Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Feb;69(2):720–727. doi: 10.1128/jvi.69.2.720-727.1995

Requirements for the self-directed replication of flock house virus RNA 1.

L A Ball 1
PMCID: PMC188634  PMID: 7815535

Abstract

The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Full Text

The Full Text of this article is available as a PDF (425.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ball L. A., Amann J. M., Garrett B. K. Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture. J Virol. 1992 Apr;66(4):2326–2334. doi: 10.1128/jvi.66.4.2326-2334.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ball L. A. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors. J Virol. 1992 Apr;66(4):2335–2345. doi: 10.1128/jvi.66.4.2335-2345.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball L. A., Li Y. cis-acting requirements for the replication of flock house virus RNA 2. J Virol. 1993 Jun;67(6):3544–3551. doi: 10.1128/jvi.67.6.3544-3551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ball L. A. Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12443–12447. doi: 10.1073/pnas.91.26.12443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ball L. A., Wohlrab B., Li Y. Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants. Arch Virol Suppl. 1994;9:407–416. doi: 10.1007/978-3-7091-9326-6_40. [DOI] [PubMed] [Google Scholar]
  6. Blumenthal T., Carmichael G. G. RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem. 1979;48:525–548. doi: 10.1146/annurev.bi.48.070179.002521. [DOI] [PubMed] [Google Scholar]
  7. Bruenn J. A. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases. Nucleic Acids Res. 1991 Jan 25;19(2):217–226. doi: 10.1093/nar/19.2.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buzayan J. M., Gerlach W. L., Bruening G. Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8859–8862. doi: 10.1073/pnas.83.23.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng R. H., Reddy V. S., Olson N. H., Fisher A. J., Baker T. S., Johnson J. E. Functional implications of quasi-equivalence in a T = 3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography. Structure. 1994 Apr 15;2(4):271–282. doi: 10.1016/s0969-2126(00)00029-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dasmahapatra B., Dasgupta R., Ghosh A., Kaesberg P. Structure of the black beetle virus genome and its functional implications. J Mol Biol. 1985 Mar 20;182(2):183–189. doi: 10.1016/0022-2836(85)90337-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dasmahapatra B., Dasgupta R., Saunders K., Selling B., Gallagher T., Kaesberg P. Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus. Proc Natl Acad Sci U S A. 1986 Jan;83(1):63–66. doi: 10.1073/pnas.83.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davison A. J., Moss B. Structure of vaccinia virus early promoters. J Mol Biol. 1989 Dec 20;210(4):749–769. doi: 10.1016/0022-2836(89)90107-1. [DOI] [PubMed] [Google Scholar]
  13. Fisher A. J., Johnson J. E. Ordered duplex RNA controls capsid architecture in an icosahedral animal virus. Nature. 1993 Jan 14;361(6408):176–179. doi: 10.1038/361176a0. [DOI] [PubMed] [Google Scholar]
  14. Friesen P. D., Rueckert R. R. Black beetle virus: messenger for protein B is a subgenomic viral RNA. J Virol. 1982 Jun;42(3):986–995. doi: 10.1128/jvi.42.3.986-995.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Friesen P. D., Rueckert R. R. Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs. J Virol. 1984 Jan;49(1):116–124. doi: 10.1128/jvi.49.1.116-124.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friesen P. D., Rueckert R. R. Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2. J Virol. 1981 Mar;37(3):876–886. doi: 10.1128/jvi.37.3.876-886.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Gallagher T. M., Friesen P. D., Rueckert R. R. Autonomous replication and expression of RNA 1 from black beetle virus. J Virol. 1983 May;46(2):481–489. doi: 10.1128/jvi.46.2.481-489.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guarino L. A., Ghosh A., Dasmahapatra B., Dasgupta R., Kaesberg P. Sequence of the black beetle virus subgenomic RNA and its location in the viral genome. Virology. 1984 Nov;139(1):199–203. doi: 10.1016/0042-6822(84)90342-8. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  23. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  24. Ling M. L., Risman S. S., Klement J. F., McGraw N., McAllister W. T. Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate. Nucleic Acids Res. 1989 Feb 25;17(4):1605–1618. doi: 10.1093/nar/17.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Novak J. E., Kirkegaard K. Coupling between genome translation and replication in an RNA virus. Genes Dev. 1994 Jul 15;8(14):1726–1737. doi: 10.1101/gad.8.14.1726. [DOI] [PubMed] [Google Scholar]
  26. Perrotta A. T., Been M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature. 1991 Apr 4;350(6317):434–436. doi: 10.1038/350434a0. [DOI] [PubMed] [Google Scholar]
  27. Selling B. H., Rueckert R. R. Plaque assay for black beetle virus. J Virol. 1984 Jul;51(1):251–253. doi: 10.1128/jvi.51.1.251-253.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES