JP2000509280A - Production of virus transfectant using nucleocapsid protein derived from recombinant DNA - Google Patents

Abstract

  (57) [Summary] The present invention provides a method of producing a viral transfectant (a virus containing a heterologous nucleic acid) that obviates the need for a purified RNP complex or a purified viral RNA polymerase. This method generally comprises the steps of (i) combining a ribonucleic acid (nucleic acid transcript) with a viral isolated nucleoprotein (NP) to form a synthetic ribonucleoprotein complex (RNP); Transfecting the host cell with a ribonucleoprotein; and (iii) maintaining (culturing) the host cell under conditions that permit replication of the virus.

Description

DETAILED DESCRIPTION OF THE INVENTION             Using nucleocapsid protein derived from recombinant DNA                     Production of virus transfectants                               Background of the Invention   The development of reverse genetics for influenza virus has Direct manipulation and production of completely new recombinant viruses not found in nature (Eg, Enami et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 3802-380). 5; Castrucci et al. (1992) J. Med. Virol., 66: 4647-4653; Li et al. Virol., 66: 3 99-404; Liu et al. (1993) Virol., 194: 403-407; Subbarao et al. Vlrol., 67: 7223-7228; and Zurcher et al. Virol., 68: 75748-5754) . Generally, the production of recombinant influenza virus is based on virion ribonucleoprotein (RNP) Combination of purified protein from core and synthetic RNA transcript, then helper Requires transfection of cells previously infected with the virus (Enami et al. (1991) J.C. Virol. 65: 2711-2713).   Purified RNP cores typically contain RNA polymer in addition to viral nucleoprotein (NP). Enzyme proteins (eg, PB1, PB2, and PA) (eg, Ishihama et al.). (1988) CRC Crit. Rev. Biochem, 23: 27-76). RN from virions Purification of P-complex proteins requires multiple gradient fractions, due in part to their complex structure. This is one of the most cumbersome aspects of the procedure that requires (Enami et al., Supra). Former wi The Luss transfection method was also complicated. Because the virus trans To achieve the transfection, viral RNA is added in addition to viral nucleoprotein. It was believed that a polymerase was required (eg, US Pat. 166,057). Viral RNA polymerase in addition to nucleoprotein (NP) The supply of enzyme requires sophisticated cloning systems or time-consuming, laborious isolation and purification procedures. (See, for example, Kimura et al. (1992) J. Virol., 1321-1328). ).                                 Summary of the Invention   The present invention eliminates the need for a purified RNP complex or a purified viral RNA polymerase A method for producing a viral transfectant. Therefore, The clear method dramatically simplifies the preparation of viral transfectants.   C) containing only viral nucleoprotein (NP) and nucleic acid transcripts (eg, RNA) When the ills RNP complex is cultured with herpes simplex virus, the virus It is a surprise of the present invention that it is sufficient to mediate the replication of (eg, influenza). It was a great discovery.   Thus, in one embodiment, the present invention provides a viral transfectant (A virus carrying a preselected ribonucleic acid). The method comprises the steps of: i) isolating the ribonucleic acid (nucleic acid transcript) from the virus; Combined with a nucleoprotein (NP) to form a synthetic ribonucleoprotein complex (RNP) Transfecting the host cells with the synthetic ribonucleoprotein complex And iii) maintaining the host cells under conditions that permit virus replication (culturing). Process. Replication of the virus from the RNP complex in the host cell depends on the virus of interest. Host cells (eg, recombinantly engineered viral RNA polymerase) Or more preferably a helper virus By providing a host cell infected with The host cell is Before, during, or after transfection with the ribonucleoprotein complex (RNP) It can be infected with a helper virus. Preferred helper viruses are live attenuated vaccines Chin parent strain (eg, A / Leningrad / 57, or A / Ann Arbor6 / 60), or laboratory Contains wild-type virus (eg, A / PR / 8/34 or A / WSN / 33) adapted to .   The isolated viral nucleoprotein (NP) is preferably isolated from other viral proteins. Isolated from the protein, specifically containing the viral RNA polymerase protein Absent. Preferably, the isolated viral nucleoprotein is recombinantly expressed. Than Preferably, a eukaryotic expression system (eg, an SP19 cell in a baculovirus vector) is used. Vesicles). Preferred viral nucleoprotein Contains NPs from influenza, parainfluenza, or measles virus . Influenza (eg, A, B, or C) NP is more preferred, and A Type Influenza NP is most preferred.   The host cell can be any cell capable of culturing a viral transfectant. Cells. Particularly preferred cells are eukaryotic cells, hen eggs or kidney Cells (eg, MDCK cells) are most preferred. Of the purpose to be transfected Viruses typically contain RNA proteins that contain a nucleoprotein complex (RNP) or equivalent structure. Including irs. Particularly preferred viruses include the genus Orthomyxovirus, and more Preferably, influenza, and paramyxoviruses (eg, Enza and measles). Influenza is most preferred.   In a particularly preferred embodiment, the virus of interest is an influenza virus Yes, nucleoprotein is recombinantly expressed with virtually no viral RNA polymerase Influenza nucleoprotein was released. And the host cell is influenza Egg or kidney cells infected with helper virus.   The isolated NP protein, when combined with RNA, is substantially a ribonucleic acid. Protects RNA from reases and directs RNA to the nucleus of host cells. Therefore, another implementation In embodiments, the present invention directs ribonucleic acids (eg, RNA transcripts) to the nucleus of a host cell. Provide a way to enter. The method comprises the following steps: i) Ribonucleic acid and virus Synthetic ribonucleoprotein complex in combination with the isolated nuclear protein (NP) (RNP) formation; and ii) triggering the host cells with the synthetic ribonucleoprotein complex. The process of transfecting. This method further sensitizes the host cells with the helper virus. Dyeing may be included. Any of the viral nucleoproteins (NPs) listed above, and Any RNA transcripts, host cells, and transfections discussed in the The law is appropriate.   In yet another embodiment, the invention relates to a method for producing a ribonucleic acid (eg, an RNA transcript) Synthetic ribosomes that can be combined to thereby mediate viral replication in host cells A transcript comprising an isolated viral nucleoprotein that forms a nucleoprotein complex. A sfection composition is provided. Any NP above and herein and RNA transcripts are appropriate.   The method of the present invention provides for transfectant host cells (ie, expressing heterologous nucleic acids). Or transgenic virus (viral transfectant) Can be used to produce. Thus, in another embodiment, the present invention provides Host cells or production transfected with ribonucleoprotein complex (RNP) Provided (derived) virus. Here, RNP is a ribonucleic acid (for example, R NA transcript) in combination with the isolated viral nucleoprotein (NP). Up Any of the viruses, host cells, NPs, and RNPs described above and herein are suitable. is there. In one embodiment, the RNA transcript is presented by a virus Expresses an antigenic determinant of a human pathogen (eg, gp120 or a subsequence thereof). The virus can be a dead (cannot infect or replicate) or live attenuated virus .   Finally, the present invention also provides a kit for performing the above method. In detail Kits are used to prepare host cells containing preselected RNA transcripts, and Are suitable for the preparation of viral transcripts. This kit contains a synthetic ribonucleic acid Includes a container containing the protein complex (RNP). Here, RNP is a ribonucleic acid (eg, RNA Transcript) in combination with the isolated viral nucleoprotein (NP). Kick The method may further include one or more of the following: using the methods described above and below herein. Instructions, buffers, host cells, culture media, helper virus, etc.Definition   As used herein, the term "virus of interest" refers to the insertion of a nucleic acid transcript into it. It is intended to refer to the virus that is performed. Therefore, the target virus is the target virus. Distinguished from helper viruses that act to promote the replication of ills.   A "helper virus" provides functions necessary for the replication of a defective virus. A virus to be provided. In the present invention, the helper virus is a synthetic ribonucleoprotein. Viruses of interest from protein complexes (eg, NPs combined with nucleic acid transcripts) Provides the functions necessary for the duplication of software. The term helper virus is also simply A combination of components of a helper virus that are necessary for the replication of the desired virus. Can be.   As used herein, the term "synthetic ribonucleoprotein complex" or "synthetic RN" `` P '' refers to its associated nucleic acid polymerase protein prior to incorporation into the complex. Refers to a ribonucleoprotein complex that includes a nucleoprotein (NP) isolated from the protein. RNP Complexes generally contain RNA, nucleoprotein (NP), and viral RNA polymerase It will be appreciated that it is used to refer to protein associations. However, in this specification The RNP complex used in is simply the association of nucleic acid transcripts and nuclear proteins (NPs) This can be done before, during, or after transfection of the host cells with RNP. NP / RNA RNP and viral RNA polymerase protein in any Quality (eg, provided by a helper virus) Recognize that it provides an RNP that can mediate the replication of ills.   