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. 1995 Apr;69(4):2412–2419. doi: 10.1128/jvi.69.4.2412-2419.1995

Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication.

Q Yu 1, R W Hardy 1, G W Wertz 1
PMCID: PMC188915  PMID: 7884888

Abstract

The RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells expressing the RS virus N, P, and L proteins. Omission of any of the three viral proteins abrogated replication, thereby defining the N, P, and L proteins as the minimal trans-acting proteins required for RNA replication. This system has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required. These features allow analysis of both cis- and trans-acting factors involved in the control of replication of RS virus RNA.

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Selected References

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  1. Barik S. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s). J Virol. 1992 Nov;66(11):6813–6818. doi: 10.1128/jvi.66.11.6813-6818.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calain P., Curran J., Kolakofsky D., Roux L. Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology. 1992 Nov;191(1):62–71. doi: 10.1016/0042-6822(92)90166-m. [DOI] [PubMed] [Google Scholar]
  3. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  4. Collins P. L., Huang Y. T., Wertz G. W. Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. J Virol. 1984 Feb;49(2):572–578. doi: 10.1128/jvi.49.2.572-578.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins P. L., Mink M. A., Hill M. G., 3rd, Camargo E., Grosfeld H., Stec D. S. Rescue of a 7502-nucleotide (49.3% of full-length) synthetic analog of respiratory syncytial virus genomic RNA. Virology. 1993 Jul;195(1):252–256. doi: 10.1006/viro.1993.1368. [DOI] [PubMed] [Google Scholar]
  6. Collins P. L., Mink M. A., Stec D. S. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9663–9667. doi: 10.1073/pnas.88.21.9663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins P. L., Wertz G. W. cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3208–3212. doi: 10.1073/pnas.80.11.3208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conzelmann K. K., Schnell M. Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. J Virol. 1994 Feb;68(2):713–719. doi: 10.1128/jvi.68.2.713-719.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De B. P., Banerjee A. K. Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3. Virology. 1993 Sep;196(1):344–348. doi: 10.1006/viro.1993.1486. [DOI] [PubMed] [Google Scholar]
  10. Dimock K., Collins P. L. Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3. J Virol. 1993 May;67(5):2772–2778. doi: 10.1128/jvi.67.5.2772-2778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emerson S. U., Wagner R. R. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J Virol. 1972 Aug;10(2):297–309. doi: 10.1128/jvi.10.2.297-309.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Enami M., Luytjes W., Krystal M., Palese P. Introduction of site-specific mutations into the genome of influenza virus. Proc Natl Acad Sci U S A. 1990 May;87(10):3802–3805. doi: 10.1073/pnas.87.10.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haltiner M., Kempe T., Tjian R. A novel strategy for constructing clustered point mutations. Nucleic Acids Res. 1985 Feb 11;13(3):1015–1025. doi: 10.1093/nar/13.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang Y. T., Collins P. L., Wertz G. W. Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein. Virus Res. 1985 Mar;2(2):157–173. doi: 10.1016/0168-1702(85)90246-1. [DOI] [PubMed] [Google Scholar]
  16. Huang Y. T., Romito R. R., De B. P., Banerjee A. K. Characterization of the in vitro system for the synthesis of mRNA from human respiratory syncytial virus. Virology. 1993 Apr;193(2):862–867. doi: 10.1006/viro.1993.1195. [DOI] [PubMed] [Google Scholar]
  17. Huang Y. T., Wertz G. W. The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species. J Virol. 1982 Jul;43(1):150–157. doi: 10.1128/jvi.43.1.150-157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. King A. M., Stott E. J., Langer S. J., Young K. K., Ball L. A., Wertz G. W. Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice. J Virol. 1987 Sep;61(9):2885–2890. doi: 10.1128/jvi.61.9.2885-2890.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Luytjes W., Krystal M., Enami M., Parvin J. D., Palese P. Amplification, expression, and packaging of foreign gene by influenza virus. Cell. 1989 Dec 22;59(6):1107–1113. doi: 10.1016/0092-8674(89)90766-6. [DOI] [PubMed] [Google Scholar]
  21. Mink M. A., Stec D. S., Collins P. L. Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA. Virology. 1991 Dec;185(2):615–624. doi: 10.1016/0042-6822(91)90532-g. [DOI] [PubMed] [Google Scholar]
  22. Park K. H., Huang T., Correia F. F., Krystal M. Rescue of a foreign gene by Sendai virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5537–5541. doi: 10.1073/pnas.88.13.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pattnaik A. K., Ball L. A., LeGrone A. W., Wertz G. W. Infectious defective interfering particles of VSV from transcripts of a cDNA clone. Cell. 1992 Jun 12;69(6):1011–1020. doi: 10.1016/0092-8674(92)90619-n. [DOI] [PubMed] [Google Scholar]
  24. Pattnaik A. K., Wertz G. W. Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. J Virol. 1990 Jun;64(6):2948–2957. doi: 10.1128/jvi.64.6.2948-2957.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Patton J. T., Davis N. L., Wertz G. W. N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus. J Virol. 1984 Feb;49(2):303–309. doi: 10.1128/jvi.49.2.303-309.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schnell M. J., Mebatsion T., Conzelmann K. K. Infectious rabies viruses from cloned cDNA. EMBO J. 1994 Sep 15;13(18):4195–4203. doi: 10.1002/j.1460-2075.1994.tb06739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schubert M., Harmison G. G., Richardson C. D., Meier E. Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7984–7988. doi: 10.1073/pnas.82.23.7984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stec D. S., Hill M. G., 3rd, Collins P. L. Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses. Virology. 1991 Jul;183(1):273–287. doi: 10.1016/0042-6822(91)90140-7. [DOI] [PubMed] [Google Scholar]
  29. Wertz G. W., Davis N. Characterization and mapping of RNase III cleavage sites in VSV genome RNA. Nucleic Acids Res. 1981 Dec 11;9(23):6487–6503. doi: 10.1093/nar/9.23.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wertz G. W., Whelan S., LeGrone A., Ball L. A. Extent of terminal complementarity modulates the balance between transcription and replication of vesicular stomatitis virus RNA. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8587–8591. doi: 10.1073/pnas.91.18.8587. [DOI] [PMC free article] [PubMed] [Google Scholar]

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