14815-20) that have specificity for tumour antigens are fused to VSV G protein. An example of such a single chain Fv fragment is one that targets the mutant EGF receptor that is found on about 80% of human breast tumour cells. [0160]
Expression of Suicide Genes Within VSV [0161]
The VSV genome is modified so that it comprises the herpes virus thymidine kinase gene or the cytosine deaminase gene. Both of these genes encode enzymes which can convert pro-drugs into toxic compounds (e.g. ganciclovir or 5-FC). Viruses modified in this way express these suicide genes, thereby permitting VSV infected cells to be killed by administration of the pro-drug. This provides two advantages since (1) it is well established that ganciclovir metabolites kill not only cell expressing HSV TK but also can cells in the immediate vicinity. This “by-stander effect” can increase the effective killing by the virus (i.e. one infected cell could result in the killing of ten or more surrounding tumour cells); and (2) having a VSV with a suicide gene could allow the elimination of virus if desired from an individual infected with the virus. [0162]
Controlling VSV Growth In Vivo [0163]
A mammalian gene product is introduced within VSV to limit VSV growth in normal cells, but this gene product does not affect VSV growth in tumour or diseased cells. [0164]
Recombinant VSVs (rVSV) comprising one or more transactivators of p53, activate apoptotic pathways in normal cells but not tumour cells. Such rVSVs limit virus spread in normal tissues but allow virus growth in tumour cells. [0165]
rVSV comprising the PKR gene limits virus replication in all normal cells, however, in cells that express PKR inhibitors, the virally encoded PKR is inactivated. An example of a cell that expresses one or more PKR inhibitors is a chronically Hepatitis C infected cell. Since Hepatitis C encodes and expresses two known inhibitors of PKR (i.e. NS5A and E2), a VSV encoded PKR gene product is be neutralized, and VSV allowed to replicate freely. [0166]

EXAMPLE 10

Progressive Loss of Interferon Responsiveness with Oncogenic Transformation

Murine fibroblasts at various stages of transformation, either pretreated with 100 units of interferon alpha or left untreated, were infected with WT Indiana VSV at an MOI of 0.1 pfu/cell. Viral production was measured 18 hours pi by standard plaque assay. MEF: mouse fibroblast primary cultures isolated from Balb/C mouse embryos. NIH 3T3 cells: immortalized mouse embryo fibroblasts. PVSrc: NIH 3T3 cells transformed with the viral src gene. MOP 8: NIH 3T3 cells transformed with the polyoma virus Large T antigen. Results are shown in Table 2. [0167]
In this example, loss of interferon responsiveness correlates with susceptibility to VSV infection and progression of the malignant phenotype. The MEF cells are mortal (ie have a limited lifespan in culture) and completely interferon responsive. NIH 3T3 cells although not tumourigenic are immortalized and are about ten thousand fold less responsive to interferon than MEFs. The PVSrc and [0168] MOP 8 cells are fully tumourigenic, support robust VSV replication and are minimally protected by interferon treatment.

TABLE 2

Viral Titre (pfu/ml)

Cell Line Untreated IFN-α

MEF (Mouse Embryonic Fibroblast) 4 × 10⁶ <10

NIH3T3 8 × 10⁷ 1 × 10⁴

PVSrc 3 × 10⁹ 2 × 10⁷

MOP 8 1 × 10⁸ 5 × 10⁶

EXAMPLE 11

Virus Yield after Overnight Infection of Various Cell Lines Either Untreated or Treated with IFN

A variety of normal and transformed cell lines were either untreated or pre-treated with 100 units of IFN-α, infected at an MOI of 0.1 pfu/ml with WT Indiana VSV and incubated for 18 hours at 37° C. Culture media from each sample was titred for VSV production. Results are shown in Table 3. [0169]

This example demonstrates that viral production is ten to ten thousand times more efficient in a range of tumour cell types as compared to normal primary tissues. In the presence of interferon alpha, virus production in normal primary cells is almost completely blocked while in tumour cells interferon has little or no effect on VSV replication.

	TABLE 3


	Viral Titre (pfu/ml)

Cell Line	Untreated	IFN-α

OSF7 (primary normal human fibroblast)	1 × 10⁶	<10
OSF12 (primary normal human fibroblast)	2 × 10⁵	<10
OSF16 (primary normal human fibroblast)	1 × 10⁵	<10
PrEC (primary normal human prostate epithelium)	8 × 10⁶	<10
HOSE (primary normal human ovarian	1 × 10⁷	<1000
surface epithelium)
A2780 (human ovarian carcinoma)	2 × 10⁸	1 × 10⁷
OVCA 420 (human ovarian carcinoma)	1 × 10⁸	3 × 10⁶
C13 (human ovarian carcinoma)	1 × 10⁸	1 × 10⁵
LC80 (human lung carcinoma)	2 × 10⁹	6 × 10⁷
SK-MEL3 (human melanoma)	1 × 10⁹	1 × 10⁹
LNCAP (human prostate carcinoma)	4 × 10⁹	5 × 10⁹
HCT116 (human colon carcinoma)	1 × 10⁹	2 × 10⁹
293T (HEK cells transformed with T antigen	1 × 10⁸	8 × 10⁷
and Ad virus E1A)

EXAMPLE 12

LD₅₀for WT and Mutant VSV Delivered Intranasally to PKR^+/−(129×Balb/c) Mice

8-10 week old female mice were anaesthetised and infected intranasally with virus diluted in 50 μl of phosphate buffered saline (PBS) into the nares of each animal (PKR[0171] ^+/−; 129×Balb/c strain). Lethal dose 50 values were calculated using the Korler-Spearman method. Results are shown in Table 4.

This example demonstrates that mutants I, II and III in particular, are attenuated as compared to the wild type Indiana strain of virus when tested for toxicity in 129×Balb/c mice.

	TABLE 4


	Virus	Intranasal LD₅₀(pfu)

	WT Indiana	1 × 10⁴
	Mutant I	1 × 10¹⁰
	Mutant II	>1 × 10¹⁰
	Mutant III	3 × 10⁸
	Mutant IV	<1 × 10⁵

EXAMPLE 13

PKR^−/− Mice are Exquisitely Sensitive to VSV Compared to Various PKR^+/+ Mouse Strains

PKR[0173] ^−/− and PKR^+/+ mice were infected intranasally at various doses and their survival monitored over time. All PKR^−/− mice succumbed to the infection between days 2 and 5 depending on the dose, while control mice remained alive beyond this point. Results are shown in Table 5.

This example demonstrates the importance of the PKR gene product in the resistance of mice to VSV infection.

TABLE 5


	Genetic Background	IN Dose (pfu)	Survival at day 5

PKR^+/+	Balb/c	5 × 10⁴	5/5
	CD-1	5 × 10⁴	5/5
	Balb/c × 129	5 × 10⁴	5/5
PKR^−/−	Balb/c × 129	5 × 10⁴	0/5
		5 × 10³	0/4
		5 × 10²	0/3
		5 × 10¹	0/3

EXAMPLE 14

AML3 Cells Die by Apopotosis Following Infection with VSV

OCI/AML3 (acute myelogenous leukemia) cells were infected with VSV at an MOI of 3.0 pfu/cell. Fourteen and twenty hours post-infection unfixed samples were analyzed. Apoptotic cells with phosphatidylserine membrane translocation were detected by flow cytometry using Annexin-V-Biotin-X/NeutrAvidin-PE red fluorescent protein (Molecular Probes). Mitochondrial membrane depolarization in early apoptotic cells were analyzed by flow cytometry using JC-1 potential-sensitive dye (Molecular Probes). JC-1 is accumulated by polarized mitochondria shifting fluorescence emission from green to red spectra. Non-viable AML3 cells were identified using Ethidium (EthD-1) homodimer-1 red fluorescent vital dye (Molecular probes). Assays were performed following the manufacturers specifications. Results are shown in Table 6. [0175]

This example demonstrates that VSV kills AML cells at least in part through a virally induced apoptotic pathway.

TABLE 6


			MOI 3.0	Net
		MOI 0.0	Percent	Positive
	Tests	Percent Positive	Positive	(Dead)

14 hrs p.i.	EthD-1	6.6	32.3	25.7
	Annexin V	14.3	52.7	38.4
	JC-1	7.5	21.4	13.4
20 hrs p.i.	EthD-1	6.4	58.5	52.1
	positive
	Annexin-V	10.8	79.6	68.8
	positive
	JC-1	3.9	43.2	39.3

EXAMPLE 15

Mutant VSV Strains Infect and Kill AML Cells

OCI/AML3 (acute myelogenous leukemia) cells were infected at an MOI of 1.0 and incubated for 23 hours. Unfixed cells were stained with Eth-D 1 (ethidium dimer, Molecular Probes) to detect non-viable cells following manufacturers specifications. Number of stained cells per 10,000 counted used to calculate percent dead. Results are shown in Table 7. [0177]
This example demonstrates that the mutant VSV strains used are as effective as the wild type Indiana strain in killing AML cells. [0178]

TABLE 7

WT

Mock IND Mut I Mut II Mut III Mut IV Mut V

Percent Dead 30.0 64.7 60.7 86.7 72.1 74.4 82.8

EXAMPLE 16

VSV and VSV Infected Cells Exhibit Antitumor Activity Against Human Melanoma Xenografts in Nude Mice

SK-MEL 3 (melanoma) derived tumours were developed in 8-10 week old female Balb/c athymic mice. On [0179] day 0, tumours were either left untreated or were infected with 10⁸pfu WT Indiana VSV in culture media or 2.5×10⁶WT Indiana VSV infected SK-MEL 3 cells (VSV producing cells). Statistical differences were calculated between treated and untreated groups at each data point with the following confidence values (b: p<0.01; c: p<0.001; d: p=0.007). Results are shown in FIG. 7. On day 3, only tumours treated with VSV producing cells were significantly smaller than untreated tumours (a: p<0.001). No statistically significant differences in tumour volumes between groups were apparent from day 0 to day 2. Data points represent means+/−SEM from multiple tumours (untreated n=8; VSV producing cells n=8; VSV alone n=4).
This example demonstrates that a single injection of VSV, directly into solid tumours profoundly affects tumour growth resulting in partial to complete regression. The use of infected tumour cells as a vehicle to deliver virus is also efficacious. [0180]

EXAMPLE 17

PKR^−/− Mice are Acutely Sensitive to Intranasal VSV Infection and Demonstrate a Deficiency in IFN Mediated Resistance

(A & B) PKR[0181] ^−/− and control mice (Balb/c×129) were infected intranasally with 5×10⁴pfu of VSV and monitored for morbidity and survival over the course of 14 days, after which remaining animals were deemed to have survived the infection. Results are shown in FIGS. 8A and 8B. PKR^−/− mice showed a severe decrease in survival compared to control mice (WT), succumbing by day 3 or 4, while all control mice survived the infection. IFN-α/β pretreatment (18 h prior to infection) with either 2×10⁴IU (FIG. 8A) or 2×10⁵IU (FIG. 8B) had no protective effect in PKR^−/− animals.
This example demonstrates that a single defect in the interferon pathway (absence of PKR gene product) is sufficient to render mice unable to resist VSV infections. This defect cannot be rescued by interferon. [0182]

EXAMPLE 18

Interferon can Protect Xenograft Bearing Nude Mice During VSV Treatment

SK-[0183] MEL 3 melanoma cells were injected intradermally into CD-1 athymic nude mice. On day 0, tumours were injected with either live WT Indiana VSV (1×10⁹pfu) or an equivalent amount of UV inactivated VSV, and measured daily. Results are shown in FIG. 9. Interferon was administered to a subset of animals (VSV IFN) at the times indicated (black arrows). (UV-VSV n=4; VSV IFN n=6; VSV n=6). In these experiments a single intratumoural injection of VSV is tumour-inhibiting in all cases. All tumours had at least a partial regression and in three of twelve mice treated a complete tumour regression. Tumours receiving UV inactivated virus continued to grow unabated until these animals were sacrificed at day 11. Nude mice not receiving interferon and injected with live virus began to die at day 10 and only two of six remained viable by day 15. In contrast, all interferon treated, infected, nude mice were protected from VSV toxicity and remained symptom free for more than 45 days.
This example demonstrates that a single intratumoural injection of live VSV is efficacious against tumours. Furthermore infected, tumour bearing, nude mice can be rescued from VSV toxicity by interferon injection. [0184]

EXAMPLE 19

VSV Infects and Kills Leukemia and Myeloma Cells

The indicated cell lines were infected with VSV Indiana HR strain at a multiplicity of infection of one plaque forming unit per cell. At 24, 48 and 72 hours post infection (p.i.) samples were taken from the infected cultures and stained directly with propidium iodide following manufacturers instructions (Molecular Probes). Samples were then analysed by flow cytometry using the FACSsort WinMDI Version 2.7 program. In Table 8 the percentage of cells dead for each leukemic cell type is shown for the indicated times post infection. [0185]

This example shows that VSV is able to infect and kill a diverse set of leukemia types. The K-562 cell is isolated from a chronic myelogenous leukemia (CML) patient while MOLT-4 is a T cell leukemia and SR and H929 are myelomas.

TABLE 8


Cell Line	24 hr. p.i.	48 hr. p.i.	72 hr. p.i.

K-562 (CML)	15.38%	52.36%	N/D
MOLT-4 (T cell	53.94%	48.80%	N/D
Leukemia)
SR (Myeloma)	32.10%	46.38%	N/D
H929(Myeloma)	10.73%	17.35%	64.41%

EXAMPLE 20

Vesicular Stomatitis Virus (VSV) Strains Including Wild Type Indiana and Five Attenuated VSV Mutants Demonstrate Selective Cytotoxicity Toward Human Prostate Carcinoma Cells Compared to Normal Human Fibroblasts

Vesicular stomatitis virus strains including wild type Indiana and attenuated mutant strains I (TR1026), II (TR1026R), III (TP3), IV (TP6) and V (G31) were obtained from Dr. Lauren Poliquin, University of Quebec at Montreal. Each of these virus strains was plaque purified five times prior to use in this experiment. [0187]
Human prostate carcinoma cells (LNCAP) and normal human cells ([0188] OSF 7 forearm fibroblast) were grown in 96-well tissue culture plates to a density of approximately 5×10⁴cells per well. Virus was added in 10-fold dilutions ranging from 5×10⁵pfu to 5 pfu. Control wells with no virus were included on each plate. The plates were incubated for 48 hours at 37° C. in 5% CO2. Cytotoxicity was quantified using a colorimetric MTS ((3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay (CellTiter 96 Aqueous, catalog #G1112, Promega Corporation, Madison Wis. 53711-5399), monitored at 490 nm, that detects mitochondrial enzyme activity. The amount of cell killing in the virus treated wells was determined by the loss in viability in the virus treated wells relative to the untreated wells. The data was plotted graphically as pfu/cell vs. percentage cell killing relative to control. The TC50 for these cells was calculated as the amount of virus in pfu/cell causing a 50% reduction in the amount of viable cells. Lower TC50 values reflect increased sensitivity of the cells to the lytic effects of the virus. The in vitro therapeutic index for each VSV strain was calculated as the ratio of TC50 for the OSF7 cells compared to the TC50 for the LNCAP cells.

The results are shown in Table 9. Wild type VSV-Indiana and each of the five mutants demonstrated a high degree of cytotoxicity toward the human prostate carcinoma cells as reflected in the low TC50 values, all less than 0.01 pfu/cell. The normal human fibroblasts cells were one to more than 3 orders of magnitude more resistant to the cytotoxic effects of all six VSV strains. All five mutants had less toxicity on the normal OSF7 fibroblasts cells and had a higher in vitro therapeutic index than the wild-type Indiana VSV.

TABLE 9


Cytotoxicity Assay Results for VSV Strains (Wild type Indiana and
Mutants I through V) Against Prostate Carcinoma Cells and Normal
Fibroblasts.

Mutant	Mutant	Mutant	Mutant	Mutant	WT
I	II	III	IV	V	Indiana

LNCAP Prostate	0.0064	0.0048	0.0014	0.0006	0.0012	0.0017
Carcinoma TC50 (pfu/cell)
OSF7 Normal Fibroblasts	>42	22	4.3	0.031	9.8	0.022
TC50 (pfu/cell)
Therapeutic Index	>6562	4583	3071	52	8167	13
(TC50 OSF7/
TC50 LNCAPP)

EXAMPLE 21

Virus Production from Tumour Cells and Normal Cells Infected with Wild Type Indiana and Various Mutant VSV Strains

HCT 116 colon carcinoma cells and [0190] OSF 7 forearm fibroblasts were grown to confluence in 35 mm tissue culture dishes. Media was removed and virus was added in a volume of 30 μl with a multiplicity of infection of 0.1 pfu/cell for the HCT 116 cells and 1.5 pfu/cell for the OSF 7 cells. After a 1 hour incubation period at 37° C., 5% CO2, 1 ml of tissue culture media was added to the dishes. Results are shown in FIGS. 10A and B. At the indicated time points, 10 μl samples of media were removed from the dishes. The virus titre of these samples was determined by a plaque assay.
This example demonstrates the rapid replication kinetics of wild type and mutant VSV strains in HCT 116 colon carcinoma cells. All four mutant VSV strains had more rapid growth in HCT116 tumor cells than the wild type VSV. Note that in the normal OSF-7 cell cultures a ten fold higher input of virus is required to attain similar replication kinetics. [0191]

EXAMPLE 22

Malignant Cells are Rapidly Killed Following VSV (WT Indiana) Infection and are not Protected by IFN-α

Monolayers of normal primary human fibroblasts (AG 1522) and several tumour cell lines were either untreated or pretreated with IFN-α (100 units) and then infected with VSV at an MOI of 0.1 pfu/ml. At 12 hours increments the infections were terminated by cell fixation and staining to determine the kinetics of cell killing. Control (CNTL) monolayers were left to grow, uninfected, over the course of the experiment and therefore stain more intensely. Results are shown in FIG. 11. LNCAP is a human prostate carcinoma; A2780 is an human ovarian epithelial carcinoma, and Sk MEL3 is a human melanoma. [0192]
This example demonstrates the rapid kinetics of tumour cell killing by VSV Indiana even in the presence of interferon alpha. While normal cells are also killed by VSV, the kinetics are slower and normal cells can be completely protected by interferon alpha. [0193]

EXAMPLE 23

VSV Induced Cytopathic Effect Visible in Human Melanoma Cells but not in Primary Human Cells with or without IFN-α

Gelatin-coated coverslips with normal human cells and SK-MEL3 cells untreated or pretreated with IFN-α (100 U/ml) were infected with WT Indiana VSV at an MOI of 0.1 pfu/ml. Results are shown in FIG. 12. The human melanoma cells (SK-MEL3) displayed cpe at 12 hours post-infection even in the presence of interferon. At 24 hours post-infection these malignant cells had died and lifted from the coverslip. Human primary cells including foreskin fibroblasts (AG1522), ovarian surface epithelial cells (HOSE) and prostate epithelial cells (PrEC) did not show CPE (cytopathic effect) until 36 hours in the absence of interferon and were completely protected in the presence of interferon beyond 72 hours post-infection. [0194]
This example demonstrates that VSV Indiana is able to rapidly destroy melanoma cells even in the presence of interferon alpha whereas normal fibroblasts and epithelial cells are slower to be killed and can be completely protected by interferon alpha. [0195]

EXAMPLE 24

VSV Selectively Kills Transformed Cells Co-Cultured with Normal Fibroblasts

Equal numbers of 293T cells (human embryo kidney cells transformed with adenovirus E1A and Large T antigen) and normal human foreskin fibroblasts were plated on gelatin-coated coverslips and infected (WT Indiana VSV) at an MOI of 0.1 both in the presence and absence of interferon. Cells were fixed at 12 (not shown), 24 and 36 hours post-infection. Fixed cells were stained with an anti-TAg antibody and DAPI. The red staining 293T cells were quickly killed as early as 12 hours post-infection, regardless of interferon treatment, with those few remaining cells displaying condensed or fragmented nuclei. The normal fibroblasts displayed altered nuclei by 36 hours post-infection in the absence of interferon but were protected from the virus in the presence of interferon beyond this time point. [0196]
This example demonstrates that in a mixed culture of normal and tumour cells, VSV Indiana preferentially replicates and kills tumour cells. Normal cells in the infected co-cultures are slower to die and can be completely rescued by interferon treatment. [0197]

EXAMPLE 25

Efficacy of a Single Intravenous Dose of Mutant VSV in Treating Human Melanoma Xenografts in Nude Mice

SK-Mel3 human melanoma xenografts were established in 5-6 week old CD-1 athymic mice. On [0198] day 0, tumours were either left untreated or were treated intravenously with 5×10⁹pfu of mutant VSV as indicated. Results are shown in FIG. 13.
This example demonstrates that mutants II and III are able to inhibit tumour growth following a single intravenous injection. Thus virus need not be administered at the tumour site to be effective in inhibiting tumour growth. Furthermore, the mutants while being attenuated for growth in normal mouse tissues, are still able to target tumour cells in vivo. [0199]

EXAMPLE 26

Selective Killing of AML Cells Co-Cultured with Normal Bone Marrow

The growth factor independent cell line OCI/AML3 was mixed 1:9 with normal bone marrow and infected for 24 hours with WT Indiana VSV. Various dilutions of cells were then plated in methylcellulose plus and minus growth factors and colony counts were performed 14 days later. Table 10 shows data for dishes receiving 10[0200] ⁴cells. The asterisk (*) signifies that no leukemic colonies were detected on the growth factor minus dishes even when 10⁵cells were plated per dish.
This example demonstrates the rapid and selective killing of leukemia cells in the presence of normal bone stem cells. Furthermore it demonstrates that bone marrow is not a dose-limiting target of VSV oncolytic therapy as it is with most other conventional cancer therapies. [0201]

TABLE 10

Multiplicity of Infection

Colony Type 0.0 1.0 5.0

Leukemic 172 0* 0*

Neutrophil 12 7 5

Mixed 6 3 4

Monocyte 10 7 5

EXAMPLE 27

VSV Sequences

The genome of VSV contains genes that encode viral proteins N, P, M, G and L. The cDNA sequences of the open reading frames (ORF) for these proteins from wild type heat resistant VSV (HR) and three mutant VSVs were determined (based on sequencing five times each) and compared with the sequences of GenBank Accession No. NC 001560 (derived from Colorado and San Juan strains of VSV). The mutants are M2 (TR1026R), M3 (TP3) and M4 (TP6). The nucleic acid sequences are shown in FIGS. 14, 16, [0202] 18, 20 and 22. The corresponding deduced amino acid sequences are shown in FIGS. 15, 17, 19, 21 and 23, respectively. Differences are indicated by highlighted letters. Dotted lines represent incomplete sequencing.
Some of the differences between the amino acid sequences are shown in Table 11 using notation based on column heading (i.e. for column heading “Differences Between GenBank and HR” notation K155R means that the amino acid at position 155 is K in GenBank and R in HR). In those cases where HR sequence is not yet available comparisons can only be made between GenBank and a particular mutant. M3* denotes a difference between [0203] mutant 3 and HR but in this case the amino acid matches the GenBank deposit at that position (i.e. mutant 3 and the Genbank sequence agree at that position while HR is different).

This data demonstrates the many differences in sequence between the HR strain and the GenBank deposit (which is primarily derived from the San Juan strain). It also demostrates some of the differences between the mutants and the HR strain from which they were derived. These genetic differences correlate with phenotypic differences.

TABLE 11


			Differences Between	Differences Between
	Differences Between	Differences Between HR and	Genbank and Mutant	Genbank and Mutant
Gene	GenBank and HR	Mutants	#	2	#4

N	D10A, K155R, N353S	A10D (M3*)
		None (M4)
P	K50R, A76V, Q77P,	None (M2, M3 and M4)
	E99D
	P110Q, S126L, S140L
	Y151H, M168I, E170K
	D237N
M	S32N, Y54H, N57H,	M51R (M3)
	T133A, I171V, I226V	None (M4)
G	H24Y, I57L, Q96H,	Q26R, R242H, S431A (all	A331V
	V141A, Y172D, G132D	M3)
	H242R, S438T, L453F	E254G (M4)
	H487Y
L	T367A, T689S, T2026I	None (M4)		I202L, K296R
	R2075K

All citations are herein incorporated by reference. [0205]
The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein. [0206]

