CA2253595A1 - Generation of viral transfectants using recombinant dna-derived nucleocapsid proteins - Google Patents

Abstract

This invention provides a method of producing viral transfectants (viruses containing a heterologous nucleic acid) that eliminates the need for purified RNP complexes or for purified viral RNA polymerases. The method generally involves: (i) combining a ribonucleic acid (nucleic acid transcript) with an isolated nucleoprotein (NP) of the virus to form a synthetic ribonucleoprotein complex (RNP); (ii) transfecting a host cell with the synthetic ribonucleoprotein complex; and (iii) holding (culturing) the host cell under conditions permitting replication of the virus.

Description

CA 02253595 l998-l0-30 WO 97/41245 rCT/US97/07277 GENERATION OF VIRAL TRANSFECTANTS USING
s RECOMBINANT DNA-DERIVED NUCLEOCAPSID PROTEINS
BACKGROUN~D OF TEIE INVENTION
The development of reverse genetics for influenza viruses has allowed the direct manipulation of virion gene products ~nd the creation of entirely new recombinant viruses not seen in nature (see, e.g., Enami el al. (1990) Proc. Natl. Acad. Sci. USA, 87:
3802-3805; Castrucci et al. (1992) J. Virol., 66: 4647-4653; Li et al. (1992) J. Virol., 66: 399-404; Liu et al. (1993) Virol., 194: 403-407; Subbarao e~ al. (1993) J. Virol., 67:
7223-7228; and Zurcher et al. (lg94) J. Virol., 68: 75748-5754). Generally, the production of recombinant influenza viruses has required the combination of purified proteins from virion ribonucleoprotein (RNP) cores with a synthetic RNA transcript followed by transfection into cells previously infected with a helper virus (Enami et al.
(1991) J. Virol. 65: 2711-2713).
The purified RNP cores t~pically include the RNA polymerase proteins (e.g., PB1, PB2 and PA) in addition to a viral nucleoprotein (NP) (see, e.g., Ishihama et al. (1988) CRC Crir. Re-. Biochem, 23: 27-76). In part, because of their complexstructure, the purification of the RNP complex proteins from virions represents one of the most labor-intensive aspects of the procedure requiring multiple gradient fractionations (Enami et al., supra.). Previous viral transfe~tion methods were also complex because it was believed that viral RNA polymerase was required in addition to the viral nucleoprotein to effect viral transf~tion (see, e.g. U.S. Patent 5,166,057). Provision of viral RNA
polymerase in addition to nucleoprotein (NP~ required an elaborate cloning system or time consuming and laborious isolation and purification procedures (see, e.g., Kimura et al.
(1992) J. Vi~ol., 13~ 1-1-.~8).

S~M~Y OF THE INVENT~ON
This invention provides a method of producing viral transfectants that tolimin~tçs the need for purified RNP complexes or for purified viral RNA polymerases.

CA 022~3~9~ l998-l0-30 WO 97/41245 PCTtUS97/07277 The methods of this invention thus dramatically simplify the preparation of viral transfectants.
It was a surprising discovery of this invention that a viral RNP complex comprising only a viral nucleoprotein (NP) and a nucleic acid transcript ~e.g~, an RNA) is 5 sufficient to mediate the replication of a virus (e.g., influenza) when cultured with a simple helper virus.
In one embodiment, this invention tl,~.crorc provides a method of pr~a,iilg a viral transfectant ( virus bearing a preselected ribonucleic acid). The method in~ludes the steps of: i) co-"binil-g the ribonucleic acid (nucleic acid ll~ls~;~ipt) with an isolated 10 nucleopl()tein (NP) of the virus to form a synthetic ribonucleopluteill complex (RNP); ii) ~ansfecting a host cell with the synthetic ribonucleoplulein complex; and iii) holding (culturing) the host cell under conditions pclll~i~Ling replication of the virus. ~2~plir~tion of the virus in the host cell from the RNP complex is accornrlich~d by providing a host c~ll capable of complementing the subject virus (e.g., a host cell recombinantly .o.ngin~red 15 to express viral RNA polymerase) or more preferably by providing a host cell infected uith a helper virus. The host cell can be infected with the helper virus before, during, or after the transfection with the synthetic ribonucleopl~,tcin complex (RMP). Plercllcd helper viruses include attenuated live vaccine parent strains (e.g., A/Leningrad/57 or A/Ann Arbor/6/60) or laboratory adapted wild-type viruses (e.g., AIPR18/34 or 20 AIWSN/33).
The isolated viral nucleoprotein (NP) is preferably isolated away from other viral proteins, in particular it is essentially free of viral RNA polymerase proteins.
Preferred isolated viral nucleoproteins are recombinantly e~lcssed, more preferably recombinantly cAp-essed in a eukaryotic expression system (e.g, in an insect system such 25 as SP19 cells in a baculovirus vector). Preferred viral nucleoproteins include MPs from influenza, parainfluenza, or measles virus, with influenza (e.g., type A, B, or C) NPs being more preferred, and influenza type A Nps being most ~,efellcd.
The host cell can be any cell in which it is possible to culture the viral transfectant. Particularly preferred cells are eukaryotic cells with cells from a hens egg or 30 kidney cells (e.g., MDCK cells) being most preferred. Subject viruses to be transfected include RNA viruses that typically contain a nucleoprotein complex (RNP) or e~uivalent structure. Particularly preferred viruses include the orthomyxovindae more preferably CA 022~3~9~ 1998-10-30 WO 97/41245 ~CTIUS97/07277 inflllçn7~ and paramyxoviruses such as par~influen7~ and measles, with infll-~n7~ being most preferred.
In a particularly plcrtll~d embodiment, the subject virus is an influlo.n7~
virus, the nucleoprotein is a recombinantly e~l)lcsscd influenza nucleoproteill substantially S free of a viral RNA polymerase and the host cell is an egg or a kidney cell infected with an influenza helper virus.
The isolated NP protein when combined with an RNA substantially protects the RNA from ribonucleases and guides the RNA to the nucleus of the host cell. Thus, in another embodiment, this invention provides a method of introducing a ribonucleic acid 10 (e.g., a RNA t~ s~lipl) into the nucleus of a host cell. The method involves the steps of: i) COIllbinillg the ribonucleic acid with an isolated nucleoprotein (NP) of a virus to forrn a synthetic ribonucleoprotein complex (RNP); and ii) transfecting the host cell with the synthetic ribonucleoprotein oo---p!~ .. The method can further include infecting the host cell with a helper virus. Any of the viral nucleoproteins (NPs) dicc lssed above, and any of the 15 RNT'. transcripts, host cells and transfection methods diccusced herein are suitable.
In still another embodiment, this invention provides a transfection composition comprising an isolated viral nucleoprotein combined with a ribonucleic acid (e.g., R~'A transcript) thereby forming a synthetic ribonucleopro~ei,l complex that is capable of mediating viral replication in a host cell. Any of the NP and RNA transcripts 20 describec above and herein are suitable.
The methods of this invention can be used to produce transfectant host cells (i.e., expressing a heterologous nucleic acid) or transgenic viruses (viral transfectants).
Thus in another embodiment this invention provides for host cells transfected with, or viruses produced (derived) from, a synthetic ribonucleoprotein complex (RNP) where the 25 RNP is an isolated viral nucleoprotein (NP) combined with a ribonucleic acid (e.g., an RNA transcript). Any of the viruses, host cells, NPs and RNPs described above and herein are suitable. In one embodiment, the RNA transcript explcsses an antigenic deterrninant of a human pathogen (e.g., gpl20 or a subsequence thereofl which isdisplayed by the virus. The virus can be dead (unable to infect or replicate) or a live 30 attenuated virus.
Finally, this invention also provides for kits for the practice of the methods described above. In particular, the kits are suitable for the preparation of host cells CA 022~3~9~ 1998-10-30 conta~ning preselected RNA transcripts or for the preparation of viral transcripts. This kits include a container containing a synthetic ribonucleoprotein complex (RNP) where the RNP is an isolaled viral nucleoprotein (NP) combined with a ribonucleic acid (e.g., an RNA transcript). The kit can additionally include one or more of the following:
5 instructions t~rhing the methods described above and below herein, buffers, host cells, culture media, helper viruses, and the like.

l:~efinitio~
The term "subject virusr, as used herein, is intencle~ to refer to the virus 10 into which is inserted a nucleic acid transcript. The subject virus is thus distinguished from the helper virus which serves to f~r~ t~te the replication of the subject virus.
A Uhelper virus" refers to a virus that provides the functions necessary for the replication of a defective virus. In the present invention, helper viruses provide functions necessary for replication of the subject virus from a synthetic ribonucleoplo~
15 complex (e.g., NP combined with a nucleic acid transcript). The term helper virus can also just refer to just the combination of components of the helper virus that are necessary to replication of the subject virus.
The term "synthetic ribonucleoprotein complex" or "synthetic RNP" as used herein referes to a ribonucleop,u~eh~ complex containing a nucleoprotein (NP) that has 20 been isolated away from its related nucleic acid polymerase proteins prior to incol~ul~ion into the complex. It will be recognized that the RNP complex is generally used tû refer to the association of an RNA, a nucleoprotein (NP), and viral RNA polymerase proteins.
~owever, as used herein the RNP complex can refer simply to the asociation of a nucleic acid transcript and a nucleoploLein (NP) recognizing that the combination of the NP/RNA
25 RNP ~,ith viral RNA polymerase proteins (e.g., supplied by a helper virus) either before, during. or after transfection of a host cell with the RNP, will provide an RNP capable of mediatinv, replication of the subject virus.
The term "complex" when used in the context of RNP complex is not intended to imply a particular structural relationship, but simply indicates the presence of 30 the RNP components, in particular an NP and an RNA, in an ~csoci~tion that permits replication of the subject virus.