When used in the context of an RNP complex, the term “complex” refers to a specific structural It is not intended to imply a relationship, but simply to a meeting that allows the replication of the virus of interest. 5 shows the presence of RNP components (especially NP and RNA) in the cell.   When used in reference to a cell, the term "recombinant" means that the cell replicates or Is expressed or encoded by a nucleic acid whose source is exogenous to the cell. Expressing a peptide or protein to be expressed. Recombinant cells Genes that are not found in the (non-recombinant) form of the cells can be expressed. Recombinant cells also contain genes found in cells in their native form (this residue). The gene can be re-directed to the cell by artificial means, for example, under the control of a heterologous promoter. ) Can be expressed.   As used with respect to nucleic acid transcripts (ie, cRNA), the term “heterologous” 1 shows a nucleic acid in an active state. Heterologous nucleic acids are modifications of the native nucleic acid (eg, (Including deletions, mutations, and insertions), or Or may be replaced by a nucleic acid sequence not found. Obedience Thus, a virus containing a heterologous nucleic acid is a nucleic acid derived from a source other than the virus, Is a nucleic acid from the same virus (where the nucleic acid is (for example, after being intentionally modified) ) Reintroduced into the virus).   The term "viral transfectant" or "transgenic virus" "Refers to a virus containing a heterologous nucleic acid.   The term "subsequence" in the context of a particular nucleic acid or polypeptide sequence A nucleic acid less than or equal to a particular nucleic acid or polypeptide Refers to the region of the polypeptide.   The term “nucleic acid” or “nucleic acid transcript” refers to a single-stranded or double-stranded form. Deoxyribonucleotide polymer or ribonucleotide poly And the same as naturally occurring nucleotides unless otherwise limited And known analogs of natural nucleotides that can function in the manner described above. In nucleic acids Bases are linked by a linkage other than a phosphodiester bond unless they interfere with the function of the nucleic acid. Can be combined. Thus, for example, in a nucleic acid, the constituent base is a phosphodiester linkage. May also be peptide nucleic acids joined by peptide bonds. In particular, the term "nucleic acid translocation" The term "transcript" or "RNA transcript" refers to heterologous RNA introduced into the virus. Used herein.   When referring to a protein (eg, a viral nucleoprotein (NP)), the phrase “substantially "Purified" or "isolated" refers to other cellular or viral components (tanks). (Usually present with these) means a chemical composition that is substantially free of I do. This can be either dry or an aqueous solution, but is preferably uniform. It is in a good condition. Purity and homogeneity are typically determined by polyacrylamide gel electrophoresis. Determined using analytical chemistry techniques such as electrophoresis or high-performance liquid chromatography Is done. The protein of the predominant species present in the preparation is isolated or substantially Purified. Generally, a substantially purified or isolated protein The quality comprises more than 80% of all macromolecular species present in the preparation. Preferably The protein was purified to show more than 90% of all macromolecular species present It is. More preferably, the protein is purified to greater than 95% and most Preferably, the protein is purified to essentially homogeneity. Where the other giant Offspring are essentially undetectable by conventional techniques.   The phrase “recombinant protein” or “recombinantly produced protein” Produced using cells that do not have an endogenous copy of the DNA capable of expressing the protein Peptide, polypeptide, or protein. The cell has the appropriate nucleic acid sequence Produce a protein because it has been genetically altered by the introduction of   The term "influenza virus" as used herein refers to Orthomyxovir Refers to members of the idae family and includes influenza A, B, and Viruses of type C, as well as tick-borne Orthomyxoviruses, include Not limited. Parainfluenza virus and measles, Paramyxoviridae Members. However, measles is sometimes referred to as measles virus.   Nucleoproteins (NPs) are closely related to nucleic acids (eg, RNA) found in viruses. A protein that is associated with and found in nature. For example, in the flu In turn, nuclear proteins associate with RNA to form helical nuclear capsids. Thus, nucleoproteins are similar to eukaryotic histone proteins. Flu Enza nucleoprotein is a type-specific antigen, and one of three antigen forms These different forms are human influenza virus types A, B , And C for classification.   A virus “derived from” the synthetic ribonucleoprotein complex of the present invention refers to a synthetic ribonucleoprotein complex of the present invention. A virus replicated from a ribonucleoprotein complex, or a synthetic ribonucleoprotein of the invention It refers to the virus that is the progeny of the virus replicated from the protein complex. Similarly, synthesis Transfect with a viral transfectant vector containing the ribonucleoprotein complex. A transfected host cell is a host cell so transfected, Alternatively, the virus RNP of the synthetic RNP-derived virus of the present invention or the virus Refers to the progeny of such a host cell that still includes any of the body.   When describing a protein, "conservative substitutions" refer to protein activity (eg, Changes in the amino acid composition of proteins that do not substantially alter nucleoprotein activity) U. Therefore, a "conservatively modified mutation" of a particular amino acid sequence is defined as a protein Amino acid substitutions for these amino acids that are not important for Similar properties such that acid substitution does not substantially alter activity (eg, acidic, basic , Positive or negative charge, polar or non-polar, etc.) Refers to substitution of amino acids. The table of conservative substitutions providing functionally similar amino acids is It is well known in the art. The following six groups each have conservative substitutions with each other: Including the amino acid substitution:         1) Alanine (A), serine (S), threonine (T);         2) aspartic acid (D), glutamic acid (E);         3) Asparagine (N), glutamine (Q);         4) Arginine (R), lysine (K);         5) 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. further A single amino acid or a small percentage of amino acids in the encoded sequence Individual substitutions, deletions, or additions that alter, add, or delete Mutations that have been artificially modified. "                               Detailed description   The present invention relates to a virus comprising a heterologous nucleic acid sequence or subsequence; A new method of producing a pectant is provided. Virus transfectants Typical production of (especially influenza virus transfectants) Purified protein and synthetic RNA transcripts from the lion ribonucleoprotein (RNP) core Combination, followed by a trip to cells previously infected with a helper virus. Transfection (Enami et al., (1991) J. Virol. 65: 2711-2713). these Purified proteins typically include components of the ribonucleoprotein complex (RNP) including.   The ribonucleoprotein complex contains RNA, nucleoprotein (NP), and viral RNA proteins. Heteroaggregates of molecules containing limerase proteins (eg, PA, PB1, PB2) . Due to the complex structure of RNP and the close association between protein and RNA components Purification of the RNP complex protein from virions, in part, has multiple gradient fractions Stations with the largest workforce of virus transfection procedures that require Show one of the faces (Enami et al., Supra). In addition to requiring considerable effort, Previous virus transfection methods were also complicated. Because For efficient viral transfection, in addition to viral nucleoprotein It was thought that viral RNA polymerase was necessary. Virus R The supply of NA polymerase requires a sophisticated cloning system in addition to nuclear protein (NP). Or requires time-consuming and laborious isolation and purification procedures (See, for example, Kimura et al. (1992) J. Virol., 1321-1328).   Thus, for the formation of a nucleoprotein complex with a nucleic acid (eg, RNA), Rus nucleoprotein (NP) alone is sufficient, and helper virus or This nucleoprotein complex, which uses an equivalent replication mechanism, is used for viral replication in host cells. It was a surprising finding of the present invention that it was sufficient to achieve the production. Therefore The present invention relates to the size required for the purification of ribonucleoprotein complex (RNP). Eliminate processes that require labor. Instead, the nucleic acid transcript is Can be simply combined with the expressed (eg, recombinantly expressed) nuclear protein; Is transfected into host cells and the target virus takes up the nucleic acid transcript. Replicating while watching can be achieved. Purified nuclear protein or nuclear protein The protein / RNA complex can be provided as a stock reagent, whereby the virus Enables routine and rapid production of transfectants.   In addition to the dramatic reduction in the effort required to prepare virus transfectants, The elimination of the need for purified RNP complex proteins Eliminates the possibility of introducing viral genes that may be present in the RNP protein. This This means that no wild-type gene exists except for those that were intentionally introduced, Allow the maintenance of the phenotype of the effector (eg, attenuated influenza). Make it easier.I. Virus transfection   As described above, the present invention relates to a virus transfectant (a virus containing a heterologous nucleic acid). Irs) is provided. In general, the methods of the present invention involve the synthesis of Nucleic acid transcripts and the isolated viral nuclear proteins are used to form the RNA complex (RNP). Combining with protein (NP). The complex is then Mediates viral replication and, in combination with Transformation into the host cell results in a multiplicity of each virion containing a copy of the nucleic acid transcript. Be effected.   A helper virus or equivalent replication mechanism requires all the proteins necessary for virus replication. That essential protein components of the target virus can be supplied. It was a surprising discovery of the present invention that only Rus nucleoprotein (NP) was present. Less than Previously, other virion proteins, especially RNA-specific RNA polymerases (eg, PA , PB1, PB2) are required for successful viral replication (See, for example, US Pat. No. 5,166,057).   Need for RNP polymerase protein from transfected virus Elimination provides a highly simplified means of producing transfected virus I do. Generally, the method involves the following steps:         i) Nucleic acid transcription to form a synthetic ribonucleoprotein complex (RNP) Combine the product (eg, RNA) with the isolated viral nucleoprotein (NP) The step of causing;         ii) Transfection of host cells with synthetic ribonucleoprotein complex About; and         iii) keep the above host cells under conditions that allow replication of the virus Process.   Of course, the preferred (transfectant) virus of interest is one whose replication is low. It is a virus mediated at least in part by nuclear proteins (NPs). like this Viruses are well known to those skilled in the art, and include, for example, influenza virus Such as orthomyxovirus, and parainfluenza virus, measles virus And members of the myxovirus, including paramyxoviruses such as virus. other Suitable viruses include Rhabdovirus (eg, rabies virus) Can be In a particularly preferred embodiment, the virus of interest is influenza virus Ills (eg, influenza A, B, or C virus).   As described above, the synthetic nucleoprotein complex is associated with a helper virus. Together, used to transfect host cells that mediate viral replication Is done. The virus culture is maintained under standard conditions, and The effector-containing host cells are selected according to standard methods in the art. Selected and isolated.   The various steps of the method are described in detail below.II . Preparation of synthetic nucleoprotein complex   As mentioned above, the method of the invention involves the preparation of a synthetic nucleoprotein complex. Honcho As used in the textbook, the synthetic nucleoprotein complex is a nucleic acid and a viral NP protein. And associations. In a preferred embodiment, the synthetic nucleoprotein The complex is composed of ribonucleic acid (eg, RNA transcript) and influenza, With an NP protein selected from Luenza, measles or rabies (most preferred Include aggregates (with influenza NP). Particularly preferred synthetic ribonucleoprotein The complex does not associate with and contains the nucleic acid polymerase of the virus of interest. Absent. Preparation of nucleoproteins, nucleic acid transcripts, and synthetic nucleoprotein complexes It is described below.   A) Preparation of viral nucleoprotein   Viral nucleoprotein (NP) is well known to those skilled in the art. For example, influenza In viruses, nucleoproteins are proteins closely associated with viral RNA It is. RNA and NP associate with each other to form a helical nucleocapsid To form Nuclear protein (NP) has a molecular weight of approximately 60 kDa and There are about 1000 molecules in a particle. Influenza NP is a type-specific antigen, It occurs in one of three forms of antigen. These different forms are A, B, and And provides criteria for the classification of human influenza viruses into strains C and C.   Proteins similar to influenza NP are known in other viruses. Thus, for example, parainfluenza, rhabdovirus, morbillivirus, etc. Contain proteins similar to influenza NP (eg, Fields Virology, Second Edition (1990) Raven Press, N.Y.).   The viral nucleoproteins of the present invention are based on the nuclear proteins of their native structure and sequence. Including quality. However, the nuclear proteins of the present invention also have deleterious effects on their activity. No, and in fact, transfection stability or efficiency, virus replication Various properties that can improve various properties, including but not limited to speed, infectivity, etc. NPs modified in the manner described above. Preferred modifications include conservative substitutions as defined above. No. Some modifications facilitate cloning, expression, or purification of nuclear proteins Can be performed. Such variants are well known to those skilled in the art, and Methionine at the amino terminus to provide a start site, or Additional restriction sites or additional codons to either end to create a stop codon Amino acid configuration. One preferred modification facilitates protein purification. Carboxyl-terminated polyhistidines (eg, His₆) (For example, Use a Ni-NTA column).   Other modifications can also be made. Thus, for example, when an amino acid substitution occurs in a nucleic acid transcript To improve the association with the virus, improve the virus packaging, or This can be done to increase the replication rate or viability of the virus. Or Non-essential regions of the NP molecule can be shortened or completely eliminated. Therefore, if there are regions of the molecule that are not themselves related to the activity of the molecule, Can be eliminated or maintain a precise spatial relationship between the active components of the molecule. Can simply be replaced with a shorter fragment supplied.   The viral nucleoprotein is well characterized and contains all amino acids and The corresponding nucleic acid sequence is known (see, for example, Cox et al. (1993) Bull. World. Health. Org., 61: 143-152 and Rota (1989) Nucl. Acids Res., 17: 3595). single Methods for preparing isolated nuclear proteins (NPs) are also well known to those of skill in the art, and Includes isolation from ills culture, novel chemical synthesis, and recombinant expression.   Means for purifying viral nucleoprotein are known to those skilled in the art (eg, US Pat. No. 5,316,910 and WO 092/16619). In a preferred embodiment, Nucleoproteins are used for other viral proteins (particularly viral RNA-specific RNA Other viruses associated with or that contain (eg, PB2, PB1, PA, etc.) Protein).   Alternatively, using amino acid sequence information known to those of skill in the art, Quality (NP) can be chemically newly synthesized in a wide variety of well-known methods. Typical In some cases, relatively short size polypeptides can be prepared using conventional techniques (eg, Merrifield ( 1963) J. Am. Chem. Soc. 85: 2149-2154; Barany and Merrifield, Solid-Phase  Peptide Synthesis; The Peptides, page 3-284: Analysis, Synthesis, Biology, Chapter 2: Special Methods in Peptide Synthesis, Part A; and Stewart et al., (198 4) Solid Phase Peptide Synthesis, 2nd edition, Pierce Chem. Co., Rockford, III), in solution or on a solid support. Various Automated synthesizers and sequencers are commercially available and comply with known protocols. Therefore it can be used. For example, Stewart and Young (1984) Solid Phase Pepti See de Synthesis, 2nd edition, Pierce Chemical Co. Longer size pen Peptides are condensed between the carboxyl and amino termini of a continuous segment Prepared by chemical synthesis of shorter segments that are attached in a reaction, Can result in a single, continuous polypeptide.   In a preferred embodiment, the viral nucleoprotein encodes a polypeptide Produced by recombinant expression of nucleic acids that are subsequently purified using standard techniques. You. Such recombination methods typically involve the insertion of a nucleic acid sequence encoding a viral NP. Providing, inserting the sequence into a vector, transducing the vector into a host cell. Culturing host cells under conditions in which nuclear proteins are expressed And isolating and purifying the expressed nuclear protein.   As shown above, viral nucleoproteins (particularly influenza, Fluenza, and measles nucleoprotein) and thus the nucleic acid sequence It is known. Nucleic acid sequences encoding these peptides can be obtained using standard recombinant techniques. Alternatively, they can be made using synthetic techniques. The present invention uses chemical technology DNA encoding nucleoprotein is obtained, for example, by the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; The phosphodiester method of Brown et al. (1979) Meth. . Enzymol. 68: 109-151; Beaucage et al., Diethylphosphoramidite method (1981) Te tra. Lett., 22: 1859-1862; and U.S. Pat. No. 4,458,066. It can be prepared by any suitable method, including any suitable method.   