REFERENCES

1. Stark, G. R., et al., How cells respond to interferons. Annu Rev Biochem, 1998. 67: p. 227-64. [0207]
2. Jaramillo, M. L., N. Abraham, and J. C. Bell, The interferon system: a review with emphasis on the role of PKR in growth control. Cancer Invest, 1995. 13(3): p. 3[0208] ²7-38.
3. Cuddihy, A. R., et al., Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol, 1999. 19(4): p. 2475-84. [0209]
4. Lee, S. B., et al., The apoptosis pathway triggered by the interferon-induced protein kinase PKR requires the third basic domain, initiates upstream of Bcl-2, and involves ICE-like proteases. Virology, 1997. 231(1): p. 81-8. [0210]
5. Lee, S. B. and M. Esteban, The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology, 1994. 199(2): p. 491-6. [0211]
6. Gale, M. J., Jr., et al., Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology, 1997. 230(2): p. 217-27. [0212]
7. McMillan, N. A., et al., HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR. Virology, 1995. 213(2): p. 413-24. [0213]
8. Kitajewski, J., et al., Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase. Cell, 1986. 45(2): p. 195-200. [0214]
9. Black, T. L., G. N. Barber, and M. G. Katze, Degradation of the interferon-induced 68,000-M(r) protein kinase by polio virus requires RNA. J Virol, 1993. 67(2): p. 791-800. [0215]
10. Melville, M. W., et al., The molecular chaperone hsp40 regulates the activity of P581PK, the cellular inhibitor of PKR. Proc Natl Acad Sci USA, 1997. 94(1): p. 9[0216] ⁷-102.
11. Swaminathan, S., et al., [0217] Simian Virus 40 Large-T Bypasses the Translational Block Imposed by the Phosphorylation of eIF-2alpha. Virology, 1996. 219(1): p. 321-3.
12. Der, S. D., et al., A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci USA, 1997. 94(7): p. 3[0218] ²79⁻⁸3.
13. Meurs, E. F., et al., Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc Natl Acad Sci USA, 1993. 90(1): p. 232-6. [0219]
14. Koromilas, A. E., et al., Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science, 1992. 257(5077): p. 1685-9. [0220]
15. Mundschau, L. J. and D. V. Faller, Oncogenic ras induces an inhibitor of double-stranded RNA-dependent [0221] eukaryotic initiation factor 2 alpha-kinase activation. J Biol Chem, 1992. 267(32): p. 23092-8.
16. Mundschau, L. J. and D. V. Faller, Endogenous inhibitors of the dsRNA-dependent eIF-2 alpha protein kinase PKR in normal and ras-transformed cells. Biochimie, 1994. 76(8): p. 792-800. [0222]
17. Strong, J. E., et al., The molecular basis of viral oncolysis: usurpation of the Ras signalling pathway by reovirus. Embo J, 1998. 17(12): p.3351-62. [0223]
18. Kumar, K. U., S. P. Srivastava, and R. J. Kaufman, Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18. Mol Cell Biol, 1999. 19(2): p. 1116-25. [0224]
19. Peralta, R. C., et al., Distinct regions of frequent loss of heterozygosity of chromosome 5p and 5q in human esophageal cancer. Int J Cancer, 1998. 78(5): p. 600-5. [0225]
20. Kirchhoff, S., et al., IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR. Oncogene, 1995. 11(3): p. 439-45. [0226]
21. Beretta, L., et al., Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q-associated leukemias. Oncogene, 1996. 12(7): p. 1593-6. [0227]
22. Lossos, I. S., et al., A novel translocation (1;2)(p34;p21-22) in acute myelomonoblastic leukemia. Cancer Genet Cytogenet, 1998. 106(1): p. 78-9. [0228]
23. Haines, G. K. d., et al., Expression of the double-stranded RNA-dependent protein kinase (p68) in squamous cell carcinoma of the head and neck region. Arch Otolaryngol Head Neck Surg, 1993. 119(10): p. 1142-7. [0229]
24. Haines, G. K., et al., Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma. Virchows Arch B Cell Pathol Incl Mol Pathol, 1993. 63(5): p. 289-95. [0230]
25. Shimada, A., et al., Aberrant expression of double-stranded RNA-dependent protein kinase in hepatocytes of chronic hepatitis and differentiated hepatocellular carcinoma. Cancer Res, 1998. 58(19): p. 4434-8. [0231]
26. Wong, L. H., et al., Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma. J Biol Chem, 1997. 272(45): p. 28779-85. [0232]
27. Petricoin, E., 3rd, et al., Human cancer cell lines express a negative transcriptional regulator of the interferon regulatory factor family of DNA binding proteins. Mol Cell Biol, 1994. 14(2): p. 1477-86. [0233]
28. Abril, E., et al., Characterization of a gastric tumor cell line defective in MHC class I inducibility by both alpha- and gamma-interferon. Tissue Antigens, 1996. 47(5): p. 391-8. [0234]
29. Abril, E., et al., Unresponsiveness to interferon associated with STAT1 protein deficiency in a gastric adenocarcinoma cell line. Cancer Immunol Immunother, 1998. 47(2): p. 113-20. [0235]
30. Sun, W. H., et al., Interferon-alpha resistance in a cutaneous T-cell lymphoma cell line is associated with lack of STAT1 expression. Blood, 1998. 91(2): p. 570-6. [0236]
31. Chelbi-Alix, M. K., et al., Resistance to virus infection conferred by the interferon-induced promyelocytic leukemia protein. J Virol, 1998. 72(2): p. 1043-51. [0237]
32. Chelbi-Alix, M. K., et al., Induction of the PML protein by interferons in normal and APL cells. Leukemia, 1995. 9(12): p. 2027-33. [0238]
33. Stadler, M., et al., Transcriptional induction of the PML growth suppressor gene by interferons is mediated through an ISRE and a GAS element. Oncogene, 1995. 11(12): p. 2565-73. [0239]
34. Koken, M. H., et al., Leukemia-associated retinoic acid receptor alpha fusion partners, PML and PLZF, heterodimerize and colocalize to nuclear bodies. Proc Natl Acad Sci USA, 1997. 94(19): p. 10255-60. [0240]
35. Abraham, N., et al., Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR. J Biol Chem, 1999. 274(9): p. 5953-62. [0241]
36. Garcia-Sastre, A., et al., Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology, 1998. 252(2): p. 324-30. [0242]
1 52 1 11 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 1 Cys Thr Thr His Arg His His Thr Ser Asn Cys 1 5 10 2 12 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 2 Cys Leu Asn Ala His Arg Thr Thr His His His Cys 1 5 10 3 11 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 3 Cys His Gly Leu His Ser Asn Met Arg His Cys 1 5 10 4 9 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 4 Cys His His His His Arg Leu Asn Cys 1 5 5 9 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 5 Cys His Ser His His His Arg Gly Cys 1 5 6 9 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 6 Cys Trp Asp His His Asn His His Cys 1 5 7 9 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 7 Cys Asp Asn Asn His His His His Cys 1 5 8 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 8 Cys His His His Arg Ile Ser Ser His Cys 1 5 10 9 1269 DNA Vesicular stomatitis virus 9 atgtctgtta cagtcaagag aatcattgac aacacagtca tagttccaaa acttcctgca 60 aatgaggatc cagtggaata cccggcagat tacttcagaa aatcaaagga gattcctctt 120 tacatcaata ctacaaaaag tttgtcagat ctaagaggat atgtctacca aggcctcaaa 180 tccggaaatg tatcaatcat acatgtcaac agctacttgt atggagcatt aaaggacatc 240 cggggtaagt tggataaaga ttggtcaagt ttcggaataa acatcgggaa agcaggggat 300 acaatcggaa tatttgacct tgtatccttg aaagccctgg acggcgtact tccagatgga 360 gtatcggatg cttccagaac cagcgcagat gacaaatggt tgcctttgta tctacttggc 420 ttatacagag tgggcagaac acaaatgcct gaatacagaa aaaagctcat ggatgggctg 480 acaaatcaat gcaaaatgat caatgaacag tttgaacctc ttgtgccaga aggtcgtgac 540 atttttgatg tgtggggaaa tgacagtaat tacacaaaaa ttgtcgctgc agtggacatg 600 ttcttccaca tgttcaaaaa acatgaatgt gcctcgttca gatacggaac tattgtttcc 660 agattcaaag attgtgctgc attggcaaca tttggacacc tctgcaaaat aaccggaatg 720 tctacagaag atgtaacgac ctggatcttg aaccgagaag ttgcagatga aatggtccaa 780 atgatgcttc caggccaaga aattgacaag gccgattcat acatgcctta tttgatcgac 840 tttggattgt cttctaagtc tccatattct tccgtcaaaa accctgcctt ccacttctgg 900 gggcaattga cagctcttct gctcagatcc accagagcaa ggaatgcccg acagcctgat 960 gacattgagt atacatctct tactacagca ggtttgttgt acgcttatgc agtaggatcc 1020 tctgccgact tggcacaaca gttttgtgtt ggagataaca aatacactcc agatgatagt 1080 accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 1140 tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaaagagca 1200 gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1260 gacaaatga 1269 10 1269 DNA Vesicular stomatitis virus 10 atgtctgtta cagtcaagag aatcattgcc aacacagtca tagttccaaa acttcctgca 60 aatgaggatc cagtggaata cccggcagat tacttcagaa aatcaaagga gattcctctt 120 tacatcaata ctacaaaaag tttgtcagat ctaagaggat atgtctacca aggcctcaaa 180 tccggaaatg tatcaatcat acatgtcaac agctacttgt atggagcatt gaaggacatc 240 cggggtaagt tggataaaga ttggtcaagt ttcggaataa acatcgggaa ggcaggggat 300 acaatcggaa tatttgacct tgtatccttg aaagccctgg acggtgtact tccagatgga 360 gtatcggatg cttccagaac cagcgcagat gacaaatggt tgcctttgta tctacttggc 420 ttatacagag tgggcagaac acaaatgcct gaatacagaa aaaggctcat ggatgggctg 480 acaaatcaat gcaaaatgat caatgaacag tttgaacctc ttgtgccaga aggtcgtgac 540 atttttgatg tgtggggaaa tgacagtaat tacacaaaaa ttgtcgctgc agtggacatg 600 ttcttccaca tgttcaaaaa acatgaatgt gcctcgttca gatacggaac tattgtttcc 660 agattcaaag attgtgctgc attggcaaca tttggacacc tctgcaaaat aaccggaatg 720 tctacagaag atgtaacgac ctggatcttg aaccgagaag ttgcagatga gatggtccaa 780 atgatgcttc caggccaaga aattgacaag gccgattcat acatgcctta tttgatcgac 840 tttggattgt cttctaagtc tccatattct tccgtcaaaa accctgcctt ccacttctgg 900 gggcaattga cagctcttct gctcagatcc accagagcaa ggaatgcccg acagcctgat 960 gacattgagt atacatctct tactacagca ggtttgttgt acgcttatgc agtaggatcc 1020 tctgctgact tggcacaaca gttttgtgtt ggagatagca aatacactcc agatgatagt 1080 accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 1140 tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaacgagca 1200 gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1260 gacaaatga 1269 11 1269 DNA Vesicular stomatitis virus 11 atgtctgtta cagtcaagag aatcattgac aacacagtca tagttccaaa acttcctgca 60 aatgaggatc cagtggaata cccggcagat tacttcagaa aatcaaagga gattcctctt 120 tacatcaata ctacaaaaag tttgtcagat ctaagaggat atgtctacca aggcctcaaa 180 tccggaaatg tatcaatcat acatgtcaac agctacttgt atggagcatt gaaggacatc 240 cggggtaagt tggataaaga ttggtcaagt ttcggaataa acatcgggaa ggcaggggat 300 acaatcggaa tatttgacct tgtatccttg aaagccctgg acggtgtact tccagatgga 360 gtatcggatg cttccagaac cagcgcagat gacaaatggt tgcctttgta tctacttggc 420 ttatacagag tgggcagaac acaaatgcct gaatacagaa aaaggctcat ggatgggctg 480 acaaatcaat gcaaaatgat caatgaacag tttgaacctc ttgtgccaga aggtcgtgac 540 atttttgatg tgtggggaaa tgacagtaat tacacaaaaa ttgtcgctgc agtggacatg 600 ttcttccaca tgttcaaaaa acatgaatgt gcctcgttca gatacggaac tattgtttcc 660 agattcaaag attgtgctgc attggcaaca tttggacacc tctgcaaaat aaccggaatg 720 tctacagaag atgtaacgac ctggatcttg aaccgagaag ttgcagatga gatggtccaa 780 atgatgcttc caggccaaga aattgacaag gccgattcat acatgcctta tttgatcgac 840 tttggattgt cttctaagtc tccatattct tccgtcaaaa accctgcctt ccacttctgg 900 gggcaattga cagctcttct gctcagatct accagagcaa ggaatgcccg acagcctgat 960 gacattgagt atacatctct tactacagca ggtttgttgt acgcttatgc agtaggatcc 1020 tctgctgact tggcacaaca gttttgtgtt ggagatagca aatacactcc agatgatagt 1080 accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 1140 tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaacgagca 1200 gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1260 gacaaatga 1269 12 1269 DNA Vesicular stomatitis virus 12 atgtctgtta cagtcaagag aatcattgac aacacagtca tagttccaaa acttcctgca 60 aatgaggatc cagtggaata cccggcagat tacttcagaa aatcaaagga gattcctctt 120 tacatcaata ctacaaaaag tttgtcagat ctaagaggat atgtctacca aggcctcaaa 180 tccggaaatg tatcaatcat acatgtcaac agctacttgt atggagcatt gaaggacatc 240 cggggtaagt tggataaaga ttggtcaagt ttcggaataa acatcgggaa ggcaggggat 300 acaatcggaa tatttgacct tgtatccttg aaagccctgg acggtgtact tccagatgga 360 gtatcggatg cttccagaac cagcgcagat gacaaatggt tgcctttgta tctacttggc 420 ttatacagag tgggcagaac acaaatgcct gaatacagaa aaaggctcat ggatgggctg 480 acaaatcaat gcaaaatgat caatgaacag tttgaacctc ttgtgccaga aggtcgtgac 540 atttttgatg tgtggggaaa tgacagtaat tacacaaaaa ttgtcgctgc agtggacatg 600 ttcttccaca tgttcaaaaa acatgaatgt gcctcgttca gatacggaac tattgtttcc 660 agattcaaag attgtgctgc attggcaaca tttggacacc tctgcaaaat aaccggaatg 720 tctacagaag atgtaacgac ctggatcttg aaccgagaag ttgcagatga gatggtccaa 780 atgatgcttc caggccaaga aattgacaag gccgattcat acatgcctta tttgatcgac 840 tttggattgt cttctaagtc tccatattct tccgtcaaaa accctgcctt ccacttctgg 900 gggcaattga cagctcttct gctcagatcc accagagcaa ggaatgcccg acagcctgat 960 gacattgagt atacatctct tactacagca ggtttgttgt acgcttatgc agtaggatcc 1020 tctgctgact tggcacaaca gttttgtgtt ggagatagca aatacactcc agatgatagt 1080 accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 1140 tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaacgagca 1200 gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1260 gacaaatga 1269 13 1029 DNA Vesicular stomatitis virus modified_base (1)..(1029) “n” represents a, t, c, g, other or unknown 13 tcaatcatac atgtcaacag ctacttgtat ggagcattga aggacatccg gggtaagttg 60 gataaagatt ggtcaagttt cggaataaac atcgggaagg caggggatac aatcggaata 120 tttgaccttg tatccttgaa agccctggac ggtgtacttc cagatggagt atcggatgct 180 tccagaacca gcgcagatga caaatggttg cctttgtatc tacttggctt atacagagtg 240 ggcagaacac aaatgcctga atacagaaaa aggctcatgg atgggctgac aaatcaatgc 300 aaaatgatca atgaacagtt tgaacctctt gtgccagaag gtcgtgacat ttttgatgtg 360 tggggaaatg acagtaatta cacaaaaatt gtcgctgcag tggacatgtt cttccacatg 420 ttcaaaaaac atgaatgtgc ctcgttcaga tacggaacta ttgtttccag attcaaagat 480 tgtgctgcat tggcaacatt tggacacctc tgcaaaataa ccggaatgtc tacagaagat 540 gtaacgacct ggatcttgaa ccgagaagtt gcagatgaga tggtccaaat gatgcttcca 600 ggccaagaaa ttgacaaggc cgattcatac atgccttatt tgatcgactt tggattgtct 660 tctaagtctc catattcttc cgtcaaaaac cctgccttcc acttctgggg gcaattgact 720 gacattgagt atacatctcn tactacagca ggtttgttgt acgcttatgc agtaggatcc 780 tctgctgact tggcacanca gttttgtgtt ggagatagca aatacactcc agatgatagt 840 accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 900 tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaacgagca 960 gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1020 gacaaatga 1029 14 422 PRT Vesicular stomatitis virus 14 Met Ser Val Thr Val Lys Arg Ile Ile Asp Asn Thr Val Ile Val Pro 1 5 10 15 Lys Leu Pro Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe 20 25 30 Arg Lys Ser Lys Glu Ile Pro Leu Tyr Ile Asn Thr Thr Lys Ser Leu 35 40 45 Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly Asn Val 50 55 60 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys Asp Ile 65 70 75 80 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly Ile Asn Ile Gly 85 90 95 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp Leu Val Ser Leu Lys Ala 100 105 110 Leu Asp Gly Val Leu Pro Asp Gly Val Ser Asp Ala Ser Arg Thr Ser 115 120 125 Ala Asp Asp Lys Trp Leu Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140 Gly Arg Thr Gln Met Pro Glu Tyr Arg Lys Lys Leu Met Asp Gly Leu 145 150 155 160 Thr Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 165 170 175 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr 180 185 190 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys Lys His 195 200 205 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 210 215 220 Cys Ala Ala Leu Ala Thr Phe Gly His Leu Cys Lys Ile Thr Gly Met 225 230 235 240 Ser Thr Glu Asp Val Thr Thr Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255 Glu Met Val Gln Met Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270 Ser Tyr Met Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 275 280 285 Tyr Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295 300 Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro Asp 305 310 315 320 Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu Leu Tyr Ala Tyr 325 330 335 Ala Val Gly Ser Ser Ala Asp Leu Ala Gln Gln Phe Cys Val Gly Asp 340 345 350 Asn Lys Tyr Thr Pro Asp Asp Ser Thr Gly Gly Leu Thr Thr Asn Ala 355 360 365 Pro Pro Gln Gly Arg Asp Val Val Glu Trp Leu Gly Trp Phe Glu Asp 370 375 380 Gln Asn Arg Lys Pro Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 385 390 395 400 Val Met Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410 415 Lys Ser Glu Phe Asp Lys 420 15 422 PRT Vesicular stomatitis virus 15 Met Ser Val Thr Val Lys Arg Ile Ile Ala Asn Thr Val Ile Val Pro 1 5 10 15 Lys Leu Pro Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe 20 25 30 Arg Lys Ser Lys Glu Ile Pro Leu Tyr Ile Asn Thr Thr Lys Ser Leu 35 40 45 Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly Asn Val 50 55 60 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys Asp Ile 65 70 75 80 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly Ile Asn Ile Gly 85 90 95 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp Leu Val Ser Leu Lys Ala 100 105 110 Leu Asp Gly Val Leu Pro Asp Gly Val Ser Asp Ala Ser Arg Thr Ser 115 120 125 Ala Asp Asp Lys Trp Leu Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140 Gly Arg Thr Gln Met Pro Glu Tyr Arg Lys Arg Leu Met Asp Gly Leu 145 150 155 160 Thr Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 165 170 175 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr 180 185 190 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys Lys His 195 200 205 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 210 215 220 Cys Ala Ala Leu Ala Thr Phe Gly His Leu Cys Lys Ile Thr Gly Met 225 230 235 240 Ser Thr Glu Asp Val Thr Thr Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255 Glu Met Val Gln Met Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270 Ser Tyr Met Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 275 280 285 Tyr Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295 300 Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro Asp 305 310 315 320 Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu Leu Tyr Ala Tyr 325 330 335 Ala Val Gly Ser Ser Ala Asp Leu Ala Gln Gln Phe Cys Val Gly Asp 340 345 350 Ser Lys Tyr Thr Pro Asp Asp Ser Thr Gly Gly Leu Thr Thr Asn Ala 355 360 365 Pro Pro Gln Gly Arg Asp Val Val Glu Trp Leu Gly Trp Phe Glu Asp 370 375 380 Gln Asn Arg Lys Pro Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 385 390 395 400 Val Met Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410 415 Lys Ser Glu Phe Asp Lys 420 16 422 PRT Vesicular stomatitis virus 16 Met Ser Val Thr Val Lys Arg Ile Ile Asp Asn Thr Val Ile Val Pro 1 5 10 15 Lys Leu Pro Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe 20 25 30 Arg Lys Ser Lys Glu Ile Pro Leu Tyr Ile Asn Thr Thr Lys Ser Leu 35 40 45 Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly Asn Val 50 55 60 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys Asp Ile 65 70 75 80 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly Ile Asn Ile Gly 85 90 95 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp Leu Val Ser Leu Lys Ala 100 105 110 Leu Asp Gly Val Leu Pro Asp Gly Val Ser Asp Ala Ser Arg Thr Ser 115 120 125 Ala Asp Asp Lys Trp Leu Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140 Gly Arg Thr Gln Met Pro Glu Tyr Arg Lys Arg Leu Met Asp Gly Leu 145 150 155 160 Thr Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 165 170 175 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr 180 185 190 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys Lys His 195 200 205 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 210 215 220 Cys Ala Ala Leu Ala Thr Phe Gly His Leu Cys Lys Ile Thr Gly Met 225 230 235 240 Ser Thr Glu Asp Val Thr Thr Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255 Glu Met Val Gln Met Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270 Ser Tyr Met Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 275 280 285 Tyr Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295 300 Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro Asp 305 310 315 320 Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu Leu Tyr Ala Tyr 325 330 335 Ala Val Gly Ser Ser Ala Asp Leu Ala Gln Gln Phe Cys Val Gly Asp 340 345 350 Ser Lys Tyr Thr Pro Asp Asp Ser Thr Gly Gly Leu Thr Thr Asn Ala 355 360 365 Pro Pro Gln Gly Arg Asp Val Val Glu Trp Leu Gly Trp Phe Glu Asp 370 375 380 Gln Asn Arg Lys Pro Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 385 390 395 400 Val Met Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410 415 Lys Ser Glu Phe Asp Lys 420 17 342 PRT Vesicular stomatitis virus MOD_RES (1)..(342) “Xaa” represents any, other or unknown amino acid 17 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys Asp Ile 1 5 10 15 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly Ile Asn Ile Gly 20 25 30 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp Leu Val Ser Leu Lys Ala 35 40 45 Leu Asp Gly Val Leu Pro Asp Gly Val Ser Asp Ala Ser Arg Thr Ser 50 55 60 Ala Asp Asp Lys Trp Leu Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 65 70 75 80 Gly Arg Thr Gln Met Pro Glu Tyr Arg Lys Arg Leu Met Asp Gly Leu 85 90 95 Thr Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 100 105 110 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr 115 120 125 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys Lys His 130 135 140 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 145 150 155 160 Cys Ala Ala Leu Ala Thr Phe Gly His Leu Cys Lys Ile Thr Gly Met 165 170 175 Ser Thr Glu Asp Val Thr Thr Trp Ile Leu Asn Arg Glu Val Ala Asp 180 185 190 Glu Met Val Gln Met Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 195 200 205 Ser Tyr Met Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 210 215 220 Tyr Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 225 230 235 240 Asp Ile Glu Tyr Thr Ser Xaa Thr Thr Ala Gly Leu Leu Tyr Ala Tyr 245 250 255 Ala Val Gly Ser Ser Ala Asp Leu Ala Xaa Gln Phe Cys Val Gly Asp 260 265 270 Ser Lys Tyr Thr Pro Asp Asp Ser Thr Gly Gly Leu Thr Thr Asn Ala 275 280 285 Pro Pro Gln Gly Arg Asp Val Val Glu Trp Leu Gly Trp Phe Glu Asp 290 295 300 Gln Asn Arg Lys Pro Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 305 310 315 320 Val Met Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 325 330 335 Lys Ser Glu Phe Asp Lys 340 18 798 DNA Vesicular stomatitis virus 18 atggataatc tcacaaaagt tcgtgagtat ctcaagtcct attctcgtct ggatcaggcg 60 gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120 caagaggatg gagtggaaga gcatactaag ccctcttatt ttcaggcagc agatgattct 180 gacacagaat ctgaaccaga aattgaagac aatcaaggtt tgtatgcaca ggatccagaa 240 gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcaga tgaggaagtg 300 gatgttgtat ttacttcgga ctggaaacca cctgagcttg aatctgacga gcatggaaag 360 accttacggt tgacatcgcc agagggttta agtggagagc agaaatccca gtggctttcg 420 acgattaaag cagtcgtgca aagtgccaaa tactggaatc tggcagagtg cacatttgaa 480 gcatcgggag aaggggtcat tatgaaggag cgccagataa ctccggatgt atataaggtc 540 actccagtga tgaacacaca tccgtcccaa tcagaagcag tatcagatgt ttggtctctc 600 tcaaagacat ccatgacttt ccaacccaag aaagcaagtc ttcagcctct caccatatcc 660 ttggatgaat tgttctcatc tagaggagag ttcatctctg tcggaggtga cggacgaatg 720 tctcataaag aggccatcct gctcggcctg agatacaaaa agttgtacaa tcaggcgaga 780 gtcaaatatt ctctgtag 798 19 798 DNA Vesicular stomatitis virus 19 atggataatc tcacaaaagt tcgtgagtat ctcaagtcct attctcgtct agatcaggcg 60 gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120 caagaggacg gagtggaaga gcatactagg ccctcttatt ttcaggcagc agatgattct 180 gacacagaat ctgaaccaga aattgaagac aatcaaggct tgtatgtacc agatccggaa 240 gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcgga tgaggacgtg 300 gatgttgtat tcacttcgga ctggaaacag cctgagcttg aatccgacga gcatggaaag 360 accttacggt tgacattgcc agagggttta agtggagagc agaaatccca gtggcttttg 420 acgattaaag cagtcgttca aagtgccaaa cactggaatc tggcagagtg cacatttgaa 480 gcatcgggag aaggggtcat cataaaaaag cgccagataa ctccggatgt atataaggtc 540 actccagtga tgaacacaca tccgtcccaa tcagaagccg tatcagatgt ttggtctctc 600 tcaaagacat ccatgacttt ccaacccaag aaagcaagtc ttcagcctct caccatatcc 660 ttggatgaat tgttctcatc tagaggagaa ttcatctctg tcggaggtaa cggacgaatg 720 tctcataaag aggccatcct gctcggtctg aggtacaaaa agttgtacaa tcaggcgaga 780 gtcaaatatt ctctgtag 798 20 570 DNA Vesicular stomatitis virus 20 atggataatc tcacaaaagt tcgtgagtat ctcaagtcct attctcgtct agatcaggcg 60 gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120 caagaggacg gagtggaaga gcatactagg ccctcttatt ttcaggcagc agatgattct 180 gacacagaat ctgaaccaga aattgaagac aatcaaggct tgtatgtacc agatccggaa 240 gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcgga tgaggacgtg 300 gatgttgtat tcacttcgga ctggaaacag cctgagcttg aatccgacga gcatggaaag 360 accttacggt tgacattgcc agagggttta agtggagagc agaaatccca gtggcttttg 420 acgattaaag cagtcgttca aagtgccaaa cactggaatc tggcagagtg cacatttgaa 480 gcatcgggag aaggggtcat cataaaaaag cgccagataa ctccggatgt atataaggtc 540 actccagtga tgaacacaca tccgtcccaa 570 21 798 DNA Vesicular stomatitis virus 21 atggataatc tcacaaaagt tcgtgagtat ctcaagtcct attctcgtct agatcaggcg 60 gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120 caagaggacg gagtggaaga gcatactagg ccctcttatt ttcaggcagc agatgattct 180 gacacagaat ctgaaccaga aattgaagac aatcaaggct tgtatgtacc agatccggaa 240 gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcgga tgaggacgtg 300 gatgttgtat tcacttcgga ctggaaacag cctgagcttg aatccgacga gcatggaaag 360 accttacggt tgacattgcc agagggttta agtggagagc agaaatccca gtggcttttg 420 acgattaaag cagtcgttca aagtgccaaa cactggaatc tggcagagtg cacatttgaa 480 gcatcgggag aaggggtcat cataaaaaag cgccagataa ctccggatgt atataaggtc 540 actccagtga tgaacacaca tccgtcccaa tcggaagccg tatcagatgt ttggtctctc 600 tcaaagacat ccatgacttt ccaacccaag aaagcaagtc ttcagcctct caccatatcc 660 ttggatgaat tgttctcatc tagaggagaa ttcatctctg tcggaggtaa cggacgaatg 720 tctcataaag aggccatcct gctcggtctg aggtacaaaa agttgtacaa tcaggcgaga 780 gtcaaatatt ctctgtag 798 22 798 DNA Vesicular stomatitis virus 22 atggataatc tcacaaaagt tcgtgagtat ctcaagtcct attctcgtct agatcaggcg 60 gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120 caagaggacg gagtggaaga gcatactagg ccctcttatt ttcaggcagc agatgattct 180 gacacagaat ctgaaccaga aattgaagac aatcaaggct tgtatgtacc agatccggaa 240 gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcgga tgaggacgtg 300 gatgttgtat tcacttcgga ctggaaacag cctgagcttg aatccgacga gcatggaaag 360 accttacggt tgacattgcc agagggttta agtggagagc agaaatccca gtggcttttg 420 acgattaaag cagtcgttca aagtgccaaa cactggaatc tggcagagtg cacatttgaa 480 gcatcgggag aaggggtcat cataaaaaag cgccagataa ctccggatgt atataaggtc 540 actccagtga tgaacacaca tccgtcccaa tcagaagccg tatcagatgt ttggtctctc 600 tcaaagacat ccatgacttt ccaacccaag aaagcaagtc ttcagcctct caccatatcc 660 ttggatgaat tgttctcatc tagaggagaa ttcatctctg tcggaggtaa cggacgaatg 720 tctcataaag aggccatcct gctcggtctg aggtacaaaa agttgtacaa tcaggcgaga 780 gtcaaatatt ctctgtag 798 23 265 PRT Vesicular stomatitis virus 23 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Lys Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Ala Gln Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Glu Val Asp Val Val Phe Thr Ser Asp Trp Lys Pro Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Ser Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Ser Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys Tyr Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Met Lys Glu Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser Arg Gly Glu Phe Ile Ser Val Gly Gly Asp Gly Arg Met 225 230 235 240 Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245 250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265 24 265 PRT Vesicular stomatitis virus 24 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Arg Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Val Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Asp Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Leu Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Leu Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys His Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Ile Lys Lys Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser Arg Gly Glu Phe Ile Ser Val Gly Gly Asn Gly Arg Met 225 230 235 240 Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245 250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265 25 190 PRT Vesicular stomatitis virus 25 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Arg Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Val Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Asp Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Leu Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Leu Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys His Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Ile Lys Lys Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln 180 185 190 26 265 PRT Vesicular stomatitis virus 26 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Arg Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Val Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Asp Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Leu Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Leu Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys His Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Ile Lys Lys Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser Arg Gly Glu Phe Ile Ser Val Gly Gly Asn Gly Arg Met 225 230 235 240 Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245 250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265 27 265 PRT Vesicular stomatitis virus 27 Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp Gly Val Glu Glu His 35 40 45 Thr Arg Pro Ser Tyr Phe Gln Ala Ala Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn Gln Gly Leu Tyr Val Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Asp Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Leu Pro Glu 115 120 125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Leu Thr Ile Lys Ala 130 135 140 Val Val Gln Ser Ala Lys His Trp Asn Leu Ala Glu Cys Thr Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Ile Lys Lys Arg Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn Thr His Pro Ser Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser Arg Gly Glu Phe Ile Ser Val Gly Gly Asn Gly Arg Met 225 230 235 240 Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245 250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265 28 690 DNA Vesicular stomatitis virus 28 atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60 gggatcgcac caccccctta tgaagaggac actagcatgg agtatgctcc gagcgctcca 120 attgacaaat cctattttgg agttgacgag atggacacct atgatccgaa tcaattaaga 180 tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240 tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300 aaacgtccct tctacaaaat cttggctttt ttgggttctt ctaatctaaa ggccactcca 360 gcggtattgg cagatcaagg tcaaccagag tatcacactc actgcgaagg cagggcttat 420 ttgccacata ggatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480 ccattcaata taggtcttta caagggaacg attgagctca caatgaccat ctacgatgat 540 gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600 ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcgtgg 660 gtcctggatt ctatcagcca cttcaaatga 690 29 690 DNA Vesicular stomatitis virus 29 atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60 gggatcgcac caccccctta tgaagaggac actaacatgg agtatgctcc gagcgctcca 120 attgacaaat cctattttgg agttgacgag atggacactc atgatccgca tcaattaaga 180 tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240 tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300 aaacgtccct tctacaagat cttggctttt ttgggttctt ctaatctaaa ggccactcca 360 gcggtattgg cagatcaagg tcaaccagag tatcacgctc actgtgaagg cagggcttat 420 ttgccacaca gaatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480 ccattcaata taggtcttta caagggaacg gttgagctca caatgaccat ctacgatgat 540 gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600 ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcttgg 660 gtcctggatt ctgtcagcca cttcaaatga 690 30 690 DNA Vesicular stomatitis virus 30 atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60 gggatcgcac caccccctta tgaagaggac actaacatgg agtatgctcc gagcgctcca 120 attgacaaat cctattttgg agttgacgag agggacactc atgatccgca tcaattaaga 180 tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240 tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300 aaacgtccct tctacaagat cttggctttt ttgggwtctt ctaatctaaa ggccactcca 360 gcggtattgg cagatcaagg tcaaccagag tatcacgctc actgtgaagg cagggcttat 420 ttgccacaca gaatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480 ccattcaata taggtcttta caagggaacg gttgagctca caatgaccat ctacgatgat 540 gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600 ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcttgg 660 gtcctggatt ctgtcagcca cttcaaatga 690 31 690 DNA Vesicular stomatitis virus 31 atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60 gggatcgcac caccccctta tgaagaggac actaacatgg agtatgctcc gagcgctcca 120 attgacaaat cctattttgg agttgacgag atggacactc atgatccgca tcaattaaga 180 tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240 tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300 aaacgtccct tctacaagat cttggctttt ttgggttctt ctaatctaaa ggccactcca 360 gcggtattgg cagatcaagg tcaaccagag tatcacgctc actgtgaagg cagggcttat 420 ttgccacaca gaatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480 ccattcaata taggtcttta caagggaacg gttgagctca caatgaccat ctacgatgat 540 gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600 ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcttgg 660 gtcctggatt ctgtcagcca cttcaaatga 690 32 229 PRT Vesicular stomatitis virus 32 Met Ser Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Ser 20 25 30 Met Glu Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45 Asp Glu Met Asp Thr Tyr Asp Pro Asn Gln Leu Arg Tyr Glu Lys Phe 50 55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Thr His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe Asn Ile Gly Leu Tyr Lys Gly Thr Ile Glu Leu Thr Met Thr 165 170 175 Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180 185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val Leu Asp Ser 210 215 220 Ile Ser His Phe Lys 225 33 229 PRT Vesicular stomatitis virus 33 Met Ser Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Asn 20 25 30 Met Glu Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45 Asp Glu Met Asp Thr His Asp Pro His Gln Leu Arg Tyr Glu Lys Phe 50 55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Ala His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe Asn Ile Gly Leu Tyr Lys Gly Thr Val Glu Leu Thr Met Thr 165 170 175 Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180 185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val Leu Asp Ser 210 215 220 Val Ser His Phe Lys 225 34 229 PRT Vesicular stomatitis virus 34 Met Ser Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Asn 20 25 30 Met Glu Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45 Asp Glu Met Asp Thr His Asp Pro His Gln Leu Arg Tyr Glu Lys Phe 50 55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Ala His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe Asn Ile Gly Leu Tyr Lys Gly Thr Val Glu Leu Thr Met Thr 165 170 175 Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180 185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val Leu Asp Ser 210 215 220 Val Ser His Phe Lys 225 35 229 PRT Vesicular stomatitis virus 35 Met Ser Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Asn 20 25 30 Met Glu Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45 Asp Glu Arg Asp Thr His Asp Pro His Gln Leu Arg Tyr Glu Lys Phe 50 55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Ala His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe Asn Ile Gly Leu Tyr Lys Gly Thr Val Glu Leu Thr Met Thr 165 170 175 Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180 185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val Leu Asp Ser 210 215 220 Val Ser His Phe Lys 225 36 1536 DNA Vesicular stomatitis virus 36 atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60 gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120 ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagccat acaagtcaaa 180 atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240 gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacagtc catccgatcc 300 ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360 ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca 420 gtgattgtcc aggtgactcc tcaccatgtg ctggttgatg aatacacagg agaatgggtt 480 gattcacagt tcatcaacgg aaaatgcagc aattacatat gccccactgt ccataactct 540 acaacctggc attctgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600 gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660 ttcagaagta actactttgc ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc 720 aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780 tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840 acctcagtgg atgtaagtct aattcaggac gttgagagga tcttggatta ttccctctgc 900 caagaaacct ggagcaaaat cagagcgggt cttccaatct ctccagtgga tctcagctat 960 cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020 tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080 ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc accatatgaa 1140 gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200 tacatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260 ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320 tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380 tggaaaagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440 gttctccgag ttggtatcca tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500 tatacagaca tagagatgaa ccgacttgga aagtaa 1536 37 885 DNA Vesicular stomatitis virus modified_base (1)..(885) “n” represents a, t, c, g, other or unknown 37 atgaagtgcc ttttgkactt agctttttta ttcatcgggg tgaattgcaa gttcaccata 60 gtttttccat acaaccaaaa aggaaactgg aaaaatgttc cttccaatta ccattattgc 120 ccgtcaagct cagatttaaa ttgncataat gacttaatag gcacagcctt acaagtcaaa 180 atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240 gtcactactt gtgatttccg ctggtacgga ccgaagtata taacacattc catccgatcc 300 ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360 ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgctgaagca 420 gcgattgtcc aggtgactcc tcaccatgtg cttgttgatg aatacacagg agaatgggtt 480 gattcacagt tcatcaacgg aaaatgcagc aatgacatat gccccactgt ccataactcc 540 acaacctggc attccgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600 gacatcacct tcttctcaga ggacggagag ctatcatccc taggaaagga gggcacaggg 660 ttcagaagta actactttgc ttatgaaact ggagacaagg cctgcaaaat gcagtactgc 720 aagcgttggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780 tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840 acctcagtgg atgtaagtct cattcaggac gttgagagga tcttg 885 38 705 DNA Vesicular stomatitis virus 38 ccatctcaga cctcagtgga tgtaagtctc attcaggacg ttgagaggat cttggattat 60 tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttcccatctc tccagtggat 120 ctcagctatc ttgctcctaa aaacccagga accggtcctg tctttaccat aatcaatggt 180 accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 240 agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggct 300 ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 360 tttcctttat atatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 420 gctcaggtgt ttgaacatcc tcacattcaa gacgctgctt cgcagcttcc tgatgatgag 480 actttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagtttgt agaaggttgg 540 ttcagtagtt ggaagagctc tattgcctct tttttcttta tcatagggtt aatcattgga 600 ctattcttgg ttctccgagt tggtatttat ctttgcatta aattaaagca caccaagaaa 660 agacagattt atacagacat agagatgaac cgacttggga agtaa 705 39 1536 DNA Vesicular stomatitis virus 39 atgaagtgcc ttttgtactt agctttttta ttcatcgggg tgaattgcaa gttcaccata 60 gtttttccat acaaccgaaa aggaaactgg aaaaatgttc cttccaatta ccattattgc 120 ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180 atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240 gtcactactt gtgatttccg ctggtacgga ccgaagtata taacacattc catccgatcc 300 ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360 ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgctgaagca 420 gcgattgtcc aggtgactcc tcaccatgtg cttgttgatg aatacacagg agaatgggtt 480 gattcacagt tcatcaacgg aaaatgcagc aatgacatat gccccactgt ccataactcc 540 acaacctggc attccgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600 gacatcacct tcttctcaga ggacggagag ctatcatccc taggaaagga gggcacaggg 660 ttcagaagta actactttgc ttatgaaact ggagacaagg cctgcaaaat gcagtactgc 720 aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780 tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840 acctcagtgg atgtaagtct cattcaggac gttgagagga tcttggatta ttccctctgc 900 caagaaacct ggagcaaaat cagagcgggt cttcccatct ctccagtgga tctcagctat 960 cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020 tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080 ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc tccatatgaa 1140 gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200 tatatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260 tttgaacatc ctcacattca agacgctght gcgcagcttc ctgatgatga gactttattt 1320 tttggtgata ctgggctatc caaaaatcca atcgagtttg tagaaggttg gttcagtagt 1380 tggaagagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440 gttctccgag ttggtattta tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500 tatacagaca tagagatgaa ccgacttggg aagtaa 1536 40 1399 DNA Vesicular stomatitis virus 40 atgaagtgcc ttttgtactt agctttttta ttcatcgggg tgaattgcaa gttcaccata 60 gtttttccat acaaccaaaa aggaaactgg aaaaatgttc cttccaatta ccattattgc 120 ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaggtcaaa 180 atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240 gtcactactt gtgatttccg ctggtacgga ccgaagtata taacacattc catccgatcc 300 ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360 ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgctgaagca 420 gcgattgtcc aggtgactcc tcaccatgtg cttgttgatg aatacacagg agaatgggtt 480 gattcacagt tcatcaacgg aaaatgcagc aatgacatat gccccactgt ccataactcc 540 acaacctggc attccgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600 gacatcacct tcttctcaga ggacggagag ctatcatccc taggaaagga gggcacaggg 660 ttcagaagta actactttgc ttatgaaact ggagacaagg cctgcaaaat gcagtactgc 720 aagcgttggg gagtcagact cccatcaggt gtctggttcg ggatggctga taaggatctc 780 tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840 acctcagtgg atgtaagtct cattcaggac gttgagagga tcttactttg agaccagata 900 catcagagtc gatattgctg ctccaatcct ctcaagaatg gtcggaatga tcagtggaac 960 taccacagaa agggaactgt gggatgactg ggctccatat gaagacgtgg aaattggacc 1020 caatggagtt ctgaggacca gttcaggata taagtttcct ttatatatga ttggacatgg 1080 tatgttggac tccgatcttc atcttagctc aaaggctcag gtgtttgaac atcctcacat 1140 tcaagacgct gcttcgcagc ttcctgatga tgagacttta ttttttggtg atactgggct 1200 atccaaaaat ccaatcgagt ttgtagaagg ttggttcagt agttggaaga gctctattgc 1260 ctcttttttc tttatcatag ggttaatcat tggactattc ttggttctcc gagttggtat 1320 ttatctttgc attaaattaa agcacaccaa gaaaagacag atttatacag acatagagat 1380 gaaccgactt gggaagtaa 1399 41 511 PRT Vesicular stomatitis virus 41 Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15 Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn 20 25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp 35 40 45 His Asn Asp Leu Ile Gly Thr Ala Ile Gln Val Lys Met Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr Gln 85 90 95 Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln 130 135 140 Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150 155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala 245 250 255 Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270 Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275 280 285 Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp 290 295 300 Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr 305 310 315 320 Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn 325 330 335 Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala 340 345 350 Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr 355 360 365 Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile 370 375 380 Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu 385 390 395 400 Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser 405 410 415 Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln 420 425 430 Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys 435 440 445 Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser 450 455 460 Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 42 295 PRT Vesicular stomatitis virus MOD_RES (1)..(295) “Xaa” represents any, other or unknown amino acid 42 Met Lys Cys Leu Leu Xaa Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15 Lys Phe Thr Ile Val Phe Pro Tyr Asn Gln Lys Gly Asn Trp Lys Asn 20 25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Xaa 35 40 45 His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95 Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Ala Ile Val Gln 130 135 140 Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150 155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Asp Ile Cys Pro Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly Asp Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys Arg Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala 245 250 255 Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270 Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275 280 285 Gln Asp Val Glu Arg Ile Leu 290 295 43 234 PRT Vesicular stomatitis virus 43 Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile Gln Asp Val Glu Arg 1 5 10 15 Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp Ser Lys Ile Arg Ala 20 25 30 Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr Leu Ala Pro Lys Asn 35 40 45 Pro Gly Thr Gly Pro Val Phe Thr Ile Ile Asn Gly Thr Leu Lys Tyr 50 55 60 Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala Ala Pro Ile Leu Ser 65 70 75 80 Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr Glu Arg Glu Leu Trp 85 90 95 Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile Gly Pro Asn Gly Val 100 105 110 Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu Tyr Met Ile Gly His 115 120 125 Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser Lys Ala Gln Val Phe 130 135 140 Glu His Pro His Ile Gln Asp Ala Ala Ser Gln Leu Pro Asp Asp Glu 145 150 155 160 Thr Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys Asn Pro Ile Glu Phe 165 170 175 Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser Ile Ala Ser Phe Phe 180 185 190 Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly 195 200 205 Ile Tyr Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln Ile Tyr 210 215 220 Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 225 230 44 511 PRT Vesicular stomatitis virus MOD_RES (1)..(511) “Xaa” represents any, other or unknown amino acid 44 Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15 Lys Phe Thr Ile Val Phe Pro Tyr Asn Arg Lys Gly Asn Trp Lys Asn 20 25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp 35 40 45 His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95 Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Ala Ile Val Gln 130 135 140 Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150 155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Asp Ile Cys Pro Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly Asp Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala 245 250 255 Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270 Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275 280 285 Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp 290 295 300 Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr 305 310 315 320 Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn 325 330 335 Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala 340 345 350 Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr 355 360 365 Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile 370 375 380 Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu 385 390 395 400 Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser 405 410 415 Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Xaa Ala Gln 420 425 430 Leu Pro Asp Asp Glu Thr Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys 435 440 445 Asn Pro Ile Glu Phe Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser 450 455 460 Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 Val Leu Arg Val Gly Ile Tyr Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510 45 465 PRT Vesicular stomatitis virus 45 Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15 Lys Phe Thr Ile Val Phe Pro Tyr Asn Gln Lys Gly Asn Trp Lys Asn 20 25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp 35 40 45 His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95 Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Ala Ile Val Gln 130 135 140 Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150 155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Asp Ile Cys Pro Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly Asp Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys Arg Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Gly Met Ala 245 250 255 Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270 Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275 280 285 Gln Asp Val Glu Arg Ile Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp 290 295 300 Ile Ala Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr 305 310 315 320 Thr Thr Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val 325 330 335 Glu Ile Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe 340 345 350 Pro Leu Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu 355 360 365 Ser Ser Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala 370 375 380 Ser Gln Leu Pro Asp Asp Glu Thr Leu Phe Phe Gly Asp Thr Gly Leu 385 390 395 400 Ser Lys Asn Pro Ile Glu Phe Val Glu Gly Trp Phe Ser Ser Trp Lys 405 410 415 Ser Ser Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu 420 425 430 Phe Leu Val Leu Arg Val Gly Ile Tyr Leu Cys Ile Lys Leu Lys His 435 440 445 Thr Lys Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly 450 455 460 Lys 465 46 6330 DNA Vesicular stomatitis virus 46 atggaagtcc acgattttga gaccgacgag ttcaatgatt tcaatgaaga tgactatgcc 60 acaagagaat tcctgaatcc cgatgagcgc atgacgtact tgaatcatgc tgattacaat 120 ttgaattctc ctctaattag tgatgatatt gacaatttga tcaggaaatt caattctctt 180 ccgattccct cgatgtggga tagtaagaac tgggatggag ttcttgagat gttaacatca 240 tgtcaagcca atcccatctc aacatctcag atgcataaat ggatgggaag ttggttaatg 300 tctgataatc atgatgccag tcaagggtat agttttttac atgaagtgga caaagaggca 360 gaaataacat ttgacgtggt ggagaccttc atccgcggct ggggcaacaa accaattgaa 420 tacatcaaaa aggaaagatg gactgactca ttcaaaattc tcgcttattt gtgtcaaaag 480 tttttggact tacacaagtt gacattaatc ttaaatgctg tctctgaggt ggaattgctc 540 aacttggcga ggactttcaa aggcaaagtc agaagaagtt ctcatggaac gaacatatgc 600 aggattaggg ttcccagctt gggtcctact tttatttcag aaggatgggc ttacttcaag 660 aaacttgata ttctaatgga ccgaaacttt ctgttaatgg tcaaagatgt gattataggg 720 aggatgcaaa cggtgctatc catggtatgt agaatagaca acctgttctc agagcaagac 780 atcttctccc ttctaaatat ctacagaatt ggagataaaa ttgtggagag gcagggaaat 840 ttttcttatg acttgattaa aatggtggaa ccgatatgca acttgaagct gatgaaatta 900 gcaagagaat caaggccttt agtcccacaa ttccctcatt ttgaaaatca tatcaagact 960 tctgttgatg aaggggcaaa aattgaccga ggtataagat tcctccatga tcagataatg 1020 agtgtgaaaa cagtggatct cacactggtg atttatggat cgttcagaca ttggggtcat 1080 ccttttatag attattacac tggactagaa aaattacatt cccaagtaac catgaagaaa 1140 gatattgatg tgtcatatgc aaaagcactt gcaagtgatt tagctcggat tgttctattt 1200 caacagttca atgatcataa aaagtggttc gtgaatggag acttgctccc tcatgatcat 1260 ccctttaaaa gtcatgttaa agaaaataca tggcccacag ctgctcaagt tcaagatttt 1320 ggagataaat ggcatgaact tccgctgatt aaatgttttg aaatacccga cttactagac 1380 ccatcgataa tatactctga caaaagtcat tcaatgaata ggtcagaggt gttgaaacat 1440 gtccgaatga atccgaacac tcctatccct agtaaaaagg tgttgcagac tatgttggac 1500 acaaaggcta ccaattggaa agaatttctt aaagagattg atgagaaggg cttagatgat 1560 gatgatctaa ttattggtct taaaggaaag gagagggaac tgaagttggc aggtagattt 1620 ttctccctaa tgtcttggaa attgcgagaa tactttgtaa ttaccgaata tttgataaag 1680 actcatttcg tccctatgtt taaaggcctg acaatggcgg acgatctaac tgcagtcatt 1740 aaaaagatgt tagattcctc atccggccaa ggattgaagt catatgaggc aatttgcata 1800 gccaatcaca ttgattacga aaaatggaat aaccaccaaa ggaagttatc aaacggccca 1860 gtgttccgag ttatgggcca gttcttaggt tatccatcct taatcgagag aactcatgaa 1920 ttttttgaga aaagtcttat atactacaat ggaagaccag acttgatgcg tgttcacaac 1980 aacacactga tcaattcaac ctcccaacga gtttgttggc aaggacaaga gggtggactg 2040 gaaggtctac ggcaaaaagg atggactatc ctcaatctac tggttattca aagagaggct 2100 aaaatcagaa acactgctgt caaagtcttg gcacaaggtg ataatcaagt tatttgcaca 2160 cagtataaaa cgaagaaatc gagaaacgtt gtagaattac agggtgctct caatcaaatg 2220 gtttctaata atgagaaaat tatgactgca atcaaaatag ggacagggaa gttaggactt 2280 ttgataaatg acgatgagac tatgcaatct gcagattact tgaattatgg aaaaataccg 2340 attttccgtg gagtgattag agggttagag accaagagat ggtcacgagt gacttgtgtc 2400 accaatgacc aaatacccac ttgtgctaat ataatgagct cagtttccac aaatgctctc 2460 accgtagctc attttgctga gaacccaatc aatgccatga tacagtacaa ttattttggg 2520 acatttgcta gactcttgtt gatgatgcat gatcctgctc ttcgtcaatc attgtatgaa 2580 gttcaagata agataccggg cttgcacagt tctactttca aatacgccat gttgtatttg 2640 gacccttcca ttggaggagt gtcgggcatg tctttgtcca ggtttttgat tagagccttc 2700 ccagatcccg taacagaaag tctctcattc tggagattca tccatgtaca tgctcgaagt 2760 gagcatctga aggagatgag tgcagtattt ggaaaccccg agatagccaa gtttcgaata 2820 actcacatag acaagctagt agaagatcca acctctctga acatcgctat gggaatgagt 2880 ccagcgaact tgttaaagac tgaggttaaa aaatgcttaa tcgaatcaag acaaaccatc 2940 aggaaccagg tgattaagga tgcaaccata tatttgtatc atgaagagga tcggctcaga 3000 agtttcttat ggtcaataaa tcctctgttc cctagatttt taagtgaatt caaatcaggc 3060 acttttttgg gagtcgcaga cgggctcatc agtctatttc aaaattctcg tactattcgg 3120 aactccttta agaaaaagta tcatagggaa ttggatgatt tgattgtgag gagtgaggta 3180 tcctctttga cacatttagg gaaacttcat ttgagaaggg gatcatgtaa aatgtggaca 3240 tgttcagcta ctcatgctga cacattaaga tacaaatcct ggggccgtac agttattggg 3300 acaactgtac cccatccatt agaaatgttg ggtccacaac atcgaaaaga gactccttgt 3360 gcaccatgta acacatcagg gttcaattat gtttctgtgc attgtccaga cgggatccat 3420 gacgtcttta gttcacgggg accattgcct gcttatctag ggtctaaaac atctgaatct 3480 acatctattt tgcagccttg ggaaagggaa agcaaagtcc cactgattaa aagagctaca 3540 cgtcttagag atgctatctc ttggtttgtt gaacccgact ctaaactagc aatgactata 3600 ctttctaaca tccactcttt aacaggcgaa gaatggacca aaaggcagca tgggttcaaa 3660 agaacagggt ctgcccttca taggttttcg acatctcgga tgagccatgg tgggttcgca 3720 tctcagagca ctgcagcatt gaccaggttg atggcaacta cagacaccat gagggatctg 3780 ggagatcaga atttcgactt tttattccaa gcaacgttgc tctatgctca aattaccacc 3840 actgttgcaa gagacggatg gatcaccagt tgtacagatc attatcatat tgcctgtaag 3900 tcctgtttga gacccataga agagatcacc ctggactcaa gtatggacta cacgccccca 3960 gatgtatccc atgtgctgaa gacatggagg aatggggaag gttcgtgggg acaagagata 4020 aaacagatct atcctttaga agggaattgg aagaatttag cacctgctga gcaatcctat 4080 caagtcggca gatgtatagg ttttctatat ggagacttgg cgtatagaaa atctactcat 4140 gccgaggaca gttctctatt tcctctatct atacaaggtc gtattagagg tcgaggtttc 4200 ttaaaagggt tgctagacgg attaatgaga gcaagttgct gccaagtaat acaccggaga 4260 agtctggctc atttgaagag gccggccaac gcagtgtacg gaggtttgat ttacttgatt 4320 gataaattga gtgtatcacc tccattcctt tctcttacta gatcaggacc tattagagac 4380 gaattagaaa cgattcccca caagatccca acctcctatc cgacaagcaa ccgtgatatg 4440 ggggtgattg tcagaaatta cttcaaatac caatgccgtc taattgaaaa gggaaaatac 4500 agatcacatt attcacaatt atggttattc tcagatgtct tatccataga cttcattgga 4560 ccattctcta tttccaccac cctcttgcaa atcctataca agccattttt atctgggaaa 4620 gataagaatg agttgagaga gctggcaaat ctttcttcat tgctaagatc aggagagggg 4680 tgggaagaca tacatgtgaa attcttcacc aaggacatat tattgtgtcc agaggaaatc 4740 agacatgctt gcaagttcgg gattgctaag gataataata aagacatgag ctatccccct 4800 tggggaaggg aatccagagg gacaattaca acaatccctg tttattatac gaccacccct 4860 tacccaaaga tgctagagat gcctccaaga atccaaaatc ccctgctgtc cggaatcagg 4920 ttgggccaat taccaactgg cgctcattat aaaattcgga gtatattaca tggaatggga 4980 atccattaca gggacttctt gagttgtgga gacggctccg gagggatgac tgctgcatta 5040 ctacgagaaa atgtgcatag cagaggaata ttcaatagtc tgttagaatt atcagggtca 5100 gtcatgcgag gcgcctctcc tgagcccccc agtgccctag aaactttagg aggagataaa 5160 tcgagatgtg taaatggtga aacatgttgg gaatatccat ctgacttatg tgacccaagg 5220 acttgggact atttcctccg actcaaagca ggcttggggc ttcaaattga tttaattgta 5280 atggatatgg aagttcggga ttcttctact agcctgaaaa ttgagacgaa tgttagaaat 5340 tatgtgcacc ggattttgga tgagcaagga gttttaatct acaagactta tggaacatat 5400 atttgtgaga gcgaaaagaa tgcagtaaca atccttggtc ccatgttcaa gacggtcgac 5460 ttagttcaaa cagaatttag tagttctcaa acgtctgaag tatatatggt atgtaaaggt 5520 ttgaagaaat taatcgatga acccaatccc gattggtctt ccatcaatga atcctggaaa 5580 aacctgtacg cattccagtc atcagaacag gaatttgcca gagcaaagaa ggttagtaca 5640 tactttacct tgacaggtat tccctcccaa ttcattcctg atccttttgt aaacattgag 5700 actatgctac aaatattcgg agtacccacg ggtgtgtctc atgcggctgc cttaaaatca 5760 tctgatagac ctgcagattt attgaccatt agcctttttt atatggcgat tatatcgtat 5820 tataacatca atcatatcag agtaggaccg atacctccga accccccatc agatggaatt 5880 gcacaaaatg tggggatcgc tataactggt ataagctttt ggctgagttt gatggagaaa 5940 gacattccac tatatcaaca gtgtttagca gttatccagc aatcattccc gattaggtgg 6000 gaggctgttt cagtaaaagg aggatacaag cagaagtgga gtactagagg tgatgggctc 6060 ccaaaagata cccgaacttc agactccttg gccccaatcg ggaactggat cagatctctg 6120 gaattggtcc gaaaccaagt tcgtctaaat ccattcaatg agatcttgtt caatcagcta 6180 tgtcgtacag tggataatca tttgaaatgg tcaaatttgc gaagaaacac aggaatgatt 6240 gaatggatca atagacgaat ttcaaaagaa gaccggtcta tactgatgtt gaagagtgac 6300 ctacacgagg aaaactcttg gagagattaa 6330 47 5327 DNA Vesicular stomatitis virus 47 cattggggtc atccttttat agattattac gctggwctag aaaaattaca ttcccaagtw 60 accatkaaga aagatattga tgtgtcatat gcraaagcac ttgcaagtga tttagctcgg 120 attgttctat ttcaacagtt caatgatcat amaaagtggt tcgtgaatgg agacttgctc 180 cctcatgatc atccctttaa aagtcatgtt aaagaaaata catggcccac agctgctcaa 240 gttcaagatt ttggagataa atggcatgaa cttccgctga ttaaatgttt tgaaataccc 300 gacttactag acccatcgat aatatactct gacaaaagtc attcaatgaa taggtcagag 360 gtgttgaaac atgtccgaat gaatccgaac actcctatcc ctagtaaaaa ggtgttgcag 420 actatgttgg acacaaaggc taccaattgg aaagaatttc ttaaagagat tgatgagaag 480 ggcttagatg atgatgatct aattattggt cttaaaggaa aggagaggga actgaagttg 540 gcaggtagat ttttctccct aatgtcttgg aaattgcgag aatactttgt aattaccgaa 600 tatttgataa agactcattt cgtccctatg tttaaaggcc tgacaatggc ggacgatcta 660 actgcagtca ttaaaaagat gttagattcc tcatccggcc aaggattgaa gtcatatgag 720 gcaatttgca tagccaatca cattgattac gaaaaatgga ataaccacca aaggaagtta 780 tcaaacggcc cagtgttccg agttatgggc cagttcttag gttatccatc cttaatcgag 840 agaactcatg aattttttga gaaaagtctt atatactaca atggaagacc agacttgatg 900 cgtgttcaca acaacacact gatcaattca acctcccaac gagtttgttg gcaaggacaa 960 gagggtggac tggaaggtct acggcaaaaa ggatggagta tcctcaatct actggttatt 1020 caaagagagg ctaaaatcag aaacactgct gtcaaagtct tggcacaagg tgataatcaa 1080 gttatttgca cacagtataa aacgaagaaa tcgagaaacg ttgtagaatt acagggtgct 1140 ctcaatcaaa tggtttctaa taatgagaaa attatgactg caatcaaaat agggacaggg 1200 aagttaggac ttttgataaa tgacgatgag actatgcaat ctgcagatta cttgaattat 1260 ggaaaaatac cgattttccg tggagtgatt agagggttag agaccaagag atggtcacga 1320 gtgacttgtg tcaccaatga ccaaataccc acttgtgcta atataatgag ctcagtttcc 1380 acaaatgctc tcaccgtagc tcattttgct gagaacccaa tcaatgccat gatacagtac 1440 aattattttg ggacatttgc tagactcttg ttgatgatgc atgatcctgc tcttcgtcaa 1500 tcattgtatg aagttcaaga taagataccg ggcttgcaca gttctacttt caaatacgcc 1560 atgttgtatt tggacccttc cattggagga gtgtcgggca tgtctttgtc caggtttttg 1620 attagagcct tcccagatcc cgtaacagaa agtctctcat tctggagatt catccatgta 1680 catgctcgaa gtgagcatct gaaggagatg agtgcagtat ttggaaaccc cgagatagcc 1740 aagtttcgaa taactcacat agacaagcta gtagaagatc caacctctct gaacatcgct 1800 atgggaatga gtccagcgaa cttgttaaag actgaggtta aaaaatgctt aatcgaatca 1860 agacaaacca tcaggaacca ggtgattaag gatgcaacca tatatttgta tcatgaagag 1920 gatcggctca gaagtttctt atggtcaata aatcctctgt tccctagatt tttaagtgaa 1980 ttcaaatcag gcactttttt gggagtcgca gacgggctca tcagtctatt tcaaaattct 2040 cgtactattc ggaactcctt taagaaaaag tatcataggg aattggatga tttgattgtg 2100 aggagtgagg tatcctcttt gacacattta gggaaacttc atttgagaag gggatcatgt 2160 aaaatgtgga catgttcagc tactcatgct gacacattaa gatacaaatc ctggggccgt 2220 acagttattg ggacaactgt accccatcca ttagaaatgt tgggtccaca acatcgaaaa 2280 gagactcctt gtgcaccatg taacacatca gggttcaatt atgtttctgt gcattgtcca 2340 gacgggatcc atgacgtctt tagttcacgg ggaccattgc ctgcttatct agggtctaaa 2400 acatctgaat ctacatctat tttgcagcct tgggaaaggg aaagcaaagt cccactgatt 2460 aaaagagcta cacgtcttag agatgctatc tcttggtttg ttgaacccga ctctaaacta 2520 gcaatgacta tactttctaa catccactct ttaacaggcg aagaatggac caaaaggcag 2580 catgggttca aaagaacagg gtctgccctt cataggtttt cgacatctcg gatgagccat 2640 ggtgggttcg catctcagag cactgcagca ttgaccaggt tgatggcaac tacagacacc 2700 atgagggatc tgggagatca gaatttcgac tttttattcc aggcaacgtt gctctatgct 2760 cagattacca ccactgttgc aagagacgga tggatcacca gttgtacaga tcattatcat 2820 attgcctgta agtcctgttt gagacccata gaagagatca ccctggactc aagtatggac 2880 tacacgcccc cagatgtatc ccatgtgctg aagacatgga ggaatgggga aggttcgtgg 2940 ggacaagaga taaaacagat ctatccttta gaagggaatt ggaagaattt agcacctgct 3000 gagcaatcct atcaagtcgg cagatgtata