CA 022~3~9~ 1998-10-30 WO 97141245 ~CT/US97/07277 The term "recombinant" when used with reference to a cell inr~ir~t~s that the cell replicates or expresses a nucleic acid, or eA~ sscs a peptide or protein encoded by a nucleic acid whose origin is exogenous to the cell. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells 5 can also express genes found in the native form of the cell wherein the genes are re-introduced into the cell by artificial means, for example under the control of aheterologous plu~ kl.
The terrn "heterologous" when used with reference to a nucleic acid transcript (i.e., a cRNA) indicates that the nucleic acid is in a non-native state. The 10 heterologous nucleic acid can be a modification (e.g., contain a deletion, mutation, insertion) of the nati~ e nucleic acid or can include or be replaced by a nucleic acid sequence not found in the resulting state in nature. Thus a virus cont~ining a heterologous nucleic acid contains a nucleic acid from a source other than that virus, or a nucleic acid from the same virus ~here the nucleic acid is reintroduced into the virus (e.g., after being 15 deliberately modified).
The terms "viral transfectant" or "transgenic virus" refer to a virus cont~ining a heterologous nucleic acid.
The term " subsequence" in the context of a particular nucleic acid or polypeptide sequ~nce refers to a region of the nucleic acid or polypeptide equal to or 20 smaller than the particular nucleic acid or polypeptide.
The term "nucleic acid" or "nucleic acid transcript" refer to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otheru ise limited, would encompass known analogs of natural nucleotides that can function in a sirnilar manner as naturally occurring nucleotides. The bases in the 25 nucleic acid may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere the function of the nucleic acid. Thus, for exarnple, the nucleic acid may be a peptide nucleic acid in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. In paticular, the terms "nucleic acid transcript" or "RNA transcript" are used herein to refer to the heterologous RNA to be introduced into a 30 virus.
The phrase "substantially purified" or "isolated" when referring to a protein (e.g., a viral nucleoprotein (NP)) means a chemical composition which is essentially free CA 022~3~9~ 1998-10-30 WO 97/41245 ~CT/US97/07277 of other cellular or viral components with which the protein normally occurs. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution.
Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high pG.rol---ance liquid chromatography. A
S protein which is the predominant species present in a p.e~ tion is isolated orsubstantially purified. Generally, a ~lb~ lially purified or isolated protein will Colll~)liS~
more than 80% of all macromolecular species present in the ~.cp~lion. Preferably, the protein is purified to IG~l~,sent greater than 90% of all macromolecular species present.
More preferably the protein is purified to greater than 95 %, and most preferably the 10 protein is purified to es.s~-nti~l homogeneity, wherein other macromolecular species are e.s~.onti~lly not ~et~t~d by conventional techniques.
The phrase "recombinant protein" or "lGco---bi~ tly produced protein~
refers to a peptide, polypeptide, or protein produced using cells that do not have an endogenous copy of DNA able to express the protein. The cells produce the protein 15 because they have been venetically altered by the introduction of the a~ o~liate nucleic acid se~uence.
The term '4influenza v;irus", as used herein, refers to members of the family Orthomyxoviridae and includes~ but is not limited to the influçn7~ type A, B, and C viruses, and the tick-borne Orthomyxoviruses. Par~inflllçn,~ viruses and measles are members ofthe 20 Paramyxoviridae although measles is sometimes referred to as a morbillivirus.A nucleoprotein (NP) is a protein naturally found in close association with the nucleic acid (e.~., R~A) found in a virus. For example in influenza, the nucleoprotein associates with RNA to form a helical structure, the nucleocapsid The nucleoproteins are thus analogous to the eukaryotic histone proteins Influenza nucleoproteins are type specific 25 antigens and occur in one of three antigenic forrns; these di~ltnt forms provide the basis for the classification of human influenza viruses into types A, B, and C
A virus "derived from" a synthetic ribonucleoprotein complex of this invention refers to a virus that was replicated from the synthetic ribonucleoprotein complex of this invention, or to a virus thaI is the progeny of a virus that was that was replicated from the 30 synthetic ribonucleoprotein complex of this invention. Similarly, a host cell transfected with a viral transfectant vector comprising a synthetic ribonucleoprotein complex refers to the host CA 022~3~9~ 1998-10-30 WO 97/41245 ~CT/US97107277 cell so transfected or to the progeny of such a host cell that still contain either a viral RNP of a virus derived from the synthe~ic RNP of this invention or the derived virus itself.
A "conservative substitution", when describing a protein refers to a change in the amino acid composition of the protein that does not substantially alter the protein's - 5 activity (e.g, nucleoprotein activity). Thus, "conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or subst~ tion of arnino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity.
Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative sukstitutions for one another:
I) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (~'), Glutamine (Q);

4) Arginine (R), Lysine (K);
S) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) Proteins W.H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations" .

DETAILED DESCRIPTION
This invention provides a new method of producing virus co-,laining a heterologous nucleic acid sequence or subsequence; a viral transfectant. Typically production of viral transfectants, particularl~ influeriza viral transfectants involves the combination of purified proteins from virion ribonucleoprotein (RNP) cores with a synthetic RNA llailsclipt followed by transfection into cell~ previously infected with a helper virus (Enami et aL (1991) J. Virol. 65: 2711-2713). These purified proteins typically include the constituents ofthe ribonucleoprotein complex (RNP).

5 ~CT/US97107277 The ribonucleoprotein complex is a heterogenous association of molecules including an RNA, a nucleoprotein (NP) and viral RNA polymerase proteins (e.g., PA, PBI, - PB2). In part, because of the complex s~ olule of the RNP and the intimate ~ccoci~til~n between the protein and RNA components, the purification of the RNP complex proteins 5 from virions ~esent~ one of the most labor-intensive aspects of the viral transfection procedure requiring multiple gradient fractionations (Enami et al., supra. ). ln addition to requiring considerable labor, previous viral l~dn~recLion methods were also co~
because it was believed that viral RNA polymerase was required in ~ tion to the viral nucleoplotein to effect viral transfection. Provision of viral RNA polymerase in addition 10 to nucleoprotein (NP) required an elaborate cloning system or time con~llming and laborious isolation and purification procedures (see, e.g., Kimura et al. (1992) J. Virol., 1321-1328).
It was thus a surprising discovery of this invention that the viral nucleoprotein (NP) alone is sufficient to form a nucleoplot~ complex with a nucleic acid 15 (e.g., RNA) and that this nucleoplot~ complex, together with a helper virus, or equivalent replication m~hinPry, is sufficient to achieve viral replicaiton in a host cell.
This invention thus çlimin~ttos the labor intensive steps required for the purification of the ribonucleoproteh~ complex (RNP). Instead, a nucleic acid transcript can simply be combined with a pre-purified (e.g., recombinantly expressed) nucleoplotein and then 20 transfected into a host cell to achieve where the subject virus replicates incol~oldLillg the nucleic acid transcript. The purified nucleoprotein or the nucleoproteinlRNA complex can be provided as stock reagents thereby allowing the routine and rapid creation of viral transfectants.
In addition to a dramatic reduction in the labor required to prepare viral 25 transfectants, çlimin~tion of the requirement for purified RNP complex proteins e.limin~t~c the possibility of introducing virus genes which might be present in the RNP proteins purified from virions. This makes it easier tO maintdin the transfectadnt phenotype (e.g. ~ dn attenuated influenza) since no wild-type genes are present except for those deliberately introduced.

WO 97/41245 ~PCT/US97/07277 I. Virl-c tr~ncfection As inAi~tPd above, this invention provides a method of pr~aling a viral transfectant (a virus cont~ining a heterologous nucleic acid). In general, the method of this invention involves combining a nucleic acid transcript with an isolated viral S nucleol". lein (NP) to form a synthetic nucleoprotein complex (RNP). The complex is then transfected into a host cell where, in colllbination with a helper virus, or equivalent replication machinery, it meAi~tPs viral replication thereby generating a multiplicity of virions each containing a copy of the nucleic acid transcript.
It was a surprising discovery of this invention that a helper virus, or equivalent replication m~chinPry, is capable of supplying all of the protein co.llponents required for viral replication and that the only necessary protein cGIllponent of the subject virus is the viral nucleoprotein (NP). It was previouslv believed that other virion proteins, in particular those that form the RNA-directed RNA polymerase (e.g., PA, PB1, PB2) were required for successful viral repli~tion (see, e.g., U.S. Patent 5,166,057).
F.limin~tion of the requhc-"ent for RNP polymerase proteins from the transfected virus provides for a greatly simplified means of producing transfected viruses.
In general, the method includes the following steps:
i) combining a nucleic acid transcripl (e.~., RNA) with an isolated nucleoprotein (NP) of the virus to form a synthetic ribonucleoprotein complex (R~P);
ii) transfecting a host cell with the s~thetic ribonucleoprotein complex;
and iii) holding said host cell under conditions permitting the replication of the virus Preferred subject (transfectant) viruses are, of course, viruses in which replication is me~ tPA, at least in part, by a nucleoprotein (NP). Such viruses are well known to those of skill in the art and include, for example members of the Myxoviridae including Orthomyxoviridae such as influenza viruses, and Paramyxoviridae such as parainfluenza viruses, measles viruses, and the like. Other suitable viruses include the Rhabdoviruses (e.g., rabies). In a particularly preferred embodiment, the subject virus is an influenza virus (e.g., an influenza type A, B, or C).

