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. 1995 Sep;69(9):5677–5686. doi: 10.1128/jvi.69.9.5677-5686.1995

RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA.

H Grosfeld 1, M G Hill 1, P L Collins 1
PMCID: PMC189426  PMID: 7637014

Abstract

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

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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., Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 1993 Aug;67(8):4822–4830. doi: 10.1128/jvi.67.8.4822-4830.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chomczynski P. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem. 1992 Feb 14;201(1):134–139. doi: 10.1016/0003-2697(92)90185-a. [DOI] [PubMed] [Google Scholar]
  4. Collins P. L., Anderson K., Langer S. J., Wertz G. W. Correct sequence for the major nucleocapsid protein mRNA of respiratory syncytial virus. Virology. 1985 Oct 15;146(1):69–77. doi: 10.1016/0042-6822(85)90053-4. [DOI] [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. 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]
  8. Crowe J. E., Jr, Bui P. T., Davis A. R., Chanock R. M., Murphy B. R. A further attenuated derivative of a cold-passaged temperature-sensitive mutant of human respiratory syncytial virus retains immunogenicity and protective efficacy against wild-type challenge in seronegative chimpanzees. Vaccine. 1994 Jul;12(9):783–790. doi: 10.1016/0264-410x(94)90286-0. [DOI] [PubMed] [Google Scholar]
  9. Crowe J. E., Jr, Bui P. T., London W. T., Davis A. R., Hung P. P., Chanock R. M., Murphy B. R. Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis. Vaccine. 1994 Jun;12(8):691–699. doi: 10.1016/0264-410x(94)90218-6. [DOI] [PubMed] [Google Scholar]
  10. Curran J., Homann H., Buchholz C., Rochat S., Neubert W., Kolakofsky D. The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation. J Virol. 1993 Jul;67(7):4358–4364. doi: 10.1128/jvi.67.7.4358-4364.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuerst T. R., Moss B. Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader. J Mol Biol. 1989 Mar 20;206(2):333–348. doi: 10.1016/0022-2836(89)90483-x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. García-Sastre A., Palese P. Genetic manipulation of negative-strand RNA virus genomes. Annu Rev Microbiol. 1993;47:765–790. doi: 10.1146/annurev.mi.47.100193.004001. [DOI] [PubMed] [Google Scholar]
  17. Gershon P. D., Ahn B. Y., Garfield M., Moss B. Poly(A) polymerase and a dissociable polyadenylation stimulatory factor encoded by vaccinia virus. Cell. 1991 Sep 20;66(6):1269–1278. doi: 10.1016/0092-8674(91)90048-4. [DOI] [PubMed] [Google Scholar]
  18. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Luo G., Palese P. Genetic analysis of influenza virus. Curr Opin Genet Dev. 1992 Feb;2(1):77–81. doi: 10.1016/s0959-437x(05)80326-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Mazumder B., Barik S. Requirement of casein kinase II-mediated phosphorylation for the transcriptional activity of human respiratory syncytial viral phosphoprotein P: transdominant negative phenotype of phosphorylation-defective P mutants. Virology. 1994 Nov 15;205(1):104–111. doi: 10.1006/viro.1994.1624. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Parvin J. D., Palese P., Honda A., Ishihama A., Krystal M. Promoter analysis of influenza virus RNA polymerase. J Virol. 1989 Dec;63(12):5142–5152. doi: 10.1128/jvi.63.12.5142-5152.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Poch O., Blumberg B. M., Bougueleret L., Tordo N. Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains. J Gen Virol. 1990 May;71(Pt 5):1153–1162. doi: 10.1099/0022-1317-71-5-1153. [DOI] [PubMed] [Google Scholar]
  28. Rohrmann G., Yuen L., Moss B. Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell. 1986 Sep 26;46(7):1029–1035. doi: 10.1016/0092-8674(86)90702-6. [DOI] [PubMed] [Google Scholar]
  29. Ryan K. W. Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques. J Virol Methods. 1992 Jan;36(1):85–90. doi: 10.1016/0166-0934(92)90159-b. [DOI] [PubMed] [Google Scholar]
  30. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  31. Satake M., Elango N., Venkatesan S. Sequence analysis of the respiratory syncytial virus phosphoprotein gene. J Virol. 1984 Dec;52(3):991–994. doi: 10.1128/jvi.52.3.991-994.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Steward M., Vipond I. B., Millar N. S., Emmerson P. T. RNA editing in Newcastle disease virus. J Gen Virol. 1993 Dec;74(Pt 12):2539–2547. doi: 10.1099/0022-1317-74-12-2539. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Yu Q., Hardy R. W., Wertz G. W. 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. J Virol. 1995 Apr;69(4):2412–2419. doi: 10.1128/jvi.69.4.2412-2419.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yuen L., Moss B. Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6417–6421. doi: 10.1073/pnas.84.18.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]

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