ggttttctat atggagactt ggcgtataga 3060 aaatctactc atgccgagga cagttctcta tttcctctat ctatacaagg tcgtattaga 3120 ggtcgaggtt tcttaaaagg gttgctagac ggattaatga gagcaagttg ctgccaagta 3180 atacaccgga gaagtctggc tcatttgaag aggccggcca acgcagtgta cggaggtttg 3240 atttacttga ttgataaatt gagtgtatca cctccattcc tttctcttac tagatcagga 3300 cctattagag acgaattaga aacgattccc cacaagatcc caacctccta tccgacaagc 3360 aaccgtgata tgggggtgat tgtcagaaat tacttcaaat accaatgccg tctaattgaa 3420 aagggaaaat acagatcaca ttattcacaa ttatggttat tctcagatgt cttatccata 3480 gacttcattg gaccattctc tatttccacc accctcttgc aaatcctata caagccattt 3540 ttatctggga aagataagaa tgagttgaga gagctggcaa atctttcttc attgctaaga 3600 tcaggagagg ggtgggaaga catacatgtg aaattcttca ccaaggacat attattgtgt 3660 ccagaggaaa tcagacatgc ttgcaagttc gggattgcta aggataataa taaagacatg 3720 agctatcccc cttggggaag ggaatccaga gggacaatta caacaatccc tgtttattat 3780 acgaccaccc cttacccaaa gatgctagag atgcctccaa gaatccaaaa tcccctgctg 3840 tccggaatca ggttgggcca gttaccaact ggcgctcatt ataaaattcg gagtatatta 3900 catggaatgg gaatccatta cagggacttc ttgagttgtg gagacggctc cggagggatg 3960 actgctgcat tactacgaga aaatgtgcat agcagaggaa tattcaatag tctgttagaa 4020 ttatcagggt cagtcatgcg aggcgcctct cctgagcccc ccagtgccct agaaacttta 4080 ggaggagata aatcgagatg tgtaaatggt gaaacatgtt gggaatatcc atctgactta 4140 tgtgacccaa ggacttggga ctatttcctc cgactcaaag caggcttggg gcttcaaatt 4200 gatttaattg taatggatat ggaagttcgg gattcttcta ctagcctgaa aattgagacg 4260 aatgttagaa attatgtgca ccggattttg gatgagcaag gagttttaat ctacaagact 4320 tatggaacat atatttgtga gagcgaaaag aatgcagtaa caatccttgg tcccatgttc 4380 aagacggtcg acttagttca aacagaattt agtagttctc aaacgtctga agtatatatg 4440 gtatgtaaag gtttgaagaa attaatcgat gaacccaatc ccgattggtc ttccatcaat 4500 gaatcctgga aaaacctgta cgcattccag tcatcagaac aggaatttgc cagagcaaag 4560 aaggttagta catactttac cttgacaggt attccctccc aattcattcc tgatcctttt 4620 gtgaacattg agactatgct acaaatattc ggagtaccca cgggtgtgtc tcatgcggct 4680 gccttaaaat catctgatag acctgcagat ttattgacca ttagcctttt ttatatggcg 4740 attatatcgt attataacat caatcatatc agagtaggac cgatacctcc gaacccccca 4800 tcagatggaa ttgcacaaaa tgtggggatc gctataactg gtataagctt ttggctgagt 4860 ttgatggaga aagacattcc actatatcaa cagtgtttag cagttatcca gcaatcattc 4920 ccgattaggt gggaggctgt ttcagtaaaa ggaggataca agcagaagtg gagtactaga 4980 ggtgatgggc tcccaaaaga tacccgaatt tcagactcct tggccccaat cgggaactgg 5040 atcagatctc tggaattggt ccgaaaccaa gttcgtctaa atccattcaa tgagatcttg 5100 ttcaatcagc tatgtcgtac agtggataat catttgaaat ggtcaaattt gcgaaaaaac 5160 acaggaatga ttgaatggat caatagacga atttcaaaag aagaccggtc tatactgatg 5220 ttgaagagtg acctacatga ggaaaactct tggagagatt aaaaaatcat gaggagactc 5280 caaactttaa gtatgaaaaa aactttgatc cttaagaccc tcttgtg 5327 48 348 DNA Vesicular stomatitis virus 48 atggaagtcc acgattttga gaccgacgag ttcaatgatt tcaatgaaga tgactatgcc 60 acaagagaat tcctgaatcc cgatgagcgc atgacgtact tgaatcatgc tgattacaat 120 ttgaattctc ctctaattag tgatgatatt gacaatttga tcaggaaatt caattctctt 180 ccgattccct cgatgtggga tagtaagaac tgggatggag ttcttgagat gttaacatca 240 tgtcaagcca atcccatctc aacatctcag atgcataaat ggatgggaag ttggttaatg 300 tctgataatc atgatgccag tcaagggtat agttttttac atgaagtg 348 49 6330 DNA Vesicular stomatitis virus modified_base (1)..(6330) “n” represents a, t, c, g, other or unknown 49 atggaagtcc acgattttga gaccgacgag ttcaatgatt tcaatgaaga tgactatgcc 60 acaagagaat tcctgaatcc cgatgagcgc atgacgtact tgaatcatgc tgattacaat 120 ttgaattctc ctctaattag tgatgatatt gacaatttga tcaggaaatt caattctctt 180 ccgattccct cgatgtggga tagtaagaac tgggatggag ttcttgagat gttaacatca 240 tgtcaagcca atcccatctc aacatctcag atgcataaat ggatgggaag ttggttaatg 300 tctgataatc atgatgccag tcaagggtat agttttttac atgaagtgga caaagaggca 360 gaaataacat ttgacgtggt ggagaccttc atccgcggct ggggcaacaa accaattgaa 420 tacatcaaaa aggaaagatg gactgactca ttcaaaattc tcgcttattt gtgtcaaaag 480 tttttggact tacacaagtt gacattaatc ttaaatgctg tctctgaggt ggaattgctc 540 aacttggcga ggactttcaa aggcaaagtc agaagaagtt ctcatggaac gaacatatgc 600 aggcttaggg ttcccagctt gggtcctact tttatttcag aaggatgggc ttacttcaag 660 aaacttgata ttctaatgga ccgaaacttt ctgttaatgg tcaaagatgt gattataggg 720 aggatgcaaa cggtgctatc catggtatgt agaatagaca acctgttctc agagcaagac 780 atcttctccc ttctaaatat ctacagaatt ggagataaaa ttgtggagag gcagggaaat 840 ttttcttatg acttgattaa aatggtggaa ccgatatgca acttgaggct gatgaaatta 900 gcaagagaat caaggccttt agtcccacaa ttccctcatt ttgaaaatca tatcaagact 960 tctgttgatg aaggggcaaa aattgaccga ggtataagat tcctccatga tcagataatg 1020 agtgtgaaaa cagtggatct cacactggtg atttatggat cgttcagaca ttggggtcat 1080 ccttttatag attattacgc tggactagaa aaattacatt cccaagtaac catgaagaaa 1140 gatattgatg tgtcatatgc aaaagcactt gcaagtgatt tagctcggat tgttctattt 1200 caacagttca atgatcataa aaagtggttc gtgaatggag acttgctccc tcatgatcat 1260 ccctttaaaa gtcatgttaa agaaaataca tggcccacag ctgctcaagt tcaagatttt 1320 ggagataaat ggcatgaact tccgctgatt aaatgttttg aaatacccga cttactagac 1380 ccatcgataa tatactctga caaaagtcat tcaatgaata ggtcagaggt gttgaaacat 1440 gtccgaatga atccgaacac tcctatccct agtaaaaagg tgttgcagac tatgttggac 1500 acaaaggcta ccaattggaa agaatttctt aaagagattg atgagaaggg cttagatgat 1560 gatgatctaa ttattggtct taaaggaaag gagagggaac tgaagttggc aggtagattt 1620 ttctccctaa tgtcttggaa attgcgagaa tactttgtaa ttaccgaata tttgataaag 1680 actcatttcg tccctatgtt taaaggcctg acaatggcgg acgatctaac tgcagtcatt 1740 aaaaagatgt tagattcctc atccggccaa ggattgaagt catatgaggc aatttgcata 1800 gccaatcaca ttgattacga aaaatggaat aaccaccaaa ggaagttatc aaacggccca 1860 gtgttccgag ttatgggcca gttcttaggt tatccatcct taatcgagag aactcatgaa 1920 ttttttgaga aaagtcttat atactacaat ggaagaccag acttgatgcg tgttcacaac 1980 aacacactga tcaattcaac ctcccaacga gtttgttggc aaggacaaga gggtggactg 2040 gaaggtctac ggcaaaaagg atggagtatc ctcaatctac tggttattca aagagaggct 2100 aaaatcagaa acactgctgt caaagtcttg gcacaaggtg ataatcaagt tatttgcaca 2160 cagtataaaa cgaagaaatc gagaaacgtt gtagaattac agggtgctct caatcaaatg 2220 gtttctaata atgagaaaat tatgactgca atcaaaatag ggacagggaa gttaggactt 2280 ttgataaatg acgatgagac tatgcaatct gcagattact tgaattatgg aaaaatacca 2340 attttccgtg gagtgattag agggttagag accaagagat ggtcacgagt gacttgtgtc 2400 accaatgacc aaatacccac ttgtgctaat ataatgagct cagtttccac aaatgctctc 2460 accgtagctc attttgctga gaacccaatc aatgccatga tacagtacaa ttattttggg 2520 acatttgcta gactcttgtt gatgatgcat gatcctgctc ttcgtcaatc attgtatgaa 2580 gttcaagata agataccggg cttgcacagt tctactttca aatacgccat gttgtatttg 2640 gacccttcca ttggaggagt gtcgggcatg tctttgtcca ggtttttgat tagagccttc 2700 ccagatcccg taacagaaag tctctcattc tggagattca tccatgtaca tgctcgaagt 2760 gagcatctga aggagatgag tgcagtattt ggaaaccccg agatagccaa gtttcgaata 2820 actcacatag acaagctagt agaagatcca acctctctga acatcgctat gggaatgagt 2880 ccagcgaact tgttaaagac tgaggttaaa aaatgcttaa tcgaatcaag acaaaccatc 2940 aggaaccagg tgattaagga tgcaaccata tatttgtatc atgaagagga tcggctcaga 3000 agtttcttat ggtcaataaa tcctctgttc cctagatttt taagtgaatt caaatcaggc 3060 acttttttgg gagtcgcaga cgggctcatc agtctatttc aaaattctcg tactattcgg 3120 aactccttta agaaaaagta tcatagggaa ttggatgatt tgattgtgag gagtgaggta 3180 tcctctttga cacatttagg gaaacttcat ttgagaaggg gatcatgtaa aatgtggaca 3240 tgttcagcta ctcatgctga cacattaaga tacaaatcct ggggccgtac agttattggg 3300 acaactgtac cccatccatt agaaatgttg ggtccacaac atcgaaaaga gactccttgt 3360 gcaccatgta acacatcagg gttcaattat gtttctgtgc attgtccaga cgggatccat 3420 gacgtcttta gttcacgggg accattgcct gcttatctag ggtctaaaac atctgaatct 3480 acatctattt tgcagccttg ggaaagggaa agcaaagtcc cactgattaa aagagctaca 3540 cgtcttagag atgctatctc ttggtttgtt gaacccgact ctaaactagc aatgactata 3600 ctttctaaca tccactcttt aacaggcgaa gaatggacca aaaggcagca tgggttcaaa 3660 agaacagggt ctgcccttca taggttttcg acatctcgga tgagccatgg tgggttcgca 3720 tctcagagca ctgcagcatt gaccaggttg atggcaacta cagacaccat gagggatctg 3780 ggagatcaga atttcgactt tttattccan gcaacgttgc tctatgctca nattaccacc 3840 actgttgcaa gagacggatg gatcaccagt tgtacagatc attatcatat tgcctgtaag 3900 tcctgtttga gacccataga agagatcacc ctggactcaa gtatggacta cacgccccca 3960 gatgtatccc atgtgctgaa gacatggagg aatggggaag gttcgtgggg acaagagata 4020 aaacagatct atcctttaga agggaattgg aagaatttag cacctgctga gcaatcctat 4080 caagtcggca gatgtatagg ttttctatat ggagacttgg cgtatagaaa atctactcat 4140 gccgaggaca gttctctatt tcctctatct atacaaggtc gtattagagg tcgaggtttc 4200 ttaaaagggt tgctagacgg attaatgaga gcaagttgct gccaagtaat acaccggaga 4260 agtctggctc atttgaagag gccggccaac gcagtgtacg gaggtttgat ttacttgatt 4320 gataaattga gtgtatcacc tccattcctt tctcttacta gatcaggacc tattagagac 4380 gaattagaaa cgattcccca caagatccca acctcctatc cgacaagcaa ccgtgatatg 4440 ggggtgattg tcagaaatta cttcaaatac caatgccgtc taattgaaaa gggaaaatac 4500 agatcacatt attcacaatt atggttattc tcagatgtct tatccataga cttcattgga 4560 ccattctcta tttccaccac cctcttgcaa atcctataca agccattttt atctgggaaa 4620 gataagaatg agttgagaga gctggcaaat ctttcttcat tgctaagatc aggagagggg 4680 tgggaagaca tacatgtgaa attcttcacc aaggacatat tattgtgtcc agaggaaatc 4740 agacatgctt gcaagttcgg gattgctaag gataataata aagacatgag ctatccccct 4800 tggggaaggg aatccagagg gacaattaca acaatccctg tttattatac gaccacccct 4860 tacccaaaga tgctagagat gcctccaaga atccaaaatc ccctgctgtc cggaatcagg 4920 ttgggccagt taccaactgg cgctcattat aaaattcgga gtatattaca tggaatggga 4980 atccattaca gggacttctt gagttgtgga gacggctccg gagggatgac tgctgcatta 5040 ctacgagaaa atgtgcatag cagaggaata ttcaatagtc tgttagaatt atcagggtca 5100 gtcatgcgag gcgcctctcc tgagcccccc agtgccctag aaactttagg aggagataaa 5160 tcgagatgtg taaatggtga aacatgttgg gaatatccat ctgacttatg tgacccaagg 5220 acttgggact atttcctccg actcaaagca ggcttggggc ttcaaattga tttaattgta 5280 atggatatgg aagttcggga ttcttctact agcctgaaaa ttgagacgaa tgttagaaat 5340 tatgtgcacc ggattttgga tgagcaagga gttttaatct acaagactta tggaacatat 5400 atttgtgaga gcgaaaagaa tgcagtaaca atccttggtc ccatgttcaa gacggtcgac 5460 ttagttcaaa cagaatttag tagttctcaa acgtctgaag tatatatggt atgtaaaggt 5520 ttgaagaaat taatcgatga acccaatccc gattggtctt ccatcaatga atcctggaaa 5580 aacctgtacg cattccagtc atcagaacag gaatttgcca gagcaaagaa ggttagtaca 5640 tactttacct tgacaggtat tccctcccaa ttcattcctg atccttttgt gaacattgag 5700 actatgctac aaatattcgg agtacccacg ggtgtgtctc atgcggctgc cttaaaatca 5760 tctgatagac ctgcagattt attgaccatt agcctttttt atatggcgat tatatcgtat 5820 tataacatca atcatatcag agtaggaccg atacctccga accccccatc agatggaatt 5880 gcacaaaatg tggggatcgc tataactggt ataagctttt ggctgagttt gatggagaaa 5940 gacattccac tatatcaaca gtgtttagca gttatccagc aatcattccc gattaggtgg 6000 gaggctgttt cagtaaaagg aggatacaag cagaagtgga gtactagagg tgatgggctc 6060 ccaaaagata cccgaatttc agactccttg gccccaatcg ggaactggat cagatctctg 6120 gaattggtcc gaaaccaagt tcgtctaaat ccattcaatg agatcttgtt caatcagcta 6180 tgtcgtacag tggataatca tttgaaatgg tcaaatttgc gaaaaaacac aggaatgatt 6240 gaatggatca atagacgaat ttcaaaagaa gaccggtcta tactgatgtt gaagagtgac 6300 ctacatgagg aaaactcttg gagagattaa 6330 50 2109 PRT Vesicular stomatitis virus 50 Met Glu Val His Asp Phe Glu Thr Asp Glu Phe Asn Asp Phe Asn Glu 1 5 10 15 Asp Asp Tyr Ala Thr Arg Glu Phe Leu Asn Pro Asp Glu Arg Met Thr 20 25 30 Tyr Leu Asn His Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45 Asp Ile Asp Asn Leu Ile Arg Lys Phe Asn Ser Leu Pro Ile Pro Ser 50 55 60 Met Trp Asp Ser Lys Asn Trp Asp Gly Val Leu Glu Met Leu Thr Ser 65 70 75 80 Cys Gln Ala Asn Pro Ile Ser Thr Ser Gln Met His Lys Trp Met Gly 85 90 95 Ser Trp Leu Met Ser Asp Asn His Asp Ala Ser Gln Gly Tyr Ser Phe 100 105 110 Leu His Glu Val Asp Lys Glu Ala Glu Ile Thr Phe Asp Val Val Glu 115 120 125 Thr Phe Ile Arg Gly Trp Gly Asn Lys Pro Ile Glu Tyr Ile Lys Lys 130 135 140 Glu Arg Trp Thr Asp Ser Phe Lys Ile Leu Ala Tyr Leu Cys Gln Lys 145 150 155 160 Phe Leu Asp Leu His Lys Leu Thr Leu Ile Leu Asn Ala Val Ser Glu 165 170 175 Val Glu Leu Leu Asn Leu Ala Arg Thr Phe Lys Gly Lys Val Arg Arg 180 185 190 Ser Ser His Gly Thr Asn Ile Cys Arg Ile Arg Val Pro Ser Leu Gly 195 200 205 Pro Thr Phe Ile Ser Glu Gly Trp Ala Tyr Phe Lys Lys Leu Asp Ile 210 215 220 Leu Met Asp Arg Asn Phe Leu Leu Met Val Lys Asp Val Ile Ile Gly 225 230 235 240 Arg Met Gln Thr Val Leu Ser Met Val Cys Arg Ile Asp Asn Leu Phe 245 250 255 Ser Glu Gln Asp Ile Phe Ser Leu Leu Asn Ile Tyr Arg Ile Gly Asp 260 265 270 Lys Ile Val Glu Arg Gln Gly Asn Phe Ser Tyr Asp Leu Ile Lys Met 275 280 285 Val Glu Pro Ile Cys Asn Leu Lys Leu Met Lys Leu Ala Arg Glu Ser 290 295 300 Arg Pro Leu Val Pro Gln Phe Pro His Phe Glu Asn His Ile Lys Thr 305 310 315 320 Ser Val Asp Glu Gly Ala Lys Ile Asp Arg Gly Ile Arg Phe Leu His 325 330 335 Asp Gln Ile Met Ser Val Lys Thr Val Asp Leu Thr Leu Val Ile Tyr 340 345 350 Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asp Tyr Tyr Thr Gly 355 360 365 Leu Glu Lys Leu His Ser Gln Val Thr Met Lys Lys Asp Ile Asp Val 370 375 380 Ser Tyr Ala Lys Ala Leu Ala Ser Asp Leu Ala Arg Ile Val Leu Phe 385 390 395 400 Gln Gln Phe Asn Asp His Lys Lys Trp Phe Val Asn Gly Asp Leu Leu 405 410 415 Pro His Asp His Pro Phe Lys Ser His Val Lys Glu Asn Thr Trp Pro 420 425 430 Thr Ala Ala Gln Val Gln Asp Phe Gly Asp Lys Trp His Glu Leu Pro 435 440 445 Leu Ile Lys Cys Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile 450 455 460 Tyr Ser Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu Lys His 465 470 475 480 Val Arg Met Asn Pro Asn Thr Pro Ile Pro Ser Lys Lys Val Leu Gln 485 490 495 Thr Met Leu Asp Thr Lys Ala Thr Asn Trp Lys Glu Phe Leu Lys Glu 500 505 510 Ile Asp Glu Lys Gly Leu Asp Asp Asp Asp Leu Ile Ile Gly Leu Lys 515 520 525 Gly Lys Glu Arg Glu Leu Lys Leu Ala Gly Arg Phe Phe Ser Leu Met 530 535 540 Ser Trp Lys Leu Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys 545 550 555 560 Thr His Phe Val Pro Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu 565 570 575 Thr Ala Val Ile Lys Lys Met Leu Asp Ser Ser Ser Gly Gln Gly Leu 580 585 590 Lys Ser Tyr Glu Ala Ile Cys Ile Ala Asn His Ile Asp Tyr Glu Lys 595 600 605 Trp Asn Asn His Gln Arg Lys Leu Ser Asn Gly Pro Val Phe Arg Val 610 615 620 Met Gly Gln Phe Leu Gly Tyr Pro Ser Leu Ile Glu Arg Thr His Glu 625 630 635 640 Phe Phe Glu Lys Ser Leu Ile Tyr Tyr Asn Gly Arg Pro Asp Leu Met 645 650 655 Arg Val His Asn Asn Thr Leu Ile Asn Ser Thr Ser Gln Arg Val Cys 660 665 670 Trp Gln Gly Gln Glu Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680 685 Thr Ile Leu Asn Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn 690 695 700 Thr Ala Val Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile Cys Thr 705 710 715 720 Gln Tyr Lys Thr Lys Lys Ser Arg Asn Val Val Glu Leu Gln Gly Ala 725 730 735 Leu Asn Gln Met Val Ser Asn Asn Glu Lys Ile Met Thr Ala Ile Lys 740 745 750 Ile Gly Thr Gly Lys Leu Gly Leu Leu Ile Asn Asp Asp Glu Thr Met 755 760 765 Gln Ser Ala Asp Tyr Leu Asn Tyr Gly Lys Ile Pro Ile Phe Arg Gly 770 775 780 Val Ile Arg Gly Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val 785 790 795 800 Thr Asn Asp Gln Ile Pro Thr Cys Ala Asn Ile Met Ser Ser Val Ser 805 810 815 Thr Asn Ala Leu Thr Val Ala His Phe Ala Glu Asn Pro Ile Asn Ala 820 825 830 Met Ile Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg Leu Leu Leu Met 835 840 845 Met His Asp Pro Ala Leu Arg Gln Ser Leu Tyr Glu Val Gln Asp Lys 850 855 860 Ile Pro Gly Leu His Ser Ser Thr Phe Lys Tyr Ala Met Leu Tyr Leu 865 870 875 880 Asp Pro Ser Ile Gly Gly Val Ser Gly Met Ser Leu Ser Arg Phe Leu 885 890 895 Ile Arg Ala Phe Pro Asp Pro Val Thr Glu Ser Leu Ser Phe Trp Arg 900 905 910 Phe Ile His Val His Ala Arg Ser Glu His Leu Lys Glu Met Ser Ala 915 920 925 Val Phe Gly Asn Pro Glu Ile Ala Lys Phe Arg Ile Thr His Ile Asp 930 935 940 Lys Leu Val Glu Asp Pro Thr Ser Leu Asn Ile Ala Met Gly Met Ser 945 950 955 960 Pro Ala Asn Leu Leu Lys Thr Glu Val Lys Lys Cys Leu Ile Glu Ser 965 970 975 Arg Gln Thr Ile Arg Asn Gln Val Ile Lys Asp Ala Thr Ile Tyr Leu 980 985 990 Tyr His Glu Glu Asp Arg Leu Arg Ser Phe Leu Trp Ser Ile Asn Pro 995 1000 1005 Leu Phe Pro Arg Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Leu Gly 1010 1015 1020 Val Ala Asp Gly Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr Ile Arg 1025 1030 1035 1040 Asn Ser Phe Lys Lys Lys Tyr His Arg Glu Leu Asp Asp Leu Ile Val 1045 1050 1055 Arg Ser Glu Val Ser Ser Leu Thr His Leu Gly Lys Leu His Leu Arg 1060 1065 1070 Arg Gly Ser Cys Lys Met Trp Thr Cys Ser Ala Thr His Ala Asp Thr 1075 1080 1085 Leu Arg Tyr Lys Ser Trp Gly Arg Thr Val Ile Gly Thr Thr Val Pro 1090 1095 1100 His Pro Leu Glu Met Leu Gly Pro Gln His Arg Lys Glu Thr Pro Cys 1105 1110 1115 1120 Ala Pro Cys Asn Thr Ser Gly Phe Asn Tyr Val Ser Val His Cys Pro 1125 1130 1135 Asp Gly Ile His Asp Val Phe Ser Ser Arg Gly Pro Leu Pro Ala Tyr 1140 1145 1150 Leu Gly Ser Lys Thr Ser Glu Ser Thr Ser Ile Leu Gln Pro Trp Glu 1155 1160 1165 Arg Glu Ser Lys Val Pro Leu Ile Lys Arg Ala Thr Arg Leu Arg Asp 1170 1175 1180 Ala Ile Ser Trp Phe Val Glu Pro Asp Ser Lys Leu Ala Met Thr Ile 1185 1190 1195 1200 Leu Ser Asn Ile His Ser Leu Thr Gly Glu Glu Trp Thr Lys Arg Gln 1205 1210 1215 His Gly Phe Lys Arg Thr Gly Ser Ala Leu His Arg Phe Ser Thr Ser 1220 1225 1230 Arg Met Ser His Gly Gly Phe Ala Ser Gln Ser Thr Ala Ala Leu Thr 1235 1240 1245 Arg Leu Met Ala Thr Thr Asp Thr Met Arg Asp Leu Gly Asp Gln Asn 1250 1255 1260 Phe Asp Phe Leu Phe Gln Ala Thr Leu Leu Tyr Ala Gln Ile Thr Thr 1265 1270 1275 1280 Thr Val Ala Arg Asp Gly Trp Ile Thr Ser Cys Thr Asp His Tyr His 1285 1290 1295 Ile Ala Cys Lys Ser Cys Leu Arg Pro Ile Glu Glu Ile Thr Leu Asp 1300 1305 1310 Ser Ser Met Asp Tyr Thr Pro Pro Asp Val Ser His Val Leu Lys Thr 1315 1320 1325 Trp Arg Asn Gly Glu Gly Ser Trp Gly Gln Glu Ile Lys Gln Ile Tyr 1330 1335 1340 Pro Leu Glu Gly Asn Trp Lys Asn Leu Ala Pro Ala Glu Gln Ser Tyr 1345 1350 1355 1360 Gln Val Gly Arg Cys Ile Gly Phe Leu Tyr Gly Asp Leu Ala Tyr Arg 1365 1370 1375 Lys Ser Thr His Ala Glu Asp Ser Ser Leu Phe Pro Leu Ser Ile Gln 1380 1385 1390 Gly Arg Ile Arg Gly Arg Gly Phe Leu Lys Gly Leu Leu Asp Gly Leu 1395 1400 1405 Met Arg Ala Ser Cys Cys Gln Val Ile His Arg Arg Ser Leu Ala His 1410 1415 1420 Leu Lys Arg Pro Ala Asn Ala Val Tyr Gly Gly Leu Ile Tyr Leu Ile 1425 1430 1435 1440 Asp Lys Leu Ser Val Ser Pro Pro Phe Leu Ser Leu Thr Arg Ser Gly 1445 1450 1455 Pro Ile Arg Asp Glu Leu Glu Thr Ile Pro His Lys Ile Pro Thr Ser 1460 1465 1470 Tyr Pro Thr Ser Asn Arg Asp Met Gly Val Ile Val Arg Asn Tyr Phe 1475 1480 1485 Lys Tyr Gln Cys Arg Leu Ile Glu Lys Gly Lys Tyr Arg Ser His Tyr 1490 1495 1500 Ser Gln Leu Trp Leu Phe Ser Asp Val Leu Ser Ile Asp Phe Ile Gly 1505 1510 1515 1520 Pro Phe Ser Ile Ser Thr Thr Leu Leu Gln Ile Leu Tyr Lys Pro Phe 1525 1530 1535 Leu Ser Gly Lys Asp Lys Asn Glu Leu Arg Glu Leu Ala Asn Leu Ser 1540 1545 1550 Ser Leu Leu Arg Ser Gly Glu Gly Trp Glu Asp Ile His Val Lys Phe 1555 1560 1565 Phe Thr Lys Asp Ile Leu Leu Cys Pro Glu Glu Ile Arg His Ala Cys 1570 1575 1580 Lys Phe Gly Ile Ala Lys Asp Asn Asn Lys Asp Met Ser Tyr Pro Pro 1585 1590 1595 1600 Trp Gly Arg Glu Ser Arg Gly Thr Ile Thr Thr Ile Pro Val Tyr Tyr 1605 1610 1615 Thr Thr Thr Pro Tyr Pro Lys Met Leu Glu Met Pro Pro Arg Ile Gln 1620 1625 1630 Asn Pro Leu Leu Ser Gly Ile Arg Leu Gly Gln Leu Pro Thr Gly Ala 1635 1640 1645 His Tyr Lys Ile Arg Ser Ile Leu His Gly Met Gly Ile His Tyr Arg 1650 1655 1660 Asp Phe Leu Ser Cys Gly Asp Gly Ser Gly Gly Met Thr Ala Ala Leu 1665 1670 1675 1680 Leu Arg Glu Asn Val His Ser Arg Gly Ile Phe Asn Ser Leu Leu Glu 1685 1690 1695 Leu Ser Gly Ser Val Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala 1700 1705 1710 Leu Glu Thr Leu Gly Gly Asp Lys Ser Arg Cys Val Asn Gly Glu Thr 1715 1720 1725 Cys Trp Glu Tyr Pro Ser Asp Leu Cys Asp Pro Arg Thr Trp Asp Tyr 1730 1735 1740 Phe Leu Arg Leu Lys Ala Gly Leu Gly Leu Gln Ile Asp Leu Ile Val 1745 1750 1755 1760 Met Asp Met Glu Val Arg Asp Ser Ser Thr Ser Leu Lys Ile Glu Thr 1765 1770 1775 Asn Val Arg Asn Tyr Val His Arg Ile Leu Asp Glu Gln Gly Val Leu 1780 1785 1790 Ile Tyr Lys Thr Tyr Gly Thr Tyr Ile Cys Glu Ser Glu Lys Asn Ala 1795 1800 1805 Val Thr Ile Leu Gly Pro Met Phe Lys Thr Val Asp Leu Val Gln Thr 1810 1815 1820 Glu Phe Ser Ser Ser Gln Thr Ser Glu Val Tyr Met Val Cys Lys Gly 1825 1830 1835 1840 Leu Lys Lys Leu Ile Asp Glu Pro Asn Pro Asp Trp Ser Ser Ile Asn 1845 1850 1855 Glu Ser Trp Lys Asn Leu Tyr Ala Phe Gln Ser Ser Glu Gln Glu Phe 1860 1865 1870 Ala Arg Ala Lys Lys Val Ser Thr Tyr Phe Thr Leu Thr Gly Ile Pro 1875 1880 1885 Ser Gln Phe Ile Pro Asp Pro Phe Val Asn Ile Glu Thr Met Leu Gln 1890 1895 1900 Ile Phe Gly Val Pro Thr Gly Val Ser His Ala Ala Ala Leu Lys Ser 1905 1910 1915 1920 Ser Asp Arg Pro Ala Asp Leu Leu Thr Ile Ser Leu Phe Tyr Met Ala 1925 1930 1935 Ile Ile Ser Tyr Tyr Asn Ile Asn His Ile Arg Val Gly Pro Ile Pro 1940 1945 1950 Pro Asn Pro Pro Ser Asp Gly Ile Ala Gln Asn Val Gly Ile Ala Ile 1955 1960 1965 Thr Gly Ile Ser Phe Trp Leu Ser Leu Met Glu Lys Asp Ile Pro Leu 1970 1975 1980 Tyr Gln Gln Cys Leu Ala Val Ile Gln Gln Ser Phe Pro Ile Arg Trp 1985 1990 1995 2000 Glu Ala Val Ser Val Lys Gly Gly Tyr Lys Gln Lys Trp Ser Thr Arg 2005 2010 2015 Gly Asp Gly Leu Pro Lys Asp Thr Arg Thr Ser Asp Ser Leu Ala Pro 2020 2025 2030 Ile Gly Asn Trp Ile Arg Ser Leu Glu Leu Val Arg Asn Gln Val Arg 2035 2040 2045 Leu Asn Pro Phe Asn Glu Ile Leu Phe Asn Gln Leu Cys Arg Thr Val 2050 2055 2060 Asp Asn His Leu Lys Trp Ser Asn Leu Arg Arg Asn Thr Gly Met Ile 2065 2070 2075 2080 Glu Trp Ile Asn Arg Arg Ile Ser Lys Glu Asp Arg Ser Ile Leu Met 2085 2090 2095 Leu Lys Ser Asp Leu His Glu Glu Asn Ser Trp Arg Asp 2100 2105 51 1753 PRT Vesicular stomatitis virus MOD_RES (1)..