CA 022~3~9~ 1998-10-30 WO 97t41245 PCT/US97/07277 As indicated above, the synthetic nucleoprotein complex is used to transfect a host cell where, in conjunction with a helper virus, it mediates viral replication. The - viral cultures are maintained under standard conditions and those host cells containing viral transfectants are selected and isolated according to standard me~hods in the art.
S The valious steps of the method are described in detail below.

. PrPp~ration of Syntlletic Nucl~ovr~ C~n~plPY.
As in~i~at~d above the methods of this invention involve l,l~d.ation of a synthetic nucleoprotein complex. As used herein, a synthetic nucleoprotein complex refers to the combination of, and association between a nucleic acid and a ~iral NP protein. In a preferred embodiment, the synthetic nucleoprotein complex involves the ~cso~i~tion of a ribonucleic acid (e.g., an RNA transcript) with an NP protein selected from influenza, parainfluenza, me~cles, or rabies, with influenza NP being most preferred. Particularly plGfellGd synthetic ribonucleoprotein complexes are not ~ccoci~t~d ~ith and do not include nucleic acid polymerases of the subject virus. Preparation of the nucleoprotein, the nucleic acid transcript and the synthetic nucleo,~"oteill complex is described below.

A) Prep~ration of viral ~u~.leoprotf~in.
Viral nuc}eoproteins (NPs) are well known to thos;e of skill in the art. For example, in influenza viruses, the nucleoprotein is a protein clos~ely associated with the viral RNA. The RNA and NP ~Ccoci~te together to form a helical sL~ucture, the nucleocapsid. The nucleoprotein (NP) has a molecular weight of approximately 60 kDa and there are approximately 1000 molecules in each virus particle. The influenza NP is a type-specific antigen and occurs in one of three antigenic forms. These different forms provide the basis for the classification of human influenza ~ iruses inlo types A, B, and C.
Proteins analogous to the influenza NP are known in other viruses. Thus, for example, parainfluenza, rhabdoviruses, morbilliviruses, and the like contain proteins analogous to the influenza NP (see, e.g., Fields Virology, 2nd ed. (1990) Raven Press, N.Y.).
The viral nucleoproteins of this invention include nucl~oproteins in their native conformation and sequence. However, the nucleoproteins of tnis invention also include NPs modified in a variety of ways that do not adversely effec: their activity and in CA 022~3~9~ 1998-10-30 fact, may improve various properties including, but not limited to transfection stability or efficiçncy, viral replication rate, infectivity, and the like. Preferred modifications will include conservative sllbstitlltions as defined above. Some modifications may be made to f~.ilit~tto the cloning, expression, or purification of the nucleoprotein. Such mo lifiç~tiQns 5 are well known to those of skill in the art and include, for example, a methionine added at the amino terrninus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons. One "eft"~d mo~ifi~tion is the ~ ition of a carboxyl terminal polyhicti~line ~e.g., His6) to f?~rilit~t~. protein purification (e.g., using an Ni-NTA column).
Other modifications can also be made. Thus, for example, amino acid substitutions may be made to improve ~Ccoci~tion with the nucleic acid transcript, to improve viral p~rl~in~, or to increase viral replication rate or viability, etc.Alternatively, non-escenti~l regions of the NP molecule may be shortened or çlimin~tçd entirely. Thus, where there are regions of the molecule that are not themselves involved in the activity of the molecule, they may be elimin~t~d or replaced with shorter s~.l~ents that merely serve to ~n~int~in the correct spatial relationships between the active components of the molecule.
Viral nucleoproteins are well characterized and the full amino acid and co", sponding nucleic acid sequences are known (see, e.g., Cox et al. (1983) Bull. World.
Health. Org., 61: 143-152 and Rota (1989) Nucl. Acids Res., 17: 3595). Methods of preparing isolated nucleoprotein (NP) are also well known to those of skill in the art and include isolation from a viral culture, de novo chemical synthesis, and recombinant expression.
Means of purifying viral nucleoprotein are known to those of s~ll in the art (see, e.g., U.S. Patent 5,316,910 and WO 92/16619)). In a preferred embodiment, the nucleoprotein is purified away from other viral proteins, espeçi~lly other viral proteins involved with or comprising the viral RNA-directed RNA polymerase (e.g., PB2, PBl, PA, etc.).
Alternatively, using the amino acid sequence information known to those of skill in the art, the viral nucleoprotein (NP) can be che~nic~lly synthesized de novo in a wide variety of well-known ways. Polypeptides of relatively short size are typically syn~hçci7çd in solution or on a solid support in accordance with conventional techniques CA 022=.3=.9=. 1998-10-30 (see, e.g., Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154; Barany and Merrifield, Solid-Phase Pep~ide Syn~hesis; pp. 3-284 in The Peptides: Ar,a~ysis, Synrhesis, Biology.
Vol. 2: Special Methods in Peptide Synthesis, Part A; and Stewart et al., (1984) Solid Phase Peptide Synthesis, 2na' ed., Pierce Chem. Co., Rockford, Ill. Various automatic synthesizers and sequencers are commercially available and can be used in accor~au~ce with known protocols. See, e.g., Stewart and Young (1984) Solid Phase Pepnde Synthesis, 2d.
ed., Pierce ( ~hemir~ Co. Peptides of longer size can be prepared by the chemical synthesis of shorter segment~ which are then coupled together in a condensation reaction between the carboxyl and amino termini of sequential segments thereby resulting in a single continuous polypeptide.
In a preferred emborlimPn~, the viral nucleoproteins are produced by recombinant expression of a nucleic acid encoding the polypeptide followed by purification using standard techniques. Such recombinant methods typically involve providing a nucleic acid sequence encoding the viral NP, inserting the sequence into a vector, transfecting a host cell with the vector, culturing the host cell under conditions where the nucleoprotien is e~ ss~ and isolating and purifying the expressed nucleop,otein.As indicated above, amino acid and hence nucleic acid sequences of viral nucleoproteins, especially influenza, parainfluenza, and measles nucleoproteins, are known. Nucleic acid sequences encoding these peptides can be made using standardrecombinant or synthetic techniques. Using chemical techniques, DNA encoding the viral nucleoproteins of this invention may be prepared by any suitable method, including, for example, methods such as the phosphotriester method of Narang et al. (1979) Meth.
Enzymol. 68: 90-99; the phosphodiester method of Brown et al., (1979) Me~h. Enymol.
68: 109-151; the diethylphosphoramidite method of Beaucage e~ al. (1981) Tetra. Let~., 22: 1859-1862; and the solid support method of U.S. Patent No. 4,458,066.
Given the nucleic acids encoding the viral nucleoprotein (NP), one of skill can construct a variety of clones containing the same, or functionally equivalent nucleic acids, such as nucleic acids which encode the same, or functionally equivalent nucleoproteins. Cloning methodologies to accomplish these ends, and sequencing methods 30 to verify the sequence of nucleic acids are well known in the art. Examples of apl)~opliate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide tO Moleclllar CA 022~3~9~ 1998-10-30 Cloning Techniques, Methods in Enymology volume 152 Ac~Aemic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3; and C~rrent Protocols in MolecularBiolog~, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Ac~oci~tes, Inc. and 5 John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Product information frommanufacturers of biological reagents and experimental equipment also provide inforrnation useful in known biological methods. Such manufacturers include the Sigma Chemical Company (Saint Louis, Missouri, USA), R&D systems (Minneapolis, Minnt~ot~, USA),Pharmacia LKB Biotechnology (Piscataway, New Jersey, USA), Clontech Labu,al(,lies, 10 Inc. (Palo Alto, California, USA), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wisconsin, USA), Glen Research, Inc., Gibco BRL Life Technologies, Inc.
(Gaithersberg, Maryland, USA), Fluka Chemica-Biochemik~ Analytika (Fluka ChernieAG, Buchs, Switzerland), Invitrogen (San Diego, California USA), and Applied Biosystems (Foster City, California, USA), as well as many other commercial sources 15 known to one of skill.
The nucleic acid sequences enco~ing the nucleop,ut~ins may be ~Ay,c~ssed in a variety of host cells, including E. coli and other bacterial hosts. However, in a y~fell~d embodiment, the nucleoproteins used in this invention are ~y~essed in various eukaryotic cells such as yeast, the COS, CHO and HeLa cells lines and myeloma cell lines, and insect 20 cell lines.
The recombinant NP gene will be operably linked to ayplolJliate expression control sequences for each host. For E. coli this includes a promoter such as the 17, trp, or lambda promoters, a ribosome binding site and yle~ldbly a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably 25 an enh~ncçr derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
Plasmids encoding the viral nucleoproteins of this invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for 30 eukaryotic cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.

