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. 1992 Dec;66(12):7293–7302. doi: 10.1128/jvi.66.12.7293-7302.1992

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

C C Kao 1, P Ahlquist 1
PMCID: PMC240433  PMID: 1433519

Abstract

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.

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Selected References

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

  1. Ahlquist P. Bromovirus RNA replication and transcription. Curr Opin Genet Dev. 1992 Feb;2(1):71–76. doi: 10.1016/s0959-437x(05)80325-9. [DOI] [PubMed] [Google Scholar]
  2. Ahlquist P., Strauss E. G., Rice C. M., Strauss J. H., Haseloff J., Zimmern D. Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. J Virol. 1985 Feb;53(2):536–542. doi: 10.1128/jvi.53.2.536-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allison R. F., Janda M., Ahlquist P. Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus. J Virol. 1988 Oct;62(10):3581–3588. doi: 10.1128/jvi.62.10.3581-3588.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allison R., Thompson C., Ahlquist P. Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1820–1824. doi: 10.1073/pnas.87.5.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Argos P. A sequence motif in many polymerases. Nucleic Acids Res. 1988 Nov 11;16(21):9909–9916. doi: 10.1093/nar/16.21.9909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barton D. J., Sawicki S. G., Sawicki D. L. Solubilization and immunoprecipitation of alphavirus replication complexes. J Virol. 1991 Mar;65(3):1496–1506. doi: 10.1128/jvi.65.3.1496-1506.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Jong W., Ahlquist P. A hybrid plant RNA virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6808–6812. doi: 10.1073/pnas.89.15.6808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dominguez G., Wang C. Y., Frey T. K. Sequence of the genome RNA of rubella virus: evidence for genetic rearrangement during togavirus evolution. Virology. 1990 Jul;177(1):225–238. doi: 10.1016/0042-6822(90)90476-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. French R., Ahlquist P. Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA. J Virol. 1988 Jul;62(7):2411–2420. doi: 10.1128/jvi.62.7.2411-2420.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. French R., Ahlquist P. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J Virol. 1987 May;61(5):1457–1465. doi: 10.1128/jvi.61.5.1457-1465.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. French R., Janda M., Ahlquist P. Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyledonous plant cells. Science. 1986 Mar 14;231(4743):1294–1297. doi: 10.1126/science.231.4743.1294. [DOI] [PubMed] [Google Scholar]
  12. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination. FEBS Lett. 1988 Aug 1;235(1-2):16–24. doi: 10.1016/0014-5793(88)81226-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haseloff J., Goelet P., Zimmern D., Ahlquist P., Dasgupta R., Kaesberg P. Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4358–4362. doi: 10.1073/pnas.81.14.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayes R. J., Buck K. W. Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell. 1990 Oct 19;63(2):363–368. doi: 10.1016/0092-8674(90)90169-f. [DOI] [PubMed] [Google Scholar]
  16. Ishikawa M., Meshi T., Motoyoshi F., Takamatsu N., Okada Y. In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus. Nucleic Acids Res. 1986 Nov 11;14(21):8291–8305. doi: 10.1093/nar/14.21.8291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kamer G., Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984 Sep 25;12(18):7269–7282. doi: 10.1093/nar/12.18.7269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kao C. C., Quadt R., Hershberger R. P., Ahlquist P. Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro. J Virol. 1992 Nov;66(11):6322–6329. doi: 10.1128/jvi.66.11.6322-6329.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kroner P. A., Young B. M., Ahlquist P. Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. J Virol. 1990 Dec;64(12):6110–6120. doi: 10.1128/jvi.64.12.6110-6120.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kroner P., Richards D., Traynor P., Ahlquist P. Defined mutations in a small region of the brome mosaic virus 2 gene cause diverse temperature-sensitive RNA replication phenotypes. J Virol. 1989 Dec;63(12):5302–5309. doi: 10.1128/jvi.63.12.5302-5309.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Mi S., Stollar V. Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype. Virology. 1990 Oct;178(2):429–434. doi: 10.1016/0042-6822(90)90340-w. [DOI] [PubMed] [Google Scholar]
  23. Pacha R. F., Ahlquist P. Use of bromovirus RNA3 hybrids to study template specificity in viral RNA amplification. J Virol. 1991 Jul;65(7):3693–3703. doi: 10.1128/jvi.65.7.3693-3703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pogue G. P., Hall T. C. The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation. J Virol. 1992 Feb;66(2):674–684. doi: 10.1128/jvi.66.2.674-684.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Quadt R., Jaspars E. M. Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase. Virology. 1990 Sep;178(1):189–194. doi: 10.1016/0042-6822(90)90393-6. [DOI] [PubMed] [Google Scholar]
  26. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  27. Scheidel L. M., Stollar V. Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1. Virology. 1991 Apr;181(2):490–499. doi: 10.1016/0042-6822(91)90881-b. [DOI] [PubMed] [Google Scholar]
  28. Traynor P., Ahlquist P. Use of bromovirus RNA2 hybrids to map cis- and trans-acting functions in a conserved RNA replication gene. J Virol. 1990 Jan;64(1):69–77. doi: 10.1128/jvi.64.1.69-77.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Traynor P., Young B. M., Ahlquist P. Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread. J Virol. 1991 Jun;65(6):2807–2815. doi: 10.1128/jvi.65.6.2807-2815.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  31. Zhu L. A., Weller S. K. The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function. J Virol. 1992 Jan;66(1):469–479. doi: 10.1128/jvi.66.1.469-479.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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