Given a nucleic acid encoding a viral nucleoprotein (NP), one skilled in the art will be able to ( The same or functionally equivalent (such as nucleic acids encoding nucleoproteins) Various clones can be constructed that contain functionally equivalent nucleic acids. Achieve these goals Cloning methods for, and sequencing methods to confirm nucleic acid sequences, It is well known in the art. Examples of suitable cloning and sequencing techniques, and The instructions sufficient for one of ordinary skill in the art to perform many cloning operations are described in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 15 Volume 2, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., (1989) Molecul. ar Cloning-A Laboratory Manual (2nd edition) volumes 1-3; and Current Protocols in  Molecular Biology, F. M. Ausubel et al., Current Protocols, Greene Publish ing Associates, Inc. and John Wiley & Sons, Inc. (1994 supplement) (Ausubel). Biological reagents and laboratory equipment manufactured by manufacturers Product information also provides useful information in known biological methods. like this Manufacturers include Sigma Chemical Company (Saint Louis, Missouri, USA), R & Dsyst ems (Minneapolis, Minnesota, USA), Pharmacia LKB Biothchnology (Piscataway , New Jersey, USA), Clontech Laboratories, Inc. (Palo Alto, California, USA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wisconsin, U.S.A.) SA), Glen Research, Inc., Gibco BRL Life Technologies, Inc. (Gaithersber g, Maryland, USA), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, B uchs, Switzerland), Invitrogen (San Diego, California USA), and Applied  Biosystems (Foster City, California, USA), and other trades known to those of skill in the art. Includes industrial sources.   The nucleic acid sequence encoding the nucleoprotein is described in E. coli. species, including E. coli and other bacterial hosts It can be expressed in various host cells. However, in a preferred embodiment, the invention The nucleoproteins used in the present invention include, for example, yeast, COS, CHO, and HeLa cells. A variety of eukaryotic cells, such as cell and myeloma cell lines, and insect cell lines Expressed in   Recombinant NP gene can work with appropriate expression control sequences for each host Linked to E. For E. coli, this is more like a T7, trp, or lambda promoter. Promoter, ribosome binding site, and preferably a transcription termination signal. Including. For eukaryotic cells, this control sequence will Preferably, enhancer derived from immunoglobulin gene, SV40, cytomegaloyl Etc., as well as containing a polyadenylation sequence, and And acceptor sequences.   Plasmids encoding the viral nucleoprotein of the present invention are E. coli. About coli For calcium chloride transformation, and for eukaryotic cells calcium phosphate treatment The selected host cells can be isolated by well-known methods such as electrophoresis or electroporation. Vesicles. Cells transformed with the plasmid are not included in the plasmid. Antibiotics conferred by included genes (eg, amp, gpt, neo and hyg genes) It can be selected according to the resistance to the substance.   Once expressed, the recombinant nucleoprotein is purified by ammonium sulfate precipitation, affinity Standards in the field, including column, column chromatography, gel electrophoresis, etc. Standard procedures (in general, R. Scopes, Protein Purification, Springer-Verlag, N . Y. (1982), Deutscher, Methods in Enzymology Volume 182: Guide to Protein Pu rification., Academic Press, Inc. N. Y. (See (1990)). Can be done. Substantially pure compositions of at least about 90-95% homogeneity are preferred And a uniformity of 98-99% or more is most preferred. As desired Once purified, partially or homogeneously, the nucleoprotein is then purified according to the invention. It can be used to form a synthetic nucleoprotein complex according to the method.   Those skilled in the art will recognize that after chemical synthesis, biological expression, or purification, Recognize that they may possess a conformation that is substantially different from their natural conformation . In this case, denaturing or reducing the polypeptide, followed by the polypeptide It may be necessary to allow the fold to refold into the preferred configuration. Ta Methods for reducing and denaturing proteins and for inducing refolding are known in the art. Is well known to the public. (Debinski et al., J. Biol. Chem., 268: 14065-14070 (1993); Krei tman and Pastan, Bioconjug. Chem., 4: 581-585 (1993); and Buchner et al., Ana. l. Biochem., 205: 263-270 (1992)). For example, Debinski et al. The denaturation and reduction of inclusion body proteins in Nidin-DTE is described. Then tongue Protein is refolded in a redox buffer containing oxidized glutathione and L-arginine It is folded.   One of skill in the art would be able to interact with the recombinant nucleoprotein without losing its biological activity. And that modifications can be made. Some modifications may be associated with nuclear protein It can be done to facilitate cloning, expression, or purification. Such a modification It is well known to those skilled in the art and can be added, for example, to the amino terminus to provide a start site. Added methionine, or a conveniently located restriction enzyme site or stop codon. Includes additional amino acids located at either end to make.   In a particularly preferred embodiment, the NPs used in the present invention are U.S. Pat. No. 910 and WO 092/16619. Used in Spodoptera frugiperda (S19) insect cells.   B) Preparation of nucleic acid transcript   Virtually all nucleic acids are used as nucleic acid transcripts for use in the present invention Can be done. Typically, it expresses a non-naturally occurring (eg, heterologous) gene product The infectivity or disease of the virus, which expresses endogenous gene products at elevated levels A nucleus that alters the origin or acts as a detectable marker An acid transcript is selected. The nucleic acid transcript can be expressed in a suitable host cell system, or Recombinant virus expressing, packaging and / or displaying a heterologous gene product Service. Gene products can be used in a variety of contexts (eg, vaccine formulations ) Can be used advantageously. When a heterologous gene is encoded by a transcript , The gene describes its effect on the virus, its effect on the host cell, Or simply a virus / host cell, particularly suitable for the expression of the gene product, Or the fact that it provides the optimal system.   Myxoviridae genome RNA is negative sense (complementary to mRNA sense). Therefore, if the nucleic acid transcript expresses the gene product, it is a negative sense It is also a product of. RNA transcripts are prepared by any of a number of methods well known to those skilled in the art. Can be done. In a preferred embodiment, the RNA template is a DNA-specific RNA poly Use a merase (for example, bacteriophage T7, T3, or Sp6 polymerase). Prepared by transcription of the appropriate DNA sequence. Using the flu For example, influenza virus polymerase binding site / promoter complementation (Ie, the complement of the 3 'end of the influenza genome segment) Constructed to encode the message sense of a heterologous gene sequence upstream of G I do. To rescue the virions, the heterologous coding sequence is Flanked by both 3 'and 5' end complements of the genomic segment It may be suitable. After transcription using a DNA-specific RNA polymerase, the resulting RNA The plate encodes the negative polarity of the heterologous gene sequence, and the viral RNA VRNA end sequences that enable specific RNA polymerases to recognize transcripts Including. Detailed protocol for the production of RNA transcripts (templates) No. 5,166,057.   However, the nucleic acid transcript need not encode the protein to be expressed. An example For example, a nucleic acid transcript may be an antigen that targets host cell DNA or mRNA by itself. It can act as a sense molecule. In addition, nucleic acid transcripts can include deletions, insertions, point mutations, And the function of the virus itself (eg, Infection, pathogenicity, etc.). These various functions are described in the sections below. Discussed in Section VI.   Primarily, RNA transcripts make up all or part of any component of the viral genome. Can be replaced. Thus, for example, in the case of influenza virus, Any one of the eight gene segments or any of the eight segments Any one of them may be replaced by a foreign sequence. But the necessary part of this equation is Defective virus (defective due to deletion or alteration of normal viral gene products) Is the ability to multiply.   There are many possible approaches to avoid this problem. In short, this These approaches involve culturing, deficient with helper virus expressing the defective gene. Culture and growth of defective viruses in cell lines engineered to complement genes Selection of natural host range systems to enable and co-culture with wild-type virus (US Patent 5,166,057), but not limited thereto, and Section I below. Considered in more detail by V.   For example, in the case of influenza, the nucleic acid transcripts are PB2, PB1, PA, NP, HA , NA, NS, or M gene segments can be replaced in whole or in part. PB2, PB1 , The gene segments encoding the PA and NP proteins have 24 to 45 non- Translated nucleotides and a single with 22-57 untranslated nucleotides at the 3 'end Including open reading frame. Exogenous inheritance into any of these segments Insertion of the codon sequence can be a complete replacement or partial replacement of the viral coding region with a foreign gene. Can be achieved by any of the following substitutions:   HA and NA proteins encoded by separate gene segments are Is a major surface glycoprotein. Therefore, these proteins are It is a major target for the humoral immune response. They are all flu The most extensively studied of the irs proteins. Because these tongues This is because both three-dimensional structures of the protein have been analyzed.   The three-dimensional structure of H3 hemagglutinin, along with sequence information for a number of variants, Enables the elucidation of antigenic sites in molecules (Webster et al., 1983, pp 127-16 0: Genetics Of Influenza Virus, edited by P.Palese and D.W.Kingsbury, Springer-V erlag, Vienna). These sites have four dispersed non-overlapping regions on the surface of HA. Classified into areas. These regions are highly variable and have insertions and deletions Has also been shown to be acceptable. Therefore, some of the foreign proteins Substitution at these sites (eg, site A; amino acids 122-147 of A / HK / 68HA) It can provide a strong humoral response to foreign proteins. Different approaches And the foreign peptide sequence is the antigenic portion without deleting any viral sequences. It can be inserted within a place. The expression product of such a construct is Recombinant virus in a vaccinated host Can avoid the problem of plant growth. Intact HA components with substitutions only at antigenic sites The offspring may enable HA function and thus allow the construction of a viable virus. Therefore, the virus is propagated without the need for additional helper functions obtain. Of course, this virus has been attenuated in other ways, Such dangers should be avoided.   Other hybrid constructs are made to express the protein on the cell surface Or can be made to allow them to be released from cells . Like surface glycoproteins, HA is an amino-terminal truncation required for transport to the cell surface It has a possible signal sequence and a carboxy terminal sequence required for membrane anchoring. Cell surface These HA signals are used to express intact foreign proteins on the surface. To make hybrid proteins. Or transport If only the signal is present and there is no membrane anchoring domain, the protein is It can be excreted out of the cell.   In the case of NA protein, the three-dimensional structure is known, but the antigenic site is located on the molecular surface. Spreading and overlapping. This means that the sequence is inserted into the NA molecule, and If it is expressed on the outer surface of NA, it indicates that it appears to be immunogenic. Sa Furthermore, as a surface glycoprotein, NA shows two significant differences from the HA protein. You. First, NA does not contain a cleavable signal sequence; The sequence acts as a membrane anchoring domain. As a result, and between the NA and HA The difference between the two is that NA is oriented at the amino terminus in the membrane, while HA is To be directed at the carboxy terminus. Therefore, in some cases It may be advantageous to construct a hybrid NA protein. Because the fusion The protein is oriented opposite the HA fusion hybrid.   The unique properties of the NS and M segments are the characteristics of the other six influenza viruses. When compared to gene segments, these segments have at least two It encodes a protein product. In each case, one protein is Is encoded by mRNA that is collinear with genomic RNA, while the other protein is , Coded by the spliced message. But splice NS1 and N1 because the donor site occurs within the coding region of the collinear transcript. The S2 protein has the same 10 amino acid amino terminus, while M1 and M2 are identical Has the amino terminus of 14 amino acids.   As a result of this unique structure, the recombinant virus can seg Construct in place, while leaving the second product intact. Can be For example, replacement of most of the NS2 or M2 coding region with a foreign gene product ( (Conserving the splicing acceptor site) is an intact NS1 or M1 It can result in expression of proteins and fusion proteins in place of NS2 or M2. Ah Alternatively, the foreign gene does not affect either NS1 or NS2 expression and It can be inserted within a gene segment.   Most NS genes have substantial duplication of the NS1 and NS2 reading frames. However, certain natural NS genes do not. For example, NS from A / Ty / Or / 71 In the gene segment, the NS1 protein is a nucleoside of the NS gene segment. Terminating at tide position 409, while the splicing acceptor site for NS2 is At nucleotide position 528 (Norton et al., (1987), Virology 156: 204-213). So Therefore, the foreign gene does not affect any of the proteins, and Between the stop codon of the region and the splicing acceptor site of the NS2 coding region. Can be placed. To ensure protein production, a splice at the 5 'end of the foreign gene sequence It may be necessary to include a licensing acceptor site (this is Encodes a hybrid protein containing an amino terminus). In this method, Recombinant virus should not be defective and requires helper function It should be able to grow without.   Insertion and / or insertion of all or part of the various viral genes mentioned above These substitutions can be made according to standard methods well known to those skilled in the art. Preferred embodiment In one embodiment, the RNA transcript of the invention is prepared by using PCR-directed mutagenesis. Can be Structure of RNA transcripts that are appropriate for the production of viral transfectants Detailed methods of construction can be found in US Patent 5,166,057.C) Synthesis of nucleoprotein (NP) and transcript to form a synthetic nucleoprotein complex combination   RNA transcripts can be obtained before, during, or after transfection by nuclear protein (N P). However, nuclear proteins are not Provides some protection from RNA transcripts to RNA transcripts, In a preferred embodiment, NP is combined with the RNA transcript prior to transfection. Be combined.   RNA transcripts and nucleoproteins are transfected by simple mixing. Can be combined before the action. Similarly, transcripts and nucleoproteins By placing both components in the transfection mixture, the transfected Can be combined during the session. These components consist of one component followed by the other. Simply transfected, and these components when present in the host cell. By allowing them to be combined, they can be combined after transfection. Can be combined.   However, in a particularly preferred embodiment, the formation of the synthetic nucleoprotein complex is Occurs in the preparation of nucleic acid transcripts. Thus, for example, the nucleoprotein is converted into a reaction mixture ( For example, amplification, reverse transcription, etc.). Nucleic acid transcripts in this reaction mixture Is produced, thereby facilitating rapid association of the NP with the transcript. NP is RNA This approach improves stability because it helps prevent degradation.   For example, in one approach, nucleic acid transcripts are expressed in the presence of a nucleoprotein. By reverse transcription from a cDNA clone encoding the desired RNA transcript in Produced. Typically, the cDNA is such that transcription can begin at the correct nucleotide. Behind a promoter located at (eg, the T7 RNA polymerase promoter) Placed in Reverse transcription is performed according to standard methods known to those skilled in the art (eg, See, eg, Sambrook et al., Supra, Ausubel et al., Supra; Van Gelder et al., (1990) Proc. Natl. Aca. d.Sci.USA, 87: 1663-1667; and Eberwine et al., Proc.Natl.Acad.Sci.USA, 89: 301. See 0-3014). This approach is illustrated in Example 1.III . Transfection of host cells   The synthetic nucleoprotein complex is transfected into a host cell where the complex Can be duplicated. Virtually any host cell can be transfected in this manner. Can be controlled. However, in a preferred embodiment, the host cell is Host cells that are compatible for infection and culture of the virus. So, for example, Preferred if the transfected virus is an influenza virus Host cells include cells commonly used to culture influenza . Such cells include cells of the amnion of hatched eggs, rhesus monkeys, baboons, chickens, Include, but are not limited to, tissue cultures of kidney tissue from various other species. Particularly preferred cells are, for example, primary chicken kidney (PCK) cells, Madin-Darby dogs Includes kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells, and the like.   As mentioned above, in one embodiment, the method of the present invention merely comprises Providing host cells containing heterologous nucleic acids, rather than for preparing sfectactants Used to In this regard, synthetic nucleoprotein complexes are simply heterologous nuclei. It can be used to direct the acid to the nucleus of the host cell. Host cells in this case Is any cell in which it is desired to transfect with a heterologous nucleic acid transcript. Can get.   The synthetic ribonucleoprotein complex can be prepared from any of a number of transfectants known to those of skill in the art. Can be transfected into a host cell according to the method of application. Suitable transformer Transfection methods include calcium chloride transformation (eg, for E. coli), Phosphate treatment, electroporation, and for eukaryotic cells, Or including ballistic transfection (eg, fast gold microspheres) , But not limited to them. In a particularly preferred embodiment, the host cell is electo ROPORATION or DEAE-Dextran DMSO protocol (eg, Al-Molish et al.) , (1973) J. Gen. Virol., 18: 189-193; and Lopata et al., (1984) Nucl. Acids R es. 12: 5707-5717). Is most preferred.IV . Virus culture   After transfection of the cells with the synthetic ribonucleoprotein complex, the cells are Preferably, it is cultured under conditions that allow the replication of the virus of interest. Those skilled in the art The ribonucleoprotein complex (RNP) containing only A and NP is commonly used Not enough to replicate alone, and additional “replication mechanisms” are typically needed Understand that it is. Furthermore, if the nucleic acid transcript contains a heterologous nucleic acid, Are often "missing." Because normal viral gene products are often lost Because they are changed or changed.   As mentioned above, a number of possible approaches exist to avoid this problem . Mutants (transfectants) of a virus (eg, influenza) Can be grown in cell lines constructed to constitutively express a "defective" gene ( See, for example, Krystal et al., (1986) Proc. Natl. Acad. Sci. USA 83: 2709-2813 That, they say, influenza viruses that are deficient in PB2 and NP proteins. Mutants are constructed to constitutively express polymerase and NP proteins Grown in cell lines). Engineered to express viral proteins These cell lines complement the deficiency in the recombinant virus, thereby (Eg, Kimura et al., (1992) J. Gen. Viro). l., 73: 1321-1328).   Alternatively, certain natural host range systems are used to propagate the recombinant virus It may be possible. An example of this approach is the natural influenza isolate CR43-3. Involved. The virus was passaged in primary chicken kidney cells (PCK) Madin-Da grows normally in some cases, but is a natural host for influenza does not proliferate in rby canine kidney cells (MDCK) (Maassab and DeBorde (1983 ) Virology, 130: 342-350). This virus is caused by a deletion of 12 amino acids. Encodes the deleted NS1 protein. PCK cells supplement defective NS1 protein Some, either complete or can completely replace the missing protein Including the activity of.   As described above, a synthetic ribonucleic protein that completely lacks the RNA polymerase of the virus of interest. The protein complex (RNP) is completely duplicated when complemented by a helper virus. What could be made was a surprising discovery and advantage of the present invention. The present invention Purified RNP protein or recombinantly engineered host cells for virus propagation Eliminate the need for   Thus, in a preferred embodiment, the virus transfectant is wild-type. Propagated through co-culture with type or helper virus. This is simply a recombination Take the virus and synthesize cells with RNP and wild-type (eg, vaccine strain) Alternatively, it can be performed by co-infection with a helper virus. Wild type or f Ruper viruses complement defective viral gene products and helper or field It should allow the growth of both live and recombinant viruses.   If the co-culture is with the wild-type virus, this is the influenza virus (Nayak et al., (1983): Genetics of Influenza)  Viruses, P.S. Palese and D. W. Kingsbury, Springer-Verlag, Vienna, 255 -279 pages). In the case of interference-defective virus, the majority of the propagated virus is wild-type virus. The conditions can be modified so that the particles are defective rather than ills. Therefore, this The approach may be useful in generating high titer stocks of recombinant virus. However, these stocks necessarily contain some wild-type virus.   In a particularly preferred embodiment, the synthetic RNP is cultured in the presence of a helper virus. Is done. Suitable helper viruses are well known to those of skill in the art and A / PR8 / 34 or A / WSN / 33) or live attenuated vaccine strains (eg, A / Leningra d / 57 or A / Ann Arbor / 6/60).   The host cell can be a synthetic ribonucleic acid into cells according to standard methods well known to those skilled in the art. Before, with, or after transfection of the protein complex, Ruper virus (see, for example, Enami M. and Palese, P. (1991) J. Am. Virol., 65: 2711). Similarly, to produce according to the method of the present invention The transgenic virus also has the appropriate complementing wild-type or helper virus Cultivated in the presence of the yeast.   In yet another approach, all eight influenza virus proteins Infection of host cells with synthetic RNPs encoding protein is infectious transgenic (Transfectant) This results in the production of virus particles. This system is a must for the selection system. Eliminate the need. Because all recombinant viruses produced have the desired genotype Because it is.   Synthetic ribonucleoprotein complexes or viral transfectants are standard Cultures, including embryonated eggs and tissue cultures.   A) Culture in embryonated eggs   The virus (eg, influenza) is in the amniotic membrane or allantoic of hatched eggs for 10-12 days. It can be cultured by species. The virus is absorbed from amniotic fluid into cells of the amniotic membrane and Here they replicate and release the newly formed virus back into the amniotic fluid. Two After three days of incubation, the virus may be present in amniotic fluid at high titers, and Aliquots of the recovered amniotic fluid are collected from chickens, turkeys, guinea pigs, Can be detected by adding to human red blood cells and observing hemagglutination. Detailed protocols for virus culture in embryonated eggs are well known to those skilled in the art, And can be found in standard references (eg, Fields Virology (previous Out)).   B) Tissue culture   The virus of interest can also be cultured in eukaryotic tissue culture according to standard methods. Can be done. For example, influenza can include dogs, rhesus monkeys, baboons, chickens, Alternatively, they can be cultured in kidney tissue cultures from various other species. Especially preferred Cells include, for example, H292 cells, primary chicken kidney (PCK) cells, Madin-Darby dog kidney Includes kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells, and the like.   Absorption (transfection) and incubation of virus-infected cells After, the newly produced virus can be detected in a number of ways. One appro Free virus released into the maintenance medium of the tissue culture can be detected. (Eg, by hemagglutination with red blood cells). Alternatively, the virus Because they are released slowly from the cell surface, erythrocytes adhere directly to these infected cells (red Blood cell adsorption), and can be detected under a microscope. Viral fiber in tissue culture Methods of retention and propagation are known to those of skill in the art (eg, Concepts and Proceedur es for Laboratory-Based Influenza Surveillance, Dept. Health and Hum. Se r., Centers for Disease Control (1982)).V. Transfectant selection   Viral transfectants (transgenic viruses) It can be identified and selected according to known standard methods. Typically, this is Cultured cells (or culture supernatant) positive for ils transfectants Identifying the host cells and / or cells containing the virus from the cells or supernatant. Or selectively growing the virus itself. Identification of host cells or supernatant Is obtained by means well known to those skilled in the art, and typically by heterologous RNA (transcript) or Includes direct or indirect detection of the presence or absence of the product.   Direct detection involves detection of the heterologous RNA (or DNA transcribed therefrom) itself. It is specific for heterologous RNA (or DNA transcribed from it) or its subsequences Nucleic acid affinity columns (including complementary probes), RNA (or Is the amplification of a particular target sequence or subsequence in the DNA transcribed from it (eg, , Via PCR), restriction analysis, etc. Can be achieved. In one preferred embodiment (exemplified by Example 1 ), Heterologous RNAs are described in Klimov et al., (1995) J. Med. Virol. Meth., 52: 41-49, PCR Detected by restriction analysis.   In other embodiments, the virus transfectant or virus tra The host cell carrying the effector is a protein expressed by a heterologous nucleic acid. Can be identified by detecting the presence or absence of the activity of the product or protein product . Thus, a heterologous nucleic acid can include sequences that confer antibiotic resistance, thereby increasing host cell specificity. It may allow for the selection of transfectants by vesicle antibiotic resistance. Or The heterologous nucleic acid may encode a detectable marker as described below.VI . Use of virus transfection.   Synthetic ribonucleoprotein complex and virus prepared according to the method of the present invention Use transfectants to simultaneously host cells with recombinant RNP and virus Transfection allows expression of the heterologous gene product in the host cell, or Alternatively, the heterologous gene may be rescued in the virus particle. Alternatively, heterologous inheritance The offspring are rescued in the transformed host cells and It may allow for complementation and rescue of different heterologous genes.   The resulting expression products and / or chimeric virions can also be used in vaccine formulations. And can be advantageously used. Viral transfector expressing a detectable label Have screened antiviral compounds and studied viral ecology. To provide an effective reporter system for research. Finally, the NP protein Provides protection against ribonuclease activity, so RNA / NP ribonuclease complex The body and the viruses derived from them are effective for the delivery of antisense RNA Provide a simple system. These various uses are discussed more fully below.   A) Expression of heterologous gene product using synthetic RNP complex.   The recombinant template prepared as described above can be used in various ways, Can express the heterologous gene product in the isolated host cell, or An expressing chimeric virus can be made. In one embodiment, the recombinant template The rate was determined using the viral polymer purified as described in Section 6 (below). Combined with the zeta complex, it can produce rRNP that is infectious. Alternatively, recombination The template was a viral polymerase complex prepared using recombinant DNA methods. (See Kingsbury et al., (1987), Virology 156: 396-403). checking). Such rRNPs can be used to transfect appropriate host cells. When used to direct expression of heterologous gene products at high levels. High level Host cell systems that provide for the expression of For example, a cell line superinfected with influenza, influenza virus function Cell lines engineered to complement).   B) Use of a detectable heterologous sequence.   In another embodiment, the heterologous RNA transcript may serve as a detectable label. Or may express a protein that is a detectable label. The detectable label is Allows quantitation of virus in a particular sample or detection of its presence or absence To Viral transfectants with detectable labels can be used in vitro. Or provide an effective indicator for antiviral screening in vivo You. For example, an organism or cell line may have a viral transcript with a detectable marker. It is infected with a spectant and then treated with one or more test compounds. After treatment Of detectable markers and the ratio of control to test samples The comparison provides a measure of the potency of the compound being tested.   The detectable label is a label that can be easily captured (eg, a nucleic acid affinity marker). Specific nucleic acid or polypeptide sequences that can be captured by the ram (eg, N1-NTA His that can be captured using ram₆If it is polyhistidine))) Rus transfectants can be used to isolate anti-viral antibodies. This implementation In an embodiment, the organism is virus under conditions that allow the immune response to be raised. The antigen can be administered with a transfectant. Virus transfectants Subsequent capture (by capture of a detectable label) may also result in any binding to the virus. Of antibodies.   Detectable labels are also used to detect one or more viral strains (eg, influenza Monitor in vitro or in vivo (eg, detection and / or determination). Amount). This can be infectious and / or infectious, alone or in combination with other viruses. Or allows for a quick assessment of the pathogenicity of a particular virus. This is on the host and And the evaluation of the effects of mutations that produce various virus strains on other virus strains. You. Other uses for viral transfectants with detectable labels are known in the art. Is known to those skilled in the art.   Detectable labels suitable for use in the present invention include, for example, certain probes. Hybridization to the capture sequence or RFLP or other finger -Any nucleic acid sequence that is detectable (e.g., via printing techniques), or Including, but not limited to, any polypeptide sequence that is detectable; an enzyme (Eg, horseradish peroxidase, alkaline phosphatase, and other Enzymes commonly used in ELISAs), and can be specifically detected and / or Or other polypeptide sequences that can be captured (eg, using an N1-NTA column). Poly His that can be caught). Means for detecting such labels are well known to those skilled in the art. .   C) Use of virus transfectants for vaccine formulation.   The resulting expression products and / or chimeric virions can be used in viral formulations. Can be used. The use of recombinant influenza for this purpose is particularly attractive. You. Because influenza shows tremendous strain variability, vaccine formulations Because it is possible to build a huge repertoire. To construct chimeric virus The ability to choose from thousands of influenza mutants for Eliminates the problem of host resistance encountered when using other viruses. further Influenza stimulates active secretory and cytotoxic T cell responses, Presentation of foreign epitopes in the influenza virus background , Can provide secretory and cell-mediated immunity induction.   Virtually any heterologous gene sequence can be used in vaccines of the invention for use in vaccines. It can be constructed within the mela (transfectant) virus. Preferably, any Epitope that induces a protective immune response against various pathogens, or Matching antigens are generated by or as part of a viral transfectant. Can be revealed. For example, to a chimeric virus of the invention for use in a vaccine Heterologous gene sequences that can be constructed include, but are not limited to: gp120 Epitopes of human immunodeficiency virus (HIV) such as hepatitis B virus Original (HBsAg); herpesvirus glycoproteins (eg, gD, gE); polio Ils VPI; antigenic determinants of non-viral pathogens (eg, bacteria and parasites) There are few, but. In another embodiment, the immunoglobulin gene All or some may be expressed. For example, anti-mimicking such epitopes The variable region of the idiotype immunoglobulin is constructed in the chimeric virus of the present invention. Can be built.   Whether a live or inactivated recombinant virus vaccine Any deviation can be prescribed. Live vaccines are preferred. Because replication in the host, Induces a prolongation of stimuli of a type and magnitude similar to those occurring in natural infections, Therefore, it provides substantially long-term immunity. Such raw recombinant wheat The manufacture of the virus vaccine formulation can be carried out in cell culture or in the allantois of chick embryos. It can be achieved using conventional methods, including purification followed by growth of the ruth.   In this regard, influenza viruses that have been genetically engineered for vaccine purposes ( Vector) may require the presence of attenuating properties in these strains. Hi Current live virus vaccine candidates for use in It is either temperature sensitive or has been passaged, resulting in attenuation. Some (six) genes are derived from avian viruses. Transfer Introduction of the appropriate mutation (eg, deletion) into the RNA transcript used in the New viruses with attenuating properties can be provided. For example, temperature sensitive or cold Certain missense mutations associated with cold adaptation can be made to deletion mutations. these Mutations are more stable than point mutations associated with cold or temperature sensitive mutants. And the return frequency should be extremely low.   Alternatively, a virus transfectant with "suicide" properties has been constructed obtain. Such viruses experience only one or several replications in the host. I do. For example, in influenza, HA cleavage allows replication to resume Is necessary for Therefore, changes in the HA cleavage site are Can produce viruses that replicate in but not in human hosts . When used as a vaccine, the recombinant virus goes through one replication cycle. Trial And induces a sufficient level of immune response, but further progresses in the human host. Does not cause disease.   Recombinant influenza lacking one or more essential influenza virus genes The virus cannot undergo the next round of replication. Such defective viruses are, for example, Reconstituted RNs lacking these genes in cell lines that constitutively express specific genes It can be produced by co-transfecting P. C lacking essential genes Ils replicates in these cell lines, but when administered to a human host One copy cannot be completed. Such preparations are sufficient to elicit an immune response. A limited number of genes can be transcribed (in this failure cycle) and translated. Alternatively, these preparations can be used as inactivated (killed) viruses, vaccines To work, larger quantities of the strain may be administered.   In an inactivated vaccine, the heterologous gene product is Preferably, it is expressed as a concomitant viral component. Such a preparation The advantage of these is that they contain native proteins and kill virus Inactivation by treatment with formalin or other agents used in the manufacture of chin Is not to receive.   In another embodiment of this aspect of the invention, the inactivated vaccine formulation comprises a virus. The transfectants can be prepared using conventional techniques to "kill" the transfectants. Inactivation Vaccines are "dead" in the sense that their infectivity has been destroyed You. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity It is. To prepare an inactivated vaccine, the virus transfectants are Can be grown in culture or in allantois of chicken embryos and purified by zonal centrifugation Can be inactivated by formaldehyde or β-propiolactone, And be pooled. The resulting vaccine is preferably inoculated intramuscularly.   Inactivated viruses are treated with appropriate adjuvants to enhance the immune response. Can be Such adjuvants may include, but are not limited to: No .: mineral gel (for example, aluminum hydroxide); surfactant (for example, Zolecithin, pluronic polyol, polyanion); peptide Oily emulsions; and potentially useful human adjuvants (eg, BCG and Cor ynebacterium parvum).   Many methods involve the above vaccine formulations in the organism of interest (eg, pigs, rabbits). , Goats, mice, rats, primates, including humans) You. These include oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal Road is included. The natural route of transmission of pathogens for which vaccines are designed Via, it may be preferred to introduce the chimeric virus vaccine formulation. Survival If a live chimeric virus vaccine preparation is used, influenza It is preferred to introduce the formulation via the natural route of transmission of the virus obtain. Strong secretion of influenza virus and ability to induce cellular immune response Force may be used to advantage. For example, respiratory tract due to chimeric influenza virus Infection is a powerful component, with concomitant protection against factors causing certain diseases. A urinary immune response can be induced (eg, in the urogenital system).   D) Regulation of intracellular activity   The virus transfectant prepared according to the method of the present invention may comprise a host It can be used to regulate the intracellular activity of a cell. This regulation is Direct or viral trafficking by the activity of RNA transcripts in the effector By the polypeptide encoded by the RNA transcript in the It can be achieved indirectly.   For example, a region of host cell genomic DNA or mRN expressed by a particular gene. An RNA transcript that is complementary (antisense) to A can be provided. RNA to host DNA Hybridization of the transcript can inhibit transcription of the target Hybridization of the mRNA depends on its translation and, therefore, on its mRNA. Can block the expression of the encoded protein. Similarly, transcription regulation Antisense (eg, promoter, start site, repressor, etc.) RNA binding can up-regulate or down-regulate transcription of specific target genes. Can be evolved. Regulate protein expression (eg, up-regulate or The use of antisense molecules to regulate) is well known to those skilled in the art. (Eg Castanotto et al. (1994) Adv. Pharmacol., 25: 289-317, WO 90/13641. And references incorporated herein by reference).   Thus, in one embodiment, the present invention provides an antisense molecule to the nucleus of a cell. Methods and compositions for the delivery of As described above, viral nuclear proteins Proteins are capable of protecting RNA transcripts from ribonucleases, and The product is directed to the nucleus of the host cell. Ribosome containing antisense RNA complexed with NP Nucleoprotein complex provides an effective vehicle for delivery of antisense molecules I do. Similarly, viral transfectants containing antisense RNA also It provides a highly effective delivery system for antisense molecules.   As described above, RNA transcription regulates intracellular activity by expressed proteins The product can be selected. The expressed protein functions as an intracellular signaling pathway Components (eg, estrogen receptor (ER), jun, fos, Elk, ATF2, Kinase, GTP-binding protein (for example, Ras. Rac, etc.), signaling molecule (Eg, JAK, PLC, and PI3K)) or, conversely, signaling It may be a protein that inhibits components of the cade. Such proteins are (Darnell and Baltimore, (1986) Molecular Cell Biol. ogy, Scientific American Books).   E) Use of viral transfectants for gene therapy   The virus transfectants of the present invention also provide suitable vectors for gene therapy. Provide a technician. The virus converts RNA that encodes a protein of therapeutic value. Includes transcripts. In addition, RNA transcripts are targeted to the genome of the host cell (organism). It may include (or encode) a segment that mediates integration of a heterologous gene. Inn Segments that mediate integration with the main genome are known to those of skill in the art and Associated virus (AAV), inverted terminal repeat (ITR), and retroviral LTR Including but not limited to: Selection of appropriate nucleic acid transcripts for gene therapy And designs are well known to those skilled in the art (eg, Freifelder Molecular Biology, 2nd Edition, 1987, Jones and Bartlett, Pub., Boston).VII . Kits for practicing the invention   The invention further provides kits for performing the methods disclosed herein. You. In a preferred embodiment, the kit comprises the above described for performing the method of the invention. A container containing the isolated viral nucleoprotein (NP). The kit also includes How to make a label or viral transfectant, and / or Instructions describing how to introduce ribonucleic acid into a host cell of the invention. NP alone Or a complex with a ribonucleic acid (eg, an RNA transcript). kit May further comprise one or more of the following elements: buffers, host cells, culture media. Earth, helper virus, target virus, etc.                                 Example   The following examples are provided to illustrate, but not limit, the invention. It is not done.                                 Example 1   Transfection assay expressing influenza virus type A NP Nuclear protein (NP) purified from insect cells infected with recombinant baculovirus It was performed using. The result shows that the virion polymerase protein was This indicates that it is not necessary for the engineered RNP complex to be transfected.   Nucleoprotein (NP) is converted to influenza A / Ann Arbor / 6/60 nucleocapsid SF9 (Spodoptera) infected with recombinant baculovirus expressing protein Purified from cells (eg, Rotal et al. (1990) J. Gen. Virol., 71: 1545-1554; R ota et al., US Pat. No. 5,316,910 and WO 92/16619). Freeze the cells again and Suspend in 0.01M Tris-HC1 (pH 7.4) containing 0.001M EDTA and 1M NaCl And dissolved. Dialysis overnight in phosphate buffered saline (PBS, pH 7.3) The lysate supernatant is then applied to the Bio-Rad A-14 Affigel matrix using the manufacturer's method. Monoclonal anti-NP IgG bound to antibodies and equilibrated in PBS (Walls et al. (19) 86) J. Amer. Clin. Microbiol., 23: 240-245). Combined tampa 1M MgCl_TwoAnd 50 mM Tris-HC1 (pH 7.6), 100 mM NaCl, 10 M Mg Cl_Two, 2 mM DTT, concentrated by dialyzing against 50% glycerol. Purity was assessed by SDS PAGE. Aliquot transfection assay Stored at -70 ° C until used.   For transfection assays, 25A-I virus, A / PR / 8/34 virus 7 Genes of Derived and Survival-Attenuated Cold-Adapted A / Leningrad / 47/57 Virus A 7 / 1ts reconstruct with the derived NS gene was used as a helper. Transcription is correct T7 RNA polymerase positioned to start on a unique nucleotide Located behind the promoter, and mung bean nuclease Has a BseAI site downstream to run off transcripts after treatment with A / PR / 8/34 viral NS gene cDNA clone Used as a source of NA. The transcription reaction was performed using 5 μg of purified baculovirus-expressed nuclei. Performed in the presence of protein (NP). The resulting synthetic RNP conjugate was purified by Li et al. (1995 ) 25A-1 reconstitution by electroporation method of Vlrus Res., 37: 153-161. Transfected into MDCK cells infected one hour before with par virus.   After 18 hours of incubation at 34 ° C, confluent After infecting MDCK cells at 37 ° C for 2 days, the culture supernatant was plaqued at 39 ° C for 3 days. . A total of 15 plaques were selected, 9 of which were chick eggs with 10-day embryos And grew. The origin of the NS gene in each clone was determined by PCR restriction analysis ( Klimov et al. Virol. Meth., 52: 41-49). Of the nine clones analyzed Four have the A / PR / 8 / 34NS gene, and thus a functional polymerase It was shown to be unnecessary in transfected synthetic RNPs. DEAE Dext Run DMSO protocol (Al-Molish et al. (1973) J. Gen. Virol., 18: 189-193; and Lo (1984) Nucl. Acids Res. 12: 5707-5717) The experiment yielded a total of 11 clones, only one of which had the A / PR / 8 / 34NS gene. Therefore, there is an element for the production of influenza virus transfectants. It showed better efficiency of crotropation.   Without being bound by any particular theory, the role of NP (protection of RNA transcripts from degradation, And helper virus transfer to the nucleus that provides the necessary transcription and replication machinery ) Are considered double.   In addition to eliminating difficult purification procedures for RNP proteins, Use is made of viral genes that may be present in RNP proteins purified from virions Eliminate the possibility of introducing For live attenuated vaccines, this is the attenuation table. Makes it easier to maintain the archetype. Because, except for what was intentionally introduced, This is because there is no wild-type gene.   The examples and embodiments described herein are for illustration only and are not intended to be limiting. Various modifications or alterations will be suggested to one skilled in the art in light of the present invention, and It is to be understood that it is included within the text and appended claims. This specification All publications, patents, and patent applications cited therein are referenced for all purposes. Incorporated herein by reference.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Hemphill, Mark El.             United States Georgia 30244             Lawrenceville, Milvale Place             911 (72) Inventor Ju, Jian             United States Georgia 30092,             Cross, Kentford Lane 3694

Claims (1)

[Claims] 1. A method for introducing ribonucleic acid into the nucleus of a host cell, the method comprising: Process:   i) combining the ribonucleic acid with the isolated nucleoprotein (NP) of the virus to synthesize Forming a ribonucleoprotein complex (RNP), wherein the nuclear protein Quality (NP) and the synthetic ribonucleoprotein complex (RNP) are substantially A step that does not contain a merase protein; and   ii) transfecting a host cell with the synthetic ribonucleoprotein complex, A method comprising: 2. The ribonucleic acid is a preselected ribonucleic acid of a virus, and further comprises a host cell. Maintaining the vesicles under conditions that allow replication of the virus. the method of. 3. The virus is influenza virus A, influenza virus B And the influenza virus type C is selected from the group consisting of: Law. 4. Further comprising the step of infecting said host cell with a helper virus. Item 1. The method of Item 1. 5. The helper virus is A / PR / 8/34, A / WSN 33, A / Lenigrad / 57, and A / 5. The method of claim 4, wherein the method is selected from the group consisting of Ann Arbor / 6/60. 6. 2. The method of claim 1, wherein said isolated nuclear protein (NP) is recombinantly expressed. 7. The nucleoprotein is influenza virus A, influenza virus Type B, influenza virus C, parainfluenza virus, measles wi A virus selected from the group consisting of Luth, Rabies, and Respiratory Synthia virus 2. The method of claim 1, which is a nuclear protein (NP). 8. The host cells are kidney cells, cells derived from chicken eggs, and primate early lung cells. 2. The method of claim 1, wherein the method is selected from the group consisting of vesicles. 9. The virus is an influenza virus; Recombinantly expressed influenza nuclei containing no viral RNA polymerase And the host cell is infected with an influenza helper virus 3. The method of claim 2, wherein the method is performed. 10. Combined with ribonucleic acid, thereby enhancing viral replication in host cells An isolated viral nuclear protein that forms a mediating synthetic ribonucleoprotein (RNP) A transfection composition comprising the nucleoprotein (NP) and And the synthetic ribonucleoprotein complex binds the viral RNA polymerase protein. A transfection composition substantially free of.