CA 022~3~9~ 1998-10-30 Once eXIJlCSS~d, the recombinant nucleoproteins can be purified accoldillg to standard procedures of the art, including ammonium sulfate ~ cipilation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R.
Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methodsin S En~mology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.
(19~0)). Substantially pure co-"po~iLions of at least about 90 to 95% homogeneity are ~lef~led, and 98 to 99% or more homogeneity are most p.e~ll~d. Once purified, partially or to homogeneity as desired, the nucleoproteins can then be used to form synthetic nucleoprotein comrl~oxes according to ehe methods of this invention.
One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the nucleoproteins may possess a confol."ation substantially different than their native conformation. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred Gonformation. Methods of reducing and denaturing proteins and indu--ing re-folding are well known to those of skill in the art. (See, Debinski el al. J. Biol. Chem., 268: 14065-14070 (1993); Kreitman and Pastan, Bioconjug. Chem., 4: 581-585 (1993);
and Buchner, et al., Anal. Biochem., 205: 263-270 (1992)). Debinski et al., for example, describe the denaturation and reduction of inclusion body proteins in gu~nitline-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
One of skill would recognize that modifications can be made to the recombinant nucleoproteins without dimini~hing their biological activity. Some modifications may be made to f~ilit~e the cloning, expression, or purification of the nuclevprotein. Such modifications are well known to those of skill in the art and include, for exarnple, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons.
In a particularly preferred embodiment, the NPs used in this invention are expressed in Spodoptera frugiperda ~S19) insect cells using a baculovirus vector as described in U.S. Patent 5,316,910 and WO 92/16619.

CA 022~3~9~ 1998-10-30 B) Preparation of nucleic acid transcript.
Virtually any nucleic acid can serve as a nucleic acid ~l ~nscl ipt for use in the present invention. Typically, nucleic acid transcripts will be chosen that express a non-naturally occurring (e.g., heterologous) gene product, that express an endogenous gene ~ S product at elevated levels, that alter the infectivity or pathogenicity of the virus, or that act as or encode detectable markers. The nucleic acid transcript can be expressed in app, O~l iate host cell systems, or can provide reco.l,~;na..L viruses that express, package, and/or presen the heterologous gene product. The gene products can be advantageously used in a variety of conteYtc. for e,.~.",~le, in vaccine fonm-l~tionc Where heterologous genes are to be encoded 10 by the transcript, the gene may be selected for its effect on the virus, for its effect on the host cell, or sirnply for the fact that the virusAlost cell provide an opportune, convenient, or particularly optimal system for expression of the gene product.
Genomic RNA of the Myxoviridae is of negative sense (complementary to that of mRNA) and thus, if the nucleic acid transcript is to express a gene product, it too should 15 be of ne~ative sense. The ~NA ~- dnsc,ilut can be prel)~, ed by any of a number of means well kno~vn to those of skill in the art. In a preferred embodiment, the RNA templates are prepared by Ll ~"s~ ,Lion of app~Opliale DNA sequences using a DNA-directed RNA
pol!merase such as bacteriophage T7, T3 or the Sp6 polymerase. Using influenza, for example, the DNA is constructed to encode the message-sense of the heterologous gene 20 sequence flanked upstream of the ATG by the complement of the viral pol~merase binding site!promoter of influenza, i.e., the complement of the 3'-terminus of a genome segment of influenza For rescue in virus particles, it may be preferred to flank the heterologous coding sequence with the complement of both the 3'-terrninus and the 5'-terrninus of a genome segment of inflllen7~ After transcription with a DNA-directed RNA polymerase, the resulting 25 RNA template will encode the negative polarity of the heterologous gene sequence and will contain the vR~A terminal sequences that enable the viral RNA-directed RNA polyrnerase to recognize the transcript. Detailed protocols for the production of RNA transcripts (templates) are provided in U.S. Patent 5,166,057.
The nucleic acid transcript, however, need not encode an expressed protein.
30 For example, the nucleic acid transcript can itself act as an antisense molecule targeting host cell DNA or mRNA. In addition, the nucleic acid transcript can alter the function (e.g, infectivit~, pathogenicity, etc.) ofthe virus itselfby introducing deletions, insertions, point .

CA 022~3~9~ 1998-10-30 ~CTIUS97/07277 mutations, and frarneshiPt mutations. These various functions are discussed below in Section VI.
In principal, the RNA transcript can replace all or part of any component of the viral genome. Thus for example, in the case of the influ~n7~ virus, one can, in principle 5 replace any one of the eight gene segm~orlt~, or part of any one of the eight segments with the foreign sequence. However, a necessary part of this equation is the ability to propagate the defective ~irus (defective because a norrnal viral gene product is missing or altered).
A number of possible approaches exist to circumvent this problem. Briefly, these approaches include, but are not limited to culture with helper ~,~iruses ~;AI~ t.~S;Ilg the 10 defective gene, culture in cell lines engineered to complement the defective gene, selection of natural host range systems that allow propagation of defective virus, and co-cultivation with wild-t~pe ~irus (see U.S. Patent 5,166,057) and are t~iscus~ed in more detail in Section IV
below.
In the case of influen7 1 for example, the nucleic acid transcript can replace all 15 or part of PB2, PB 1, PA, NP, HA, NA, NS, or M gene segmerl~c The gene segments coding for the PB2, PB 1, PA and NP proteins contain a single open reading frarne with 24~5 untranslated nucleotides at their 5'-end, and 22-57 untranslated nucleotides at their 3'-end.
Insertion of a foreicrn gene sequence into any of these segm~lts could be accomplished by either a complete replacement of the viral coding region with the foreign _ene or by a partial 20 replacement.
The HA and NA proteins, coded for by separate gene segments, are the major surface glycoproteins, of the virus. Consequently, these proteins are the major targets for the humoral irnmune response after infection. They have been the most widely-studied of all the infl~lPn7~ viral proteins as the three-dimensional structures of both these proteins have been 25 solved.
Th~ three-dimensional structure of the H3 hemagglutinin along with se4uence information on large numbers of variants has allowed for the elucidation of the antigenic sites on the H~ moleeule (Webster et al., 1983, pp 127-160 In: GeneJics Of InJ72~enza Vims, P.
Palese and D. U'. Kingsbury, eds., Springer-Verlag, Vienna). These sites fall into four discrete 30 non-overlapping regions on the surface of the HA. These regions are highly variable and have also been shown to be able to accept insertions and deletions. Therefore, substitution of these sites within HA (e.g, site A; amino acids 122-147 of the AMV68 HA) wi~h a portion of a CA 022~3~9~ l998-l0-30 WO 97/41245 ~CT/US97107277 foreign protein may provide for a vigorous humoral response against this foreign protein. In a di~e, e"l approach, the foreign peptide sequence may be inserted within the antigenic site without deleting any viral sequences. Expression products of such constructs may be useful in vaccines against the forei~n antigen, and may circumvent the problem of pl opagaLion of the 5 1 eco".b;nant virus in the vaccin~ted host. An intact HA molecule with a substitution only in antigenic sites may allow for HA function and thus allow for the construction of a viable virus. Therefore, this virus can be grown without the need for additional helper functions. Of course, the virus should be ~ n--~ted in other ways to avoid any danger of ~ccident~l escape.
Other hybrid constructions can be made to express proteins on the cell surface 10 or enable them to be released from the cell. As a surface glycoprotein, the HA has an amino-terrninal cleavable signal sequence necessa~ y for transport to the cell surface, and a carboxy-terminal se~quence nesecs~ry for ~--~,."b,~ne anchoring. In order to express an intact foreign protein on the cell surface it may be necessary to use these HA signals to create a hybrid protein. Alternatively, if only the transport signals are present and the membrane 15 anchoring domain is absent, the protein may be excreted out of the cell.
In the case of the NA protein, the three-dimensional structure is known but the antigenic sites are spread out over the surface of the molecule and are overlapping. This indicates that if a se~quence is inserted within the NA molecule and it is expressed on the outside surface of the 1~ . it is likely to be immunogenic. Additionally, as a surface 20 glycoprotein, the N.~ exhibits tWO striking differences from the HA protein. Firstly, the NA
does not contain a cleavable signal sequence; in fact, the amino-terminal signal sequence acts as a membrane anchorinv domain. ~he consequence of this, and the second difference between the NA and H~ is that the NA is orientated with the amino-terminus in the membrane while the HA is orientated with the carboxy-terminus in the membrane. Therefore 25 it may be advantageous in some cases to construct a hybrid NA protein, since the fusion protein will be orientated opposite of a HA-fusion hybrid.
The unique property of the NS and M segments as compared to the other six gene segments of influenza virus is that these segments code for at least two protein products.
In each case, one protein is coded for by an mRNA which is co-linear with genomic RNA
30 while the other protein is coded for by a spliced message. However, since the splice donor site occurs within the coding region for the co-linear Ir~nsc~ ipt, the NS I and NS2 proteins CA 022~3~9~ l998-l0-30 ~8 --have an identical 10 amino abd ar~ino terminus while Ml and M2 have an identical 14 amino acid amino terminus.
As a result of this unique structure, recombinant viruses may be constructed so as to replace one gene product ~vith'in the Segmtont while leaving the second product intact.
S For instance, rep}acement of the buLk of the NS2 or M2 coding region with a foreign gene product (keeping the sp1ice acceptor site) could result in the expression of an intact NS 1 or M I protein and a fusion protein instead of NS2 or M2. Alternatively, a foreign gene may be inserted within the NS gene secrm~nt without affecting either NS 1 or NS2 ~ ., esa;on.
Although most NS genes contain a substantial overlap of NS 1 and NS2 10 reading frames, certain na~ural NS genes do not. For example, in the NS gene seYm~nt from A/Ty/Or/71 the NS I protein terminates at nucleotide position 409 of the NS gene s~ nt while the splice acceptor site for the NS2 is at nucleotide position 528 (Norton et aL, ~1987), Virology 156: 204-213). Therefore, a foreign gene could be placed between the tel...; ~ ;or codon of the NS 1 coding region and the splice acceptor site of the NS2 coding region I5 without affecting either protein. It may be necessary to include a splice acceptor site at the 5' end of the foreign gene sequence to ensure protein production (this would encode a hybrid protein containing the amino-terminus of NSl). In this way, the reco~"l)ina"l virus should not be defective and should be able to be propagated without need of helper functions.
The insertions into andJor replacements of all or part of various viral genes 20 described above, can be perforrned according to standard methods well known to those of skill in the art. In a preferred embodiment, the RNA transcripts of this invention can be prepared through the use of PCR-directed mutagenesis. Detailed methods for the construction of RNA transcripts suitàble for the production of viral transfectants can be found in U.S. Patent 5,166,057.
C) Combination of transcript with nucleoprotein to form synthetic nucleprotein complex.
The R~TA transcript can be combined with the nucleoprotein (NP) before, during, or after transfection. However, because the nucleoprotein affords the RNA transcript 30 some protection from ribonucleases during transfection, in a preferred embodiment, the NP is combined with the RNA transcript before L~nsre-;Lion.