(1753) “Xaa” represents any, other or unknown amino acid 51 His Trp Gly His Pro Phe Ile Asp Tyr Tyr Ala Gly Leu Glu Lys Leu 1 5 10 15 His Ser Gln Val Thr Xaa Lys Lys Asp Ile Asp Val Ser Tyr Ala Lys 20 25 30 Ala Leu Ala Ser Asp Leu Ala Arg Ile Val Leu Phe Gln Gln Phe Asn 35 40 45 Asp His Xaa Lys Trp Phe Val Asn Gly Asp Leu Leu Pro His Asp His 50 55 60 Pro Phe Lys Ser His Val Lys Glu Asn Thr Trp Pro Thr Ala Ala Gln 65 70 75 80 Val Gln Asp Phe Gly Asp Lys Trp His Glu Leu Pro Leu Ile Lys Cys 85 90 95 Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile Tyr Ser Asp Lys 100 105 110 Ser His Ser Met Asn Arg Ser Glu Val Leu Lys His Val Arg Met Asn 115 120 125 Pro Asn Thr Pro Ile Pro Ser Lys Lys Val Leu Gln Thr Met Leu Asp 130 135 140 Thr Lys Ala Thr Asn Trp Lys Glu Phe Leu Lys Glu Ile Asp Glu Lys 145 150 155 160 Gly Leu Asp Asp Asp Asp Leu Ile Ile Gly Leu Lys Gly Lys Glu Arg 165 170 175 Glu Leu Lys Leu Ala Gly Arg Phe Phe Ser Leu Met Ser Trp Lys Leu 180 185 190 Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys Thr His Phe Val 195 200 205 Pro Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu Thr Ala Val Ile 210 215 220 Lys Lys Met Leu Asp Ser Ser Ser Gly Gln Gly Leu Lys Ser Tyr Glu 225 230 235 240 Ala Ile Cys Ile Ala Asn His Ile Asp Tyr Glu Lys Trp Asn Asn His 245 250 255 Gln Arg Lys Leu Ser Asn Gly Pro Val Phe Arg Val Met Gly Gln Phe 260 265 270 Leu Gly Tyr Pro Ser Leu Ile Glu Arg Thr His Glu Phe Phe Glu Lys 275 280 285 Ser Leu Ile Tyr Tyr Asn Gly Arg Pro Asp Leu Met Arg Val His Asn 290 295 300 Asn Thr Leu Ile Asn Ser Thr Ser Gln Arg Val Cys Trp Gln Gly Gln 305 310 315 320 Glu Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp Ser Ile Leu Asn 325 330 335 Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn Thr Ala Val Lys 340 345 350 Val Leu Ala Gln Gly Asp Asn Gln Val Ile Cys Thr Gln Tyr Lys Thr 355 360 365 Lys Lys Ser Arg Asn Val Val Glu Leu Gln Gly Ala Leu Asn Gln Met 370 375 380 Val Ser Asn Asn Glu Lys Ile Met Thr Ala Ile Lys Ile Gly Thr Gly 385 390 395 400 Lys Leu Gly Leu Leu Ile Asn Asp Asp Glu Thr Met Gln Ser Ala Asp 405 410 415 Tyr Leu Asn Tyr Gly Lys Ile Pro Ile Phe Arg Gly Val Ile Arg Gly 420 425 430 Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val Thr Asn Asp Gln 435 440 445 Ile Pro Thr Cys Ala Asn Ile Met Ser Ser Val Ser Thr Asn Ala Leu 450 455 460 Thr Val Ala His Phe Ala Glu Asn Pro Ile Asn Ala Met Ile Gln Tyr 465 470 475 480 Asn Tyr Phe Gly Thr Phe Ala Arg Leu Leu Leu Met Met His Asp Pro 485 490 495 Ala Leu Arg Gln Ser Leu Tyr Glu Val Gln Asp Lys Ile Pro Gly Leu 500 505 510 His Ser Ser Thr Phe Lys Tyr Ala Met Leu Tyr Leu Asp Pro Ser Ile 515 520 525 Gly Gly Val Ser Gly Met Ser Leu Ser Arg Phe Leu Ile Arg Ala Phe 530 535 540 Pro Asp Pro Val Thr Glu Ser Leu Ser Phe Trp Arg Phe Ile His Val 545 550 555 560 His Ala Arg Ser Glu His Leu Lys Glu Met Ser Ala Val Phe Gly Asn 565 570 575 Pro Glu Ile Ala Lys Phe Arg Ile Thr His Ile Asp Lys Leu Val Glu 580 585 590 Asp Pro Thr Ser Leu Asn Ile Ala Met Gly Met Ser Pro Ala Asn Leu 595 600 605 Leu Lys Thr Glu Val Lys Lys Cys Leu Ile Glu Ser Arg Gln Thr Ile 610 615 620 Arg Asn Gln Val Ile Lys Asp Ala Thr Ile Tyr Leu Tyr His Glu Glu 625 630 635 640 Asp Arg Leu Arg Ser Phe Leu Trp Ser Ile Asn Pro Leu Phe Pro Arg 645 650 655 Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Leu Gly Val Ala Asp Gly 660 665 670 Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr Ile Arg Asn Ser Phe Lys 675 680 685 Lys Lys Tyr His Arg Glu Leu Asp Asp Leu Ile Val Arg Ser Glu Val 690 695 700 Ser Ser Leu Thr His Leu Gly Lys Leu His Leu Arg Arg Gly Ser Cys 705 710 715 720 Lys Met Trp Thr Cys Ser Ala Thr His Ala Asp Thr Leu Arg Tyr Lys 725 730 735 Ser Trp Gly Arg Thr Val Ile Gly Thr Thr Val Pro His Pro Leu Glu 740 745 750 Met Leu Gly Pro Gln His Arg Lys Glu Thr Pro Cys Ala Pro Cys Asn 755 760 765 Thr Ser Gly Phe Asn Tyr Val Ser Val His Cys Pro Asp Gly Ile His 770 775 780 Asp Val Phe Ser Ser Arg Gly Pro Leu Pro Ala Tyr Leu Gly Ser Lys 785 790 795 800 Thr Ser Glu Ser Thr Ser Ile Leu Gln Pro Trp Glu Arg Glu Ser Lys 805 810 815 Val Pro Leu Ile Lys Arg Ala Thr Arg Leu Arg Asp Ala Ile Ser Trp 820 825 830 Phe Val Glu Pro Asp Ser Lys Leu Ala Met Thr Ile Leu Ser Asn Ile 835 840 845 His Ser Leu Thr Gly Glu Glu Trp Thr Lys Arg Gln His Gly Phe Lys 850 855 860 Arg Thr Gly Ser Ala Leu His Arg Phe Ser Thr Ser Arg Met Ser His 865 870 875 880 Gly Gly Phe Ala Ser Gln Ser Thr Ala Ala Leu Thr Arg Leu Met Ala 885 890 895 Thr Thr Asp Thr Met Arg Asp Leu Gly Asp Gln Asn Phe Asp Phe Leu 900 905 910 Phe Gln Ala Thr Leu Leu Tyr Ala Gln Ile Thr Thr Thr Val Ala Arg 915 920 925 Asp Gly Trp Ile Thr Ser Cys Thr Asp His Tyr His Ile Ala Cys Lys 930 935 940 Ser Cys Leu Arg Pro Ile Glu Glu Ile Thr Leu Asp Ser Ser Met Asp 945 950 955 960 Tyr Thr Pro Pro Asp Val Ser His Val Leu Lys Thr Trp Arg Asn Gly 965 970 975 Glu Gly Ser Trp Gly Gln Glu Ile Lys Gln Ile Tyr Pro Leu Glu Gly 980 985 990 Asn Trp Lys Asn Leu Ala Pro Ala Glu Gln Ser Tyr Gln Val Gly Arg 995 1000 1005 Cys Ile Gly Phe Leu Tyr Gly Asp Leu Ala Tyr Arg Lys Ser Thr His 1010 1015 1020 Ala Glu Asp Ser Ser Leu Phe Pro Leu Ser Ile Gln Gly Arg Ile Arg 1025 1030 1035 1040 Gly Arg Gly Phe Leu Lys Gly Leu Leu Asp Gly Leu Met Arg Ala Ser 1045 1050 1055 Cys Cys Gln Val Ile His Arg Arg Ser Leu Ala His Leu Lys Arg Pro 1060 1065 1070 Ala Asn Ala Val Tyr Gly Gly Leu Ile Tyr Leu Ile Asp Lys Leu Ser 1075 1080 1085 Val Ser Pro Pro Phe Leu Ser Leu Thr Arg Ser Gly Pro Ile Arg Asp 1090 1095 1100 Glu Leu Glu Thr Ile Pro His Lys Ile Pro Thr Ser Tyr Pro Thr Ser 1105 1110 1115 1120 Asn Arg Asp Met Gly Val Ile Val Arg Asn Tyr Phe Lys Tyr Gln Cys 1125 1130 1135 Arg Leu Ile Glu Lys Gly Lys Tyr Arg Ser His Tyr Ser Gln Leu Trp 1140 1145 1150 Leu Phe Ser Asp Val Leu Ser Ile Asp Phe Ile Gly Pro Phe Ser Ile 1155 1160 1165 Ser Thr Thr Leu Leu Gln Ile Leu Tyr Lys Pro Phe Leu Ser Gly Lys 1170 1175 1180 Asp Lys Asn Glu Leu Arg Glu Leu Ala Asn Leu Ser Ser Leu Leu Arg 1185 1190 1195 1200 Ser Gly Glu Gly Trp Glu Asp Ile His Val Lys Phe Phe Thr Lys Asp 1205 1210 1215 Ile Leu Leu Cys Pro Glu Glu Ile Arg His Ala Cys Lys Phe Gly Ile 1220 1225 1230 Ala Lys Asp Asn Asn Lys Asp Met Ser Tyr Pro Pro Trp Gly Arg Glu 1235 1240 1245 Ser Arg Gly Thr Ile Thr Thr Ile Pro Val Tyr Tyr Thr Thr Thr Pro 1250 1255 1260 Tyr Pro Lys Met Leu Glu Met Pro Pro Arg Ile Gln Asn Pro Leu Leu 1265 1270 1275 1280 Ser Gly Ile Arg Leu Gly Gln Leu Pro Thr Gly Ala His Tyr Lys Ile 1285 1290 1295 Arg Ser Ile Leu His Gly Met Gly Ile His Tyr Arg Asp Phe Leu Ser 1300 1305 1310 Cys Gly Asp Gly Ser Gly Gly Met Thr Ala Ala Leu Leu Arg Glu Asn 1315 1320 1325 Val His Ser Arg Gly Ile Phe Asn Ser Leu Leu Glu Leu Ser Gly Ser 1330 1335 1340 Val Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala Leu Glu Thr Leu 1345 1350 1355 1360 Gly Gly Asp Lys Ser Arg Cys Val Asn Gly Glu Thr Cys Trp Glu Tyr 1365 1370 1375 Pro Ser Asp Leu Cys Asp Pro Arg Thr Trp Asp Tyr Phe Leu Arg Leu 1380 1385 1390 Lys Ala Gly Leu Gly Leu Gln Ile Asp Leu Ile Val Met Asp Met Glu 1395 1400 1405 Val Arg Asp Ser Ser Thr Ser Leu Lys Ile Glu Thr Asn Val Arg Asn 1410 1415 1420 Tyr Val His Arg Ile Leu Asp Glu Gln Gly Val Leu Ile Tyr Lys Thr 1425 1430 1435 1440 Tyr Gly Thr Tyr Ile Cys Glu Ser Glu Lys Asn Ala Val Thr Ile Leu 1445 1450 1455 Gly Pro Met Phe Lys Thr Val Asp Leu Val Gln Thr Glu Phe Ser Ser 1460 1465 1470 Ser Gln Thr Ser Glu Val Tyr Met Val Cys Lys Gly Leu Lys Lys Leu 1475 1480 1485 Ile Asp Glu Pro Asn Pro Asp Trp Ser Ser Ile Asn Glu Ser Trp Lys 1490 1495 1500 Asn Leu Tyr Ala Phe Gln Ser Ser Glu Gln Glu Phe Ala Arg Ala Lys 1505 1510 1515 1520 Lys Val Ser Thr Tyr Phe Thr Leu Thr Gly Ile Pro Ser Gln Phe Ile 1525 1530 1535 Pro Asp Pro Phe Val Asn Ile Glu Thr Met Leu Gln Ile Phe Gly Val 1540 1545 1550 Pro Thr Gly Val Ser His Ala Ala Ala Leu Lys Ser Ser Asp Arg Pro 1555 1560 1565 Ala Asp Leu Leu Thr Ile Ser Leu Phe Tyr Met Ala Ile Ile Ser Tyr 1570 1575 1580 Tyr Asn Ile Asn His Ile Arg Val Gly Pro Ile Pro Pro Asn Pro Pro 1585 1590 1595 1600 Ser Asp Gly Ile Ala Gln Asn Val Gly Ile Ala Ile Thr Gly Ile Ser 1605 1610 1615 Phe Trp Leu Ser Leu Met Glu Lys Asp Ile Pro Leu Tyr Gln Gln Cys 1620 1625 1630 Leu Ala Val Ile Gln Gln Ser Phe Pro Ile Arg Trp Glu Ala Val Ser 1635 1640 1645 Val Lys Gly Gly Tyr Lys Gln Lys Trp Ser Thr Arg Gly Asp Gly Leu 1650 1655 1660 Pro Lys Asp Thr Arg Ile Ser Asp Ser Leu Ala Pro Ile Gly Asn Trp 1665 1670 1675 1680 Ile Arg Ser Leu Glu Leu Val Arg Asn Gln Val Arg Leu Asn Pro Phe 1685 1690 1695 Asn Glu Ile Leu Phe Asn Gln Leu Cys Arg Thr Val Asp Asn His Leu 1700 1705 1710 Lys Trp Ser Asn Leu Arg Lys Asn Thr Gly Met Ile Glu Trp Ile Asn 1715 1720 1725 Arg Arg Ile Ser Lys Glu Asp Arg Ser Ile Leu Met Leu Lys Ser Asp 1730 1735 1740 Leu His Glu Glu Asn Ser Trp Arg Asp 1745 1750 52 2109 PRT Vesicular stomatitis virus MOD_RES (1)..(2109) “Xaa” represents any, other or unknown amino acid 52 Met Glu Val His Asp Phe Glu Thr Asp Glu Phe Asn Asp Phe Asn Glu 1 5 10 15 Asp Asp Tyr Ala Thr Arg Glu Phe Leu Asn Pro Asp Glu Arg Met Thr 20 25 30 Tyr Leu Asn His Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45 Asp Ile Asp Asn Leu Ile Arg Lys Phe Asn Ser Leu Pro Ile Pro Ser 50 55 60 Met Trp Asp Ser Lys Asn Trp Asp Gly Val Leu Glu Met Leu Thr Ser 65 70 75 80 Cys Gln Ala Asn Pro Ile Ser Thr Ser Gln Met His Lys Trp Met Gly 85 90 95 Ser Trp Leu Met Ser Asp Asn His Asp Ala Ser Gln Gly Tyr Ser Phe 100 105 110 Leu His Glu Val Asp Lys Glu Ala Glu Ile Thr Phe Asp Val Val Glu 115 120 125 Thr Phe Ile Arg Gly Trp Gly Asn Lys Pro Ile Glu Tyr Ile Lys Lys 130 135 140 Glu Arg Trp Thr Asp Ser Phe Lys Ile Leu Ala Tyr Leu Cys Gln Lys 145 150 155 160 Phe Leu Asp Leu His Lys Leu Thr Leu Ile Leu Asn Ala Val Ser Glu 165 170 175 Val Glu Leu Leu Asn Leu Ala Arg Thr Phe Lys Gly Lys Val Arg Arg 180 185 190 Ser Ser His Gly Thr Asn Ile Cys Arg Leu Arg Val Pro Ser Leu Gly 195 200 205 Pro Thr Phe Ile Ser Glu Gly Trp Ala Tyr Phe Lys Lys Leu Asp Ile 210 215 220 Leu Met Asp Arg Asn Phe Leu Leu Met Val Lys Asp Val Ile Ile Gly 225 230 235 240 Arg Met Gln Thr Val Leu Ser Met Val Cys Arg Ile Asp Asn Leu Phe 245 250 255 Ser Glu Gln Asp Ile Phe Ser Leu Leu Asn Ile Tyr Arg Ile Gly Asp 260 265 270 Lys Ile Val Glu Arg Gln Gly Asn Phe Ser Tyr Asp Leu Ile Lys Met 275 280 285 Val Glu Pro Ile Cys Asn Leu Arg Leu Met Lys Leu Ala Arg Glu Ser 290 295 300 Arg Pro Leu Val Pro Gln Phe Pro His Phe Glu Asn His Ile Lys Thr 305 310 315 320 Ser Val Asp Glu Gly Ala Lys Ile Asp Arg Gly Ile Arg Phe Leu His 325 330 335 Asp Gln Ile Met Ser Val Lys Thr Val Asp Leu Thr Leu Val Ile Tyr 340 345 350 Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asp Tyr Tyr Ala Gly 355 360 365 Leu Glu Lys Leu His Ser Gln Val Thr Met Lys Lys Asp Ile Asp Val 370 375 380 Ser Tyr Ala Lys Ala Leu Ala Ser Asp Leu Ala Arg Ile Val Leu Phe 385 390 395 400 Gln Gln Phe Asn Asp His Lys Lys Trp Phe Val Asn Gly Asp Leu Leu 405 410 415 Pro His Asp His Pro Phe Lys Ser His Val Lys Glu Asn Thr Trp Pro 420 425 430 Thr Ala Ala Gln Val Gln Asp Phe Gly Asp Lys Trp His Glu Leu Pro 435 440 445 Leu Ile Lys Cys Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile 450 455 460 Tyr Ser Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu Lys His 465 470 475 480 Val Arg Met Asn Pro Asn Thr Pro Ile Pro Ser Lys Lys Val Leu Gln 485 490 495 Thr Met Leu Asp Thr Lys Ala Thr Asn Trp Lys Glu Phe Leu Lys Glu 500 505 510 Ile Asp Glu Lys Gly Leu Asp Asp Asp Asp Leu Ile Ile Gly Leu Lys 515 520 525 Gly Lys Glu Arg Glu Leu Lys Leu Ala Gly Arg Phe Phe Ser Leu Met 530 535 540 Ser Trp Lys Leu Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys 545 550 555 560 Thr His Phe Val Pro Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu 565 570 575 Thr Ala Val Ile Lys Lys Met Leu Asp Ser Ser Ser Gly Gln Gly Leu 580 585 590 Lys Ser Tyr Glu Ala Ile Cys Ile Ala Asn His Ile Asp Tyr Glu Lys 595 600 605 Trp Asn Asn His Gln Arg Lys Leu Ser Asn Gly Pro Val Phe Arg Val 610 615 620 Met Gly Gln Phe Leu Gly Tyr Pro Ser Leu Ile Glu Arg Thr His Glu 625 630 635 640 Phe Phe Glu Lys Ser Leu Ile Tyr Tyr Asn Gly Arg Pro Asp Leu Met 645 650 655 Arg Val His Asn Asn Thr Leu Ile Asn Ser Thr Ser Gln Arg Val Cys 660 665 670 Trp Gln Gly Gln Glu Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680 685 Ser Ile Leu Asn Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn 690 695 700 Thr Ala Val Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile Cys Thr 705 710 715 720 Gln Tyr Lys Thr Lys Lys Ser Arg Asn Val Val Glu Leu Gln Gly Ala 725 730 735 Leu Asn Gln Met Val Ser Asn Asn Glu Lys Ile Met Thr Ala Ile Lys 740 745 750 Ile Gly Thr Gly Lys Leu Gly Leu Leu Ile Asn Asp Asp Glu Thr Met 755 760 765 Gln Ser Ala Asp Tyr Leu Asn Tyr Gly Lys Ile Pro Ile Phe Arg Gly 770 775 780 Val Ile Arg Gly Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val 785 790 795 800 Thr Asn Asp Gln Ile Pro Thr Cys Ala Asn Ile Met Ser Ser Val Ser 805 810 815 Thr Asn Ala Leu Thr Val Ala His Phe Ala Glu Asn Pro Ile Asn Ala 820 825 830 Met Ile Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg Leu Leu Leu Met 835 840 845 Met His Asp Pro Ala Leu Arg Gln Ser Leu Tyr Glu Val Gln Asp Lys 850 855 860 Ile Pro Gly Leu His Ser Ser Thr Phe Lys Tyr Ala Met Leu Tyr Leu 865 870 875 880 Asp Pro Ser Ile Gly Gly Val Ser Gly Met Ser Leu Ser Arg Phe Leu 885 890 895 Ile Arg Ala Phe Pro Asp Pro Val Thr Glu Ser Leu Ser Phe Trp Arg 900 905 910 Phe Ile His Val His Ala Arg Ser Glu His Leu Lys Glu Met Ser Ala 915 920 925 Val Phe Gly Asn Pro Glu Ile Ala Lys Phe Arg Ile Thr His Ile Asp 930 935 940 Lys Leu Val Glu Asp Pro Thr Ser Leu Asn Ile Ala Met Gly Met Ser 945 950 955 960 Pro Ala Asn Leu Leu Lys Thr Glu Val Lys Lys Cys Leu Ile Glu Ser 965 970 975 Arg Gln Thr Ile Arg Asn Gln Val Ile Lys Asp Ala Thr Ile Tyr Leu 980 985 990 Tyr His Glu Glu Asp Arg Leu Arg Ser Phe Leu Trp Ser Ile Asn Pro 995 1000 1005 Leu Phe Pro Arg Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Leu Gly 1010 1015 1020 Val Ala Asp Gly Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr Ile Arg 1025 1030 1035 1040 Asn Ser Phe Lys Lys Lys Tyr His Arg Glu Leu Asp Asp Leu Ile Val 1045 1050 1055 Arg Ser Glu Val Ser Ser Leu Thr His Leu Gly Lys Leu His Leu Arg 1060 1065 1070 Arg Gly Ser Cys Lys Met Trp Thr Cys Ser Ala Thr His Ala Asp Thr 1075 1080 1085 Leu Arg Tyr Lys Ser Trp Gly Arg Thr Val Ile Gly Thr Thr Val Pro 1090 1095 1100 His Pro Leu Glu Met Leu Gly Pro Gln His Arg Lys Glu Thr Pro Cys 1105 1110 1115 1120 Ala Pro Cys Asn Thr Ser Gly Phe Asn Tyr Val Ser Val His Cys Pro 1125 1130 1135 Asp Gly Ile His Asp Val Phe Ser Ser Arg Gly Pro Leu Pro Ala Tyr 1140 1145 1150 Leu Gly Ser Lys Thr Ser Glu Ser Thr Ser Ile Leu Gln Pro Trp Glu 1155 1160 1165 Arg Glu Ser Lys Val Pro Leu Ile Lys Arg Ala Thr Arg Leu Arg Asp 1170 1175 1180 Ala Ile Ser Trp Phe Val Glu Pro Asp Ser Lys Leu Ala Met Thr Ile 1185 1190 1195 1200 Leu Ser Asn Ile His Ser Leu Thr Gly Glu Glu Trp Thr Lys Arg Gln 1205 1210 1215 His Gly Phe Lys Arg Thr Gly Ser Ala Leu His Arg Phe Ser Thr Ser 1220 1225 1230 Arg Met Ser His Gly Gly Phe Ala Ser Gln Ser Thr Ala Ala Leu Thr 1235 1240 1245 Arg Leu Met Ala Thr Thr Asp Thr Met Arg Asp Leu Gly Asp Gln Asn 1250 1255 1260 Phe Asp Phe Leu Phe Xaa Ala Thr Leu Leu Tyr Ala Xaa Ile Thr Thr 1265 1270 1275 1280 Thr Val Ala Arg Asp Gly Trp Ile Thr Ser Cys Thr Asp His Tyr His 1285 1290 1295 Ile Ala Cys Lys Ser Cys Leu Arg Pro Ile Glu Glu Ile Thr Leu Asp 1300 1305 1310 Ser Ser Met Asp Tyr Thr Pro Pro Asp Val Ser His Val Leu Lys Thr 1315 1320 1325 Trp Arg Asn Gly Glu Gly Ser Trp Gly Gln Glu Ile Lys Gln Ile Tyr 1330 1335 1340 Pro Leu Glu Gly Asn Trp Lys Asn Leu Ala Pro Ala Glu Gln Ser Tyr 1345 1350 1355 1360 Gln Val Gly Arg Cys Ile Gly Phe Leu Tyr Gly Asp Leu Ala Tyr Arg 1365 1370 1375 Lys Ser Thr His Ala Glu Asp Ser Ser Leu Phe Pro Leu Ser Ile Gln 1380 1385 1390 Gly Arg Ile Arg Gly Arg Gly Phe Leu Lys Gly Leu Leu Asp Gly Leu 1395 1400 1405 Met Arg Ala Ser Cys Cys Gln Val Ile His Arg Arg Ser Leu Ala His 1410 1415 1420 Leu Lys Arg Pro Ala Asn Ala Val Tyr Gly Gly Leu Ile Tyr Leu Ile 1425 1430 1435 1440 Asp Lys Leu Ser Val Ser Pro Pro Phe Leu Ser Leu Thr Arg Ser Gly 1445 1450 1455 Pro Ile Arg Asp Glu Leu Glu Thr Ile Pro His Lys Ile Pro Thr Ser 1460 1465 1470 Tyr Pro Thr Ser Asn Arg Asp Met Gly Val Ile Val Arg Asn Tyr Phe 1475 1480 1485 Lys Tyr Gln Cys Arg Leu Ile Glu Lys Gly Lys Tyr Arg Ser His Tyr 1490 1495 1500 Ser Gln Leu Trp Leu Phe Ser Asp Val Leu Ser Ile Asp Phe Ile Gly 1505 1510 1515 1520 Pro Phe Ser Ile Ser Thr Thr Leu Leu Gln Ile Leu Tyr Lys Pro Phe 1525 1530 1535 Leu Ser Gly Lys Asp Lys Asn Glu Leu Arg Glu Leu Ala Asn Leu Ser 1540 1545 1550 Ser Leu Leu Arg Ser Gly Glu Gly Trp Glu Asp Ile His Val Lys Phe 1555 1560 1565 Phe Thr Lys Asp Ile Leu Leu Cys Pro Glu Glu Ile Arg His Ala Cys 1570 1575 1580 Lys Phe Gly Ile Ala Lys Asp Asn Asn Lys Asp Met Ser Tyr Pro Pro 1585 1590 1595 1600 Trp Gly Arg Glu Ser Arg Gly Thr Ile Thr Thr Ile Pro Val Tyr Tyr 1605 1610 1615 Thr Thr Thr Pro Tyr Pro Lys Met Leu Glu Met Pro Pro Arg Ile Gln 1620 1625 1630 Asn Pro Leu Leu Ser Gly Ile Arg Leu Gly Gln Leu Pro Thr Gly Ala 1635 1640 1645 His Tyr Lys Ile Arg Ser Ile Leu His Gly Met Gly Ile His Tyr Arg 1650 1655 1660 Asp Phe Leu Ser Cys Gly Asp Gly Ser Gly Gly Met Thr Ala Ala Leu 1665 1670 1675 1680 Leu Arg Glu Asn Val His Ser Arg Gly Ile Phe Asn Ser Leu Leu Glu 1685 1690 1695 Leu Ser Gly Ser Val Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala 1700 1705 1710 Leu Glu Thr Leu Gly Gly Asp Lys Ser Arg Cys Val Asn Gly Glu Thr 1715 1720 1725 Cys Trp Glu Tyr Pro Ser Asp Leu Cys Asp Pro Arg Thr Trp Asp Tyr 1730 1735 1740 Phe Leu Arg Leu Lys Ala Gly Leu Gly Leu Gln Ile Asp Leu Ile Val 1745 1750 1755 1760 Met Asp Met Glu Val Arg Asp Ser Ser Thr Ser Leu Lys Ile Glu Thr 1765 1770 1775 Asn Val Arg Asn Tyr Val His Arg Ile Leu Asp Glu Gln Gly Val Leu 1780 1785 1790 Ile Tyr Lys Thr Tyr Gly Thr Tyr Ile Cys Glu Ser Glu Lys Asn Ala 1795 1800 1805 Val Thr Ile Leu Gly Pro Met Phe Lys Thr Val Asp Leu Val Gln Thr 1810 1815 1820 Glu Phe Ser Ser Ser Gln Thr Ser Glu Val Tyr Met Val Cys Lys Gly 1825 1830 1835 1840 Leu Lys Lys Leu Ile Asp Glu Pro Asn Pro Asp Trp Ser Ser Ile Asn 1845 1850 1855 Glu Ser Trp Lys Asn Leu Tyr Ala Phe Gln Ser Ser Glu Gln Glu Phe 1860 1865 1870 Ala Arg Ala Lys Lys Val Ser Thr Tyr Phe Thr Leu Thr Gly Ile Pro 1875 1880 1885 Ser Gln Phe Ile Pro Asp Pro Phe Val Asn Ile Glu Thr Met Leu Gln 1890 1895 1900 Ile Phe Gly Val Pro Thr Gly Val Ser His Ala Ala Ala Leu Lys Ser 1905 1910 1915 1920 Ser Asp Arg Pro Ala Asp Leu Leu Thr Ile Ser Leu Phe Tyr Met Ala 1925 1930 1935 Ile Ile Ser Tyr Tyr Asn Ile Asn His Ile Arg Val Gly Pro Ile Pro 1940 1945 1950 Pro Asn Pro Pro Ser Asp Gly Ile Ala Gln Asn Val Gly Ile Ala Ile 1955 1960 1965 Thr Gly Ile Ser Phe Trp Leu Ser Leu Met Glu Lys Asp Ile Pro Leu 1970 1975 1980 Tyr Gln Gln Cys Leu Ala Val Ile Gln Gln Ser Phe Pro Ile Arg Trp 1985 1990 1995 2000 Glu Ala Val Ser Val Lys Gly Gly Tyr Lys Gln Lys Trp Ser Thr Arg 2005 2010 2015 Gly Asp Gly Leu Pro Lys Asp Thr Arg Ile Ser Asp Ser Leu Ala Pro 2020 2025 2030 Ile Gly Asn Trp Ile Arg Ser Leu Glu Leu Val Arg Asn Gln Val Arg 2035 2040 2045 Leu Asn Pro Phe Asn Glu Ile Leu Phe Asn Gln Leu Cys Arg Thr Val 2050 2055 2060 Asp Asn His Leu Lys Trp Ser Asn Leu Arg Lys Asn Thr Gly Met Ile 2065 2070 2075 2080 Glu Trp Ile Asn Arg Arg Ile Ser Lys Glu Asp Arg Ser Ile Leu Met 2085 2090 2095 Leu Lys Ser Asp Leu His Glu Glu Asn Ser Trp Arg Asp 2100 2105