. .

CA 022~3~9~ 1998-10-30 The RNA Ll al-sc- ipl and the nucleoprotein can be combined before transfection by simple admixture. Sirnilarly, the Llal1s~ Jt and nucleoprotein can be corl~b;,lcd during Ll ~hsrecLion by placing both components in the transfection mixture. The col-lponenLs can be combined after transfection by sirnply transfecting one component followed by the 5 other component and allowing the components to combine when inside the host cell.
In a particularly pl~.led embodiment, however, the formation ofthe synthetic nucleo~lo~e;.- complex occurs with preparation ofthe nucleic acid L~i,s~,.ipt. Thus, for example, the nucleoprotein can be added to the reaction mixture (e.g., ~mplifi~tion, reverse tlans~ "ion, e~c.) in which the nucleic acid transcript is produced thereby f~Cilit~ting 10 the rapid ~C~oci~tion ofthe MP with the Llal-sc~ipL. As NP helps prevent RNA degradation, this approach improves the stability.
For example, in one approach, the nucleic acid transcript is produced by reverse Ll ai)Scl i~,Lion from a cDNA clone encoding the desired RNA Ll ansc, ipl in the p. ~sence of a nucleoprotein. Typically the cDNA is placed behind a promoter (e.g., a T;' RNA
15 polymerase promoter~ situated to allow tlansclil)tion to begin on the correct nucleotide.
Reverse transcription is pe. rOl ~l~ed accordh~g to standard methcl~ls known to those of skill (see, e.g., Sambrook et al., supra, Ausubel et al., supra.; Van Gelder, et aL, (1990) Proc.
Na~l. Acad. Sci. ~rsA~ 87: 1663-1667; and Eberwine et al. Proc. Na~l. Acad. Sci. USA, 89:
3010-3014). This approach is illustrated in Example 1.
m. Transfection of EIost Cell.
The synthetic nucleprotein complex is transfected into a host cell where it is allowed to replicate. Virtually any host cell can be transfected in this manner. However, in a preferred embodiment, the host cell is one that is compatible with infection and culture of the 25 particular subject virus. Thus, for example where the t~ ~n~ ed virus is an inflnçn7~ virus, preferred host cells include those cells commonly used to culture influçn7~ Such cells include, but are not limited to cells of the arnniotic membrane of embryonated eggs, tissue cultures of kidney tissue from rhesus monkeys, baboons, chicks, or a variety of other species.
Particularly preferred cells include, for example, primary chick kidney (~CK) cells, Madin-30 Darby canine kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells and the like.
As indicated above, in one embodiment, the methods of this invention are not used simply to prepare viral transfectants, but rather to provide host cells co."ai..i"g a CA 022~3~9~ 1998-10-30 heterologous nucleic acid. ln this context. the synthetic nucleoprotein complex can be used simply to direct the heterologous nucleic acid to the nucleus of the host cell. The host cell in this case, can be any cell it is desired to transfect with the heterologous nucleic acid transcript.
The synthetic ribonucleoprotein complex can be tl ar,s~e-,led into the host cellaccording to any of a number of ll ~ r.~r~ ~,lion methods well known those of skill in the art.
Suitable L,al1s~ecLion methods include, but are not limited to calcium chloride tran~rol..,alion e.g., for E. coli, and calcium phosphate trea~nent, ele~lul)oldtion, or ballistic t~nsfection (e.g., high velocity gold micl~,s~h~s) for eukaryotic cells. In a particularly ~ Çelled embodiment, the host cells are transfe~ted by electroporation or by the DEAE-dextran DMSO protocol ~see, e.g., Al-Molish el al. (1973) J. Gen. Virol., 18: 189-193; and Lopata et al. (1984) NlJcL AcidsRes. 12: 5707-5717), with electroporation being most plef~"ed.

IV. Viral Culture.
After transfection of aells by the synthetic ribonucleoprotein complex, the cells are preferably cultured under conditions that permit replication of the subject virus. One of skill in the art will appreciate that a n~onucleoprotein complex (RNP) comprising just an RNA and an NP is generally insufficient to repLicate the subject virus alone and additional "replication m~-~hinery" is typically required. In addition, where the nucleic acid l,~ns~;.i~,l - 20 includes a heterologous nucleic acid, the ~irus is o~en "defective" because a normal viral gene product is o~en missing or altered.
As indicated above, a number of possible approaches exist to circumvent this problem. Mutants (transfectants) of the virus (e.g., influenza) can be grown in cell lines constructed to constitutively express the ' defective" gene (see, e.g., Krystal et al., (1986) Proc. Natl. Acad Sci. USA 83: 2709-281i who grew mutants of infl~lçn7~ virus defective in the PB~ and NP proteins in cell lines which were constructed to constitutively express the polymerase and NP proteins). These cell lines which are made to express the viral protein may be used to complement the defect in Lr~e re~ombinant virus and thereby propagate it (see, e.g., Kimura et al. (1992)J. Gen. Virol., 7i: 13~1-1328).
Alternatively, certain natur21 host range systems may be available to propagate recombinant virus. An example of this approach concerns the natural influPn7~ isolate CR43-3. This virus will grow norrnally when passaged in primary chick kidney cells (PCK) CA 022~3~9~ 1998-10-30 but will not grow in Madin-Darby canine kidney cells (r-fDCK), a natural host for influenza (Maassab & DeBorde (1983) ~irology, 130: ~42-3~0). This virus codes for a defective NS1 - protein caused by a deletion of 12 amino acids. The PCK cells contain some activity which either complements the defective NS I protein or can completely substitute for the defective protein.
As intlicated above, it was a surprising discovery and advantage of this invention that synthetic ribonucleoprotein complexes (R~Ps) completely lacking RNA
polymerase of the subject virus are fully capable of replicating when complemented by a helper virus. This invention thereby elirninates the requirement for purified RNP proteins or recombinal-~ly engineered host cells for viral propagation.
Thus, in a p,~r~ d embodiment, the viral l~ars~Lalll is propagated through involve co-cultivation with wild-type or helper virus. This can be done by simply taking lecul"binallL virus and co-infecting cells ~ith the svnthetic RNP and a wild-type (e.g., a vaccine strain) or a helper virus. The wild-type or helper virus should complement for the defective virus gene product and allow growth of both the helper or wild-type and reco",binall~ virus.
Where co-cultivation is with wild-t~e virus~ this is analogous to the propagation of defective-interfering particles o~ infl~:enza virus (Nayak et al., (1983) In:
Genetic* of InJ7uenza Vir71se~; P. Palese and D W. Iiingsbury, eds., Springer-Verlag, Vienna, pp. 255-279). In the case of defective-interfenng ~i~uses. conditions can be modified such that the majority of the propagated virus i~ the defeceive particle rather than the wild-type virus. Therefore this approach may be useful in gene~ating high titer stocks of recombinant virus. However, these stocks would necessaril-- con~ain some wild-type virus.
In a particularly preferred embo~imerlt, the synthetic RNPs are cultured in the presence of a helper virus. Suitable helper viru~es a~e well known to those of skill and include; but are not limited to wild-type (e.g., A/PR 3/34 or A/WSN/33) or live ~ttenl~ted vaccine strains (~.g., A/Leningrad/57 or A'Ann Arbor/6/60).
The host cell can be infected with the helper virus prior to, along with, or after transfection of the synthetic ribonucleopro~ein comp ex into the cell according to standard methods well known to those of skill in the art (see, ~.g., Enami M. and Palese, P. (1991) J.
Virol., 6S: 2711). Similarly, transgenic viruses produced according to the method ofthis CA 022~3~9~ l998-l0-30 invention can also be cultured in the pltsence of appropliate colllplc.l,enting wild-type or helper virus.
- In still yet another approach, infection of ho~ cells with synthetic RNPs encoding all eight influen2a virus proteins results in the production of infectious transgenic 5 (transfectant) virus particles. This system would eliminate the need for a selection system, as all I eco-nbinal-l virus produced would be of the desired genotype.
The synthetic ribonucleoprotein complexes or the viral transr~ a.lL~ can be cultured in standard culture systems inrludin~ for example, embryonated eggs and tissue culture.
A) Culture in embryonated eggs The virus (e.g, influen7~) can be cultured bv amniotic or ~ ntoic inoculation of 10-12 day embryonated eggs. The virus is absorbed from the fluid of the amniotic cavity onto the cells of the amniotic nle.lll l ane in which the y multiply, releasing newly formed virus 15 back into the amniotic fluids. A~er two to three days' incubation, virus can be present in high titre in the amniotic fluid and can be detected by adding aliquots of harvested amniotic fluid to chick, turkey, guinea-pig or human erythrocytes and observing haem~gglutin~tion. Detailed protocols for viral culture in embryonated eggs are well kno~ n to those of skill in the art and can be found in standard references (see, e.g., Fields Virolon, s~pra.).
B) Tissue culture.
The subject viruses can also be cultured in eu~;aryotic tissue cultures according to standard methods. For example, influl~n7~ can be culture is tissue cultures of kidney from dogs, rhesus monkeys, baboons, chicks, or a variety of other species. Particularly preferred 25 cells include, for example H292 cells, primary chick kidney (PCK) cells, Madin-Darby canine kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells, and the like.
A~er absorption (~la--~re-;lion) and incubation of virus-infected cells, newly produced virus can be detected in a number of ways. In one approach, free virus released into the maintenance medium of the tissue culture can be det~cted (e.g, by haem~gglutin~tion 30 with erythrocytes). Alternatively, since virus is slowly released from the cell surface of infected cells, erythrocytes will adhere directly to these infected cells (haemadsorption) and can be detected under the microscope. Methods of viral main[enance and propagation in CA 022~3~9~ 1998-10-30 tissue culture are known to those of skill in the art (see, e.~., Concepts and Proceeduresfor Laboratory-Based lnJ7uenza Surveillance, Dept. Health and Hum. Ser.~ Centers for Disease Control (1982)).

V. Selection of Transfectants.
Viral ~l~n~e-,lanLs (tr~nC~.~nic viruses) can be identified and selected according to standard methods known to those of skill in the art. Typically this involves identifying culture cells (or culture supernate) positive for the viral transfectant, and selectively prop~g~ting the host cell containing the virus and/or the virus itself from the cell or supernatant. Identification ofthe host cell or supe.llala"l is by means well known to those of skill in the art and typically involves direct or indirect detection of the presence or absence of the heterologous RNA (transcript) or its product.
Direct detection involves detection of the heterologous RNA (or DNA
Ll ~ns-,- il,ed therefrom) itself. This can be accomplished by a number of means incltl~in~ but not limited to detection using a nucleic acid affinity column specific to (containing probes complementary to) the heterologous RNA (or DNA transcribed therefrom) or to subsequences thereof, amplification (e.g., via PCR) of particular target sequences or subsequences in the RNA (or DNA transcribed therefrom), restriction analysis, and so forth.
In one preferred embodiment, illustrated by Example 1, the heterologous R~A is detected by PCR restriction analysis according to the method of Klimov et al. ( 199~) J. T irol. Me~h., 52:
4 1 -49.
In other embodiments, the viral transfectants, or host celis harboring the viraltransfectants, can be identified by detection of the presence or absence of a protein product of activity of the protein product expressed by the heterologous nucleic acid. Thus, the heterologous nucleic acid can include a sequence that confers antibiotic resistance thereby allowing selection of transfectants by antibiotic r~ci~t~nce of the host cells. Alternatively, the heterologous nucleic acid can encode a detectable marker as described below.

VI. Uses of viral transfection.
The synthetic ribonucleoprotein complexes and the ~iral ~ransfectants made according to the methods of this invention can be used to express heterologous gene products in host cells or to rescue the heterologous gene in virus particles by cotransfection of host CA 022~3~9~ 1998-10-30 cells with reco.,-bioalll RNPs and virus. Alternatively, heterologous gene can be rescued in host cells transformed to allow for compl~ment~tion and rescue of the heterologous gene in virus particles.
The expression products and/or chimeric virions obtained may also be advantageously be utilized in vaccine formulations. Viral transfectants expressing detect~hle labels provide effective reporter systems for screening anti- iral compounds and investig~ting viral ecology. Finally, because the NP protein confers protection against ribonllcle~ce activity the RNA/NP ribonuclease complex and viruses derived therefrom provide an effective system for the delivery of ~nti~ence RNA. These various uses are rliscussed more fully below.
A) Expression of heterologous gene products using svnthetic RNP complexes.
The recombinant templ~tes prepared as described abo~e can be used in a variety of ways to express the heterologous gene products in appropriate host cells or to create chimeric viruses that express the heterologous gene products. In one embodiment, the recombinant templ~te can be combined with viral polymerase complex purified as described in Section 6, infra, to produce rRNPs which are infectious. Alternativel~l the reco.--l inal"
template may be mixed with viral polymerase complex prepared usin_ recombinant DNA
methods (see, e.g., Kingsbury e~ al., (1987), Virolog~ 156: 396~03). Such r~Ps, when used to transfect appropriate host cells, may direct the expression of the heterologous gene product at high levels. Host cell systems which provide for high levels of expression include continuous cell lines that supply viral functions such as cell lines superinfected with influenza, cell lines engineered to complement inflll~n7~ viral functions, etc.

B) Use of detect?~ble hel~. ulo~ous sequences.
In another embodiment, the heterologous R~A transcript can act as a detectable label or can express a protein that is a detectable label. The detectable label allows quantification, or detection of the presence or absence, of the virus in a particular sample. A
viral transfectant bearing a detect~hle label provides an effective indicator for screening for anti-viral drugs i~ vi~ro or i~1 vivo. For example, the organism or cell line is infected with the viral transfectant bearing the detect~hle marker and then treated with one or more test compounds. Quantification of the detectable marker after treatment and comparison of test with control samples provides a measure of the efficacy of the compound tested.

CA 022~3~9~ 1998-10-30 WO 97/41245 PCT/US97tO7277 Where the detectable label is one that can be easily captured (e.g., a particular nucleic acid sequence that can be captured by a nucleic acid affinity column or a polypeptide - sequence (e.g, polyhistidine such as His6 that can be captured using an Ni-NTA column) the viral transfectant can be used to isolate anti-viral antibodies. In this embodiment, an o-~;ani;,l,.
5 is challenged with the viral transfectant under conditions that allow the organism to mount an immune response. Subsequent capture of the viral transfectant (through capture of the detectable label) will also capture any antibody binding to the virus.
The detectable label can also be used to monitor (e.g., detect and/or quantify) one or more strains of virus (e.g., infl~len7~) in vltro or in vivo. This perrnits rapid ~Cce~"~
10 of the infectivity and/or the pathogenicity of a particular virus, alone or in combination with other viruses. This permits the ~csess" ~e-~l of the effects of mutations producing various viral strains on the host and on other viral strains. Other uses of viral transfectants bearing detectable labels will be known to those of skill in the art.
Detectable labels suitable for use in the present invention include any 15 nucleic acid sequence that is detectable (e.g., via hybridi_ation to a particular probe or capture sequence, or through RFLP or other fingerprinting technique, etc.) or any polypeptide sequence that is detectable including, but not limited to enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), an other polypeptide sequences that can be specifically detected and/or captured (e.g., 20 polyHis that can be captured using an Ni-NTA column). Means of detecting such labels are well known to those of skill in the art.

C) Use of viral transfectants for vaccine formulations.
The expression products and/or chimeric virions obtained can be used in 25 vaccine formulations. The use of reco",bh~a,l~ influen_a for this purpose is especially attractive since influenza demonstrates tremendous strain variability allo~ving for the construction of a vast repertoire of vaccine formulations. The ability to select from thousands of influenza variants for constructing chimeric viruses obviates the problem of host resistance encountered when using other viruses such as vaccinia. In addition, since influenza s~im~ tes 30 a vigorous secretory and cytotoxic T cell response. the presentation of foreign epitopes in the inflllen7~ virus background may also provide for the induction of secretory immunity and cell-mediated immunity.

CA 022~3~9~ l998-l0-30 WO 97/41245 PCT/U~97/07277 Virtually any heterologous gene sequence may be constructed into the chimeric (transfectant) viruses of the invention for use in vaccines. Preferably, epitopes that induce a protective immune response to any of a variety of pathogens, or antigens that bind neutralizing antibodies may be ~;AI.11 essed by or as part of the viral Ll dn~re~;lants. For example, 5 heterologous gene sequences that can be constructed into the chimeric viruses ofthe invention for use in vaccines include but are not limited to epitopes of human immunodeficiency virus (HIV) such as gpl20; hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpes virus (e.g, gD, gE); ~I of poliovirus; antigenic dete. ~ni..~..ls of non-viral pathogens such as bacteria and parasites, to name but a few. In another 10 embodiment, all or portions of immunoglobulin genes may be e, ~- essed. For example, variable regions of anti-idiotypic immunoglobulins that mimic such epitopes may be constructed into the chimeric viruses of the invention.
Either a live reco..-bi"allt viral vaccine or an inactiviated recombinant viral vaccine can be formulated. A live vaccine may be pre~erred because multiplication in the host leads to a prolonged stimul~ls of similar kind and rn~gnitude to that occurring in natural infections, and therefore, confers substantial, long-lasting immunity. Production of such live recol~ ina-~l virus vaccine formulations may be accomplished using conventional methods involving propagation of the virus in cell culture or in the allantois of the chick embryo followed by purification.
In this regard, the use of genetically engineered influenza virus (vectors) for vaccine purposes may require the presence of ~ttçnu~tion characteristics in these strains. Current live virus vaccine can~idates for use in humans are either cold adapted, temperature sensitive, or passaged so that they derive several (six) genes from ~vian viruses, which results in ~tt~n~l~tion. The introduction of appropriate mutations (e.g, deletions) into the RNA
transcripts used for transfection may provide the novel viruses with attenuationchàracteristics. For example, specific missense mutations which are associated with temperature sensitivity or cold adaption can be made into deletion mutations. These mutations should be more stable than the point mutations associated with cold or temperature sensitive mutants and reversion frequencies should be extremely low.
Alternatively, viral transfectants with "suicide" characteristics may be constructed. Such viruses would go through only one or a few rounds of replication in the host. For example, in influenza, cleavage of HA is necessary to allow for re-initiation of CA 022~3~9~ l998-l0-30 replication. Therefore, changes in the HA cleavage site can produce a virus that replicates in an appropriate cell system but not in the human host. When used as a vaccine, the . eco,nbinant virus goes through a single replication cycle and induces a sufficient level of immune response but fails to progress further in the human host and cause disease.
Reco.. l,i.. alll influ~n7~ viruses lacking one or more of the ess~n*~l infiuenza virus genes would not be able to undergo successive rounds of replication. Such defective viruses can be produced, for e~a--ll~le, by co-transfecting reconstituted RNPs lacking specific gene(s) into cell lines which perrnanently express these gene(s). Viruses lacking essential gene(s) will be replicated in these cell lines but when administered to the human host will not 10 be able to complete a round of replication. Such pre~,a. ~tions may transcribe and translate -in this abortive cycle - a sufficient number of genes to induce an immune response.
Alternatively, larger quantities of the strains could be administered, so that these preparations serve as inactivated (killed) virus, vaccines.
In inactivated vaccines, it is preferred that the heterologous gene product be 15 expressed as a viral component, so that the gene product is associated with the virion. The advantage of such p-el~a-~lions is that they contain native proteins and do not undergo inactivation by ll eal---ent with forrnalin or other agents used in the m~n~1f~c.turing of killed virus vaccines.
In another embodiment of this aspect of the invention, inactivated vaccine 20 formulations may be prepared using conventional techniques to "kill" the viral transfectants.
Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity. In order to prepare inactivated vaccines, the viral transfectant may be grown in cell culture or in the allantois of the chick embryo, purified by zonal ultracentrifugation, inactivated by 25 formaldehyde or beta-propiolactone, and pooled. The resulting vaccine is p. ere- ~bly inoculated intramuscularly.
Inactivated viruses may be formulated with a suitable adjuvant in order to enhance the immunological response. Such adjuvants may include but are not limited to mineral gels, e.g., ~ mirlllm hydroxide; surface active substances such as Iysolecithin, 30 pluronic polyols, polyanions; peptides; oil emulsions; and potentially useful human adjuvants such as BCG and C.orynebacteri1lm parvum.

CA 022~3~9~ 1998-10-30 WO 97t41245 PCT/US97107277 r-Iany methods may be used to introduce the vaccine formu}ations described above, into the subject organism (e.g., pig, rabbit, goat, mouse, rat, primate inciuding human, e~c.). These include but are not limited to oral, intradermal, intr~ml-cc~ r, h~ pelilonea intravenous, subcutaneous, and intranasal routes. It may be preferable to introduce the 5 chimeric virus vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed. Where a live chimeric virus vaccine preparation is used, it may be preferable to introduce the forrnulation via the natural route of infection for influ~n7~ virus.
The ability of influen7~q virus to induce a vigorous secretory and cellular immune response can be used advanta_eously. For example, infection of the respiratory tract by chimeric influen7~
10 viruses may induce a strong secretory immune response, for example in the urogenital system, with concornitant protection against a particular disease causing agent.

D) I~Iodulation of intracellul~r activity.
The viral transfectants prepared according to the methods of this invention can I5 be used to modulate the intracellular activity of the host cell. This modulation can be accomplished directly through the activity of the RNA transcript in the viral transfectant or indirectly through a polypeptide encoded by the RNA transcript in the viral trans~,Lallt.
For example, the RNA transcript can be provided that is comple,~ aly (antisense) to re_ions of the genomic DNA of the host cell or to an rnRNA expressed by a 20 particular gene. Hybridization of the RNA transcript to the host DNA can inhibit transcription of the target region while hybridization to an mRNA can block the translation of that mRNA and hence the expression of the protein encoded by the mRNA. Similarly binding of the antisense RNA to transcription regulators (e.g., promoters, initiation sites, repressors, e~c. ) can up-regulate or downregulate 11 ~ns.,l il.lion of particular targeted genes. The use of 25 antisense molecules to modulate (e.g, upregulate or downregulate) protein expression is well known to those of skill in the art (see, e.g., Castanotto e~ al. (1994) Adv. Pharmacol., 25:
289-317, WO 90,'13641, and references cited therein).
Thus, in one embodiment this invention provides for methods and compositions for the delivery of antisense molecules into the nucleus of a cell. As indicated 30 above, the viral nucleoprotein affords the RNA ll al~SCI ipt protection from ribonucleases and directs the R~A transcript into the nucleus of the host cell. The ribonucleoprotein complexes comprising an antisense RNA complexed with an NP provide an effective vehicle for delivery CA 022~3~9~ 1998-10-30 W O 97/41245 ~CT~US97/07277 of the antisense molecule. Similarly, viral transfectants containing the antisense RNA also provide a highly effective delivery system for the ~ntisence molecule.
As indicated above, the RNA transcripts can be selected that modulate intracellular acti~ity throu~h an G,.l"essed protein. The expressed protein can be a functional S component of an intracellular si~n~iing pathway (e.g, an estrogen receptor (ER), jun, fos Elk, ATF2, a tyrosine kinase, a GTP binding protein such as Ras, Rac, and the like, si~ ng molecules such as JAE~s, PLCs, and PI3K) or, conversely can be a protein that inhibits a component of a sign~ling cascade. Such proteins are well known to those of skill in the art (see, e.g., Darnell and Baltimore, ( l 986) Molecular Cell Biology, Scientif c American 10 Books).

E) Use of viral transfectants for gene therapy.
The viral transfectants of this invention also provide suitable vectors for genetherapy. The virus, containing an RNA transcript that encodes a protein of therapeutic value.
15 In addition, the RNA transcript can include (or encode) segments that mediate integration of the heterologous gene of interest into the genome of the host cell (organism). Seg",~ L~ that mediate integration wi~h the host genome are known to those of skill in the art and include, but are not limite~ to 2deno associated viruses (AAVs), inverted terminal repeats (ITRs), and retroviral LTRs. The selection and design of nucleic acid Llansc~ )ts suitable for gene therapy 20 is well known to those of s~;ill in the art (see, e.g., Freifelder Molecular Biology, 2nd ed., l987, Jones and Bartle.t, Pub., Boston) VII. Kits for the practice of this invention.
This in~ ention additionally provides for kits for the practice of the methods 25 disclosed herein. In a preferred embodiment, the kit contains a container co-,L~;nlng an isolated viral nucleoprotein (NP), as described above, for the practice of the methods of this invention. The kits additionally include labels or instructions describing the methods of making viral transfecta.nts andlor the metho-l~ of introducing a ribonucleic acid into a host cell described above. The ~Tp can be provided alone or complexed with a ribonucleic acid (e.g., 30 RNA transcript). The ~;it can additionally include one or more of the following elements buffers, host cells, culture media, helper viruses, subject viruses, and the like.