Claims

What is claimed is:

1. A method of reducing the viability of a tumor cell, comprising administering to the tumor cell a virus, wherein said virus is not a common human pathogen and said tumor cell is a melanoma.

2. The method of claim 1, wherein the tumor cell has substantially no PKR activity.

3. The method of claim 1, wherein the tumor cell is PKR−/−; STAT1−/−; or both PKR−/− and STAT1−/−.

4. The method of claim 1, wherein the virus is a Rhabdovirus or a picornavirus.

5. The method of claim 4, wherein the virus is a Rhabdovirus.

6. The method of claim 5, wherein the Rhabdovirus is a vesicular stomatitis virus.

7. The method of claim 6, wherein the virus is unable to inactivate PKR activity within the tumor cell.

8. The method of claim 6, wherein the virus is an attenuated strain of vesicular stomatitis virus.

9. The method of claim 8, wherein the virus is vesicular stomatitis virus strain M1.

10. The method of claim 8, wherein the virus is vesicular stomatitis virus strain M2.

11. The method of claim 8, wherein the virus is vesicular stomatitis virus strain M3.

12. The method of claim 8, wherein the virus is vesicular stomatitis virus strain M4.

13. The method of claim 8, wherein the virus is vesicular stomatitis virus strain M5.

14. The method of claim 1, wherein the tumor cell is in a mammalian subject and the virus is administered to the tumor cell by intravenous, intranasal, intraperitoneal or intratumoral administration to the subject.

15. The method of claim 14, wherein the mammalian subject is a human or a non-human mammal.

16. The method of claim 14, wherein the virus is contained in cell line infected with the virus and the administration comprises administering the virus-infected cell line to the subject by a route selected from intratumorally, intravenously or intraperitoneally.

17. A method of reducing the viability of a tumor cell within a population of tumor cells and non-tumor cells comprising administering a vesicular stomatitis virus to the population of cells, wherein the tumor cells are melanoma cells and the virus is able to selectively infect and kill the tumor cell.

18. The method of claim 17, wherein the virus is unable to inactivate PKR activity in the tumor cell.

19. The method of claim 18, further comprising treating the population of cells with interferon prior to administering the virus.