EXAMPLES
The follo~ ing examples are offered to illustrate, but not to limit the present invention.
E~ample 1 Transfection assays were cond~cted using nucleprotein (NP) punfied from insect cells infected with a recol~.bi,~ baculovirus expressing type A influenza NP. The results de~llor~sLI~le that ~irion polyrnerase proteins are unnecessary n the artificial RNP
complexes transfected into cells.
Nucleoprotein (~P was purified from SF9 (Spodoptera) cells infected with a recombinant baculovirus expressing the influPn7~ A/Ann Arbor/6/60 nucleocapsid protein (see, e.g, Rotal et al. (1990) J. Gen. Virol., 71: 1545-1554; Rota ef al., U.S. Patent No.
5,316,910 and WO 92/16619). The cells were Iysed by refreezing and suspended in 0.01 M
Tris-HCI, pH 7.4, containing 0.001 ~ EDTA and 1 M NaCI. After overnight dialysis against phosphate buffered saline (PBS. pH 7.3), the supernate of the Iysate was fractionated on a column of monoclonal anti-NP IgG (Walls ef al. ( 1986) J. C.lin. MicrobioL, 23: 240-245) coupled to Bio-Rad A-14 ~ffi_el matrix using the rn~nllf~-~.tl-rer's methods and equilibrated in PBS. bound protein was eluted in I M MgCl2 and concellLlaled by dialysis against 50 mM
Tris-HCI, pH 7.6, 100 mr-~ NaCl, 10 M MgCI2, 2 mM DTT, 50% Glycerol. Purity was verified by SDS PAGE. Aliquots were stored at -70~C until used in transfection assays.
- For transfection assays, the 25A-1 virus, a 7/1 fs reassortment having seven genes from A/PR/8/34 viru, and the ~S gene from the live-attenuated, cold-adapted A/Leningrad/47/57 virus w as used as a helper. A cDNA clone of the A/PRt8/34 virus NS
gene placed behind a T7 R~TA polymerase promoter situated to allow transcription to begin on the correct nucleotide and ha~ng a BseAI site downstream to allow run offtranscripts after treatment with mung bean nuclease was used as the source of RNA for transfection.
Transcription reactions were conducted in the presence of S ~,lg of purified baculo-eA~- essed nucleoprotein (NP). The resulting synthetic RNP complexes were transfected into MDCK
cells that had been infected one nour previously with the 25A-I reassortant helper virus by the electroporation method of Li ef al. (1995) VinJsRes., 37: 153-161.
After incubalion at 34~C for 18 hours, culture supernates were used to infect confluent MDCK cells for two days at 37~C before plaquing the culture supernates at 39~C
for three days. A total of 15 plaques were picked, nine of which grew in 10 day embryonated CA 022~3~9~ 1998-10-30 hens' eggs. The origin of the NS gene in each clone was determined by PCR restriction analysis (Klimov et al. (199i~ J. ~ ;rol. M~h., 52: 41-49). Of the nine clones analyzed, four had the A/PR18/34 NS gene thus demonstrating that functional polymerases are not necessary in the synthetic RNPs transfected into cells. Transfection experiments using DEAE-dextran DMSO protocol (Al-Molish et al. (1973)J. Ge7~. Virol., 18: 189-193; and ~opata etal.
(1984) Nucl. Acids Res. 12: 5707-5717), yielded a total of eleven clones, only one of which had the A/PR18134 NS gene, thus demonstrating the superior efficiency of electroporation for the generation of inflllen7~ virus transfectants.
Without beinQ bound to a particular theory, it is believed the role of NP is two-fold; protection of the RNA transcript from degradation and transport to the cell nucleus where the helper virus supplies the necessary transcription and replication m~rllinery.
In addition to ~iimin~tinv the laborious purification procedure for RNP
proteins, the use of recombinant D~A-derived NP çlimin~tes the possibility of introducing virus genes which might be present in the R~TP proteins purified from virions. In the case of live-~ttenuated vaccines, this will make it easier to m~int~in the ~ttenuated phenotype since no wild-type genes are present, except for those deliberately introduced.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that v arious modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a virus bearing a preselected ribonucleic acid, said method comprising the steps of:
i) combining said ribonucleic acid with an isolated nucleoprotein (NP) of said virus to form a synthetic ribonucleoprotein complex (RNP), wherein said nucleoprotein (NP) and said synthetic ribonucleoprotein complex (RNP) are substantially free of viral RNA
polymerase proteins;
ii) transfecting a host cell with said synthetic ribonucleoprotein complex; and iii) holding said host cell under conditions permitting the replication of said virus.

2. The method of claim 1, wherein said host cell is infected with a helper virus.

3. The method of claim 2, wherein said helper virus is a laboratory-adapted wild-type virus or a live-attenuated vaccine strain.

4. The method of claim 3, wherein said helper virus is selected from the group consisting of A/PR/8/34, A/WSN 33, A/Lenigrad/57, and A/Ann Arbor/6/60.

6. The method of claim 1, wherein said isolated nucleoprotein (NP) is recombinantly expressed.

7. The method of claim 1, wherein said isolated nucleoprotein (NP) is recombinantly expressed in a eukaryotic expression system.

8. The method of claim 1, wherein said virus is selected from the group consisting of an influenza type A virus, an influenza type B virus, and an influenza type C virus.

9. The method of claim 1, wherein said nucleoprotein is a nucleoprotein (NP) of a virus selected from the group consisting of an influenza type A virus, an influenza type B virus, and an influenza type C virus.

10. The method of claim 1, wherein said host cell is selected from the group consisting of a kidney cell, a cell from a hen's egg, and a primate primary lung cell.

11. The method of claim 1, wherein said virus is an influenza virus;
said nucleoprotein is a recombinantly expressed influenza nucleoprotein substantially free of a viral RNA polymerase; and said host cell is infected with an influenza helper virus.

12. A method of introducing a ribonucleic acid into the nucleus of a host cell, said method comprising the steps of:
i) combining said ribonucleic acid with an isolated nucleoprotein (NP) of a virus to form a synthetic ribonucleoprotein complex (RNP), wherein said nucleoprotein (NP) and said synthetic ribonucleoprotein complex (RNP) are substantially free of viral RNA
polymerase proteins; and ii) transfecting a host cell with said synthetic ribonucleoprotein complex.

13. The method of claim 12, further comprising infecting said host cell with a helper virus.

14. The method of claim 13, wherein said helper virus is a laboratory-adapted wild-type virus or a live-attenuated vaccine strain.

15. The method of claim 14, wherein said helper virus is selected from the group consisting of A/PR18/34, A/WSN 33, A/Lenigrad/57, and A/Ann Arbor/6/60.
17. The method of claim 12, wherein said isolated nucleoprotein (NP) is recombinantly expressed.

18. The method of claim 12, wherein said nucleoprotein is a nucleoprotein (NP) of a virus is selected from the group consisting of an influenza type A virus, an influenza type B virus, and an influenza type C virus.

19. The method of claim 12, wherein said host cell is selected from the group consisting of a kidney cell, a cell from a hen's egg, and a primate primary lung cell.

20. A transfection composition comprising an isolated viral nucleoprotein combined with a ribonucleic acid thereby forming a synthetic ribonucleoprotein complex (RNP) that mediates replication of a virus in a host cell, wherein said nucleoprotein (NP) and said synthetic ribonucleoprotein complex (RNP) are substantially free of viral RNA
polymerase proteins.

22. The composition of claim 20, wherein said isolated nucleoprotein (NP) is recombinantly expressed.

23. The composition of claim 20, wherein said nucleoprotein is a nucleoprotein (NP) of a virus selected from the group consisting of an influenza type A virus, an influenza type B virus, and an influenza type C virus.

24. A virus expressed from a synthetic ribonucleoprotein complex (RNP), said synthetic ribonucleoprotein complex comprising an isolated nucleoprotein (NP) combined with a ribonucleic acid (RNA), wherein said nucleoprotein (NP) and said synthetic ribonucleoprotein complex (RNP) are substantially free of viral RNA polymerase proteins prior to transfection into a host cell.
26. The virus of claim 24, wherein said isolated nucleoprotein (NP) is recombinantly expressed.

27. The virus of claim 24, wherein said virus is selected from the group consisting of an influenza type A virus, an influenza type B virus, and an influenza type C virus.

28. The virus of claim 24, wherein said nucleoprotein is a nucleoprotein (NP) of a virus selected from the group consisting of parainfluenza virus, a measles virus, rabies, and respiratory syncytial virus.

29. The virus of claim 24, wherein the virus is a live-attenuated virus.

30. A eukaryotic cell comprising a synthetic ribonucleoprotein complex or a virus derived from said synthetic ribonucleoprotein complex wherein said synthetic ribonucleoprotein complex comprises an isolated nucleoprotein (NP) combined with a ribonucleic acid (RNA), wherein said nucleoprotein (NP) and said synthetic ribonucleoprotein complex (RNP) are substantially free of viral RNA polymerase proteins.

31. The eurkaryotic cell of claim 30, wherein said ribonucleoprotein complex is an influenza ribonucleoprotein complex.

32. A kit for the transfection of a virus or a cell, said kit comprising a container containing an isolated viral nucleoprotein (NP) and a label or instructions describing the method of claims 1 or 11, wherein said wherein said viral nucleoprotein (NP) is substantially free of viral RNA polymerase proteins.

33. The kit of claim 32, wherein said NP is provided combined with a ribonucleoprotein thereby forming a synthetic ribonucleoprotein complex, wherein said synthetic ribonucleoprotein complex (RNP) is substantially free of viral RNA polymerase proteins.