Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Nov;71(11):8482–8489. doi: 10.1128/jvi.71.11.8482-8489.1997

Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.

D J Barton 1, J B Flanegan 1
PMCID: PMC192311  PMID: 9343205

Abstract

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Ambros V., Baltimore D. Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine. J Biol Chem. 1978 Aug 10;253(15):5263–5266. [PubMed] [Google Scholar]
  2. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andino R., Rieckhof G. E., Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. doi: 10.1016/0092-8674(90)90170-j. [DOI] [PubMed] [Google Scholar]
  4. Andino R., Rieckhof G. E., Trono D., Baltimore D. Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region. J Virol. 1990 Feb;64(2):607–612. doi: 10.1128/jvi.64.2.607-612.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baltera R. F., Jr, Tershak D. R. Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C. J Virol. 1989 Oct;63(10):4441–4444. doi: 10.1128/jvi.63.10.4441-4444.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barton D. J., Black E. P., Flanegan J. B. Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA. J Virol. 1995 Sep;69(9):5516–5527. doi: 10.1128/jvi.69.9.5516-5527.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barton D. J., Flanegan J. B. Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus. J Virol. 1993 Feb;67(2):822–831. doi: 10.1128/jvi.67.2.822-831.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barton D. J., Morasco B. J., Flanegan J. B. Assays for poliovirus polymerase, 3D(Pol), and authentic RNA replication in HeLa S10 extracts. Methods Enzymol. 1996;275:35–57. doi: 10.1016/s0076-6879(96)75005-x. [DOI] [PubMed] [Google Scholar]
  9. Bienz K., Egger D., Pfister T., Troxler M. Structural and functional characterization of the poliovirus replication complex. J Virol. 1992 May;66(5):2740–2747. doi: 10.1128/jvi.66.5.2740-2747.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bienz K., Egger D., Troxler M., Pasamontes L. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. J Virol. 1990 Mar;64(3):1156–1163. doi: 10.1128/jvi.64.3.1156-1163.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cho M. W., Teterina N., Egger D., Bienz K., Ehrenfeld E. Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells. Virology. 1994 Jul;202(1):129–145. doi: 10.1006/viro.1994.1329. [DOI] [PubMed] [Google Scholar]
  12. Egger D., Pasamontes L., Bolten R., Boyko V., Bienz K. Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis. J Virol. 1996 Dec;70(12):8675–8683. doi: 10.1128/jvi.70.12.8675-8683.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Flanegan J. B., Baltimore D. Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proc Natl Acad Sci U S A. 1977 Sep;74(9):3677–3680. doi: 10.1073/pnas.74.9.3677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flanegan J. B., Petterson R. F., Ambros V., Hewlett N. J., Baltimore D. Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate RNAs of poliovirus. Proc Natl Acad Sci U S A. 1977 Mar;74(3):961–965. doi: 10.1073/pnas.74.3.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giachetti C., Semler B. L. Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis. J Virol. 1991 May;65(5):2647–2654. doi: 10.1128/jvi.65.5.2647-2654.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorbalenya A. E., Blinov V. M., Donchenko A. P., Koonin E. V. An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication. J Mol Evol. 1989 Mar;28(3):256–268. doi: 10.1007/BF02102483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. A conserved NTP-motif in putative helicases. Nature. 1988 May 5;333(6168):22–22. doi: 10.1038/333022a0. [DOI] [PubMed] [Google Scholar]
  18. Gorbalenya A. E., Koonin E. V. Viral proteins containing the purine NTP-binding sequence pattern. Nucleic Acids Res. 1989 Nov 11;17(21):8413–8440. doi: 10.1093/nar/17.21.8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harris K. S., Xiang W., Alexander L., Lane W. S., Paul A. V., Wimmer E. Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. J Biol Chem. 1994 Oct 28;269(43):27004–27014. [PubMed] [Google Scholar]
  20. Jacobson S. J., Konings D. A., Sarnow P. Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification. J Virol. 1993 Jun;67(6):2961–2971. doi: 10.1128/jvi.67.6.2961-2971.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kadaré G., Haenni A. L. Virus-encoded RNA helicases. J Virol. 1997 Apr;71(4):2583–2590. doi: 10.1128/jvi.71.4.2583-2590.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee Y. F., Nomoto A., Detjen B. M., Wimmer E. A protein covalently linked to poliovirus genome RNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):59–63. doi: 10.1073/pnas.74.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mirzayan C., Wimmer E. Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity. Virology. 1994 Feb 15;199(1):176–187. doi: 10.1006/viro.1994.1110. [DOI] [PubMed] [Google Scholar]
  24. Mirzayan C., Wimmer E. Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C. Virology. 1992 Aug;189(2):547–555. doi: 10.1016/0042-6822(92)90578-d. [DOI] [PubMed] [Google Scholar]
  25. Molla A., Paul A. V., Wimmer E. Cell-free, de novo synthesis of poliovirus. Science. 1991 Dec 13;254(5038):1647–1651. doi: 10.1126/science.1661029. [DOI] [PubMed] [Google Scholar]
  26. Paul A. V., Molla A., Wimmer E. Studies of a putative amphipathic helix in the N-terminus of poliovirus protein 2C. Virology. 1994 Feb 15;199(1):188–199. doi: 10.1006/viro.1994.1111. [DOI] [PubMed] [Google Scholar]
  27. Pettersson R. F., Ambros V., Baltimore D. Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA. J Virol. 1978 Aug;27(2):357–365. doi: 10.1128/jvi.27.2.357-365.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pilipenko E. V., Maslova S. V., Sinyakov A. N., Agol V. I. Towards identification of cis-acting elements involved in the replication of enterovirus and rhinovirus RNAs: a proposal for the existence of tRNA-like terminal structures. Nucleic Acids Res. 1992 Apr 11;20(7):1739–1745. doi: 10.1093/nar/20.7.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pincus S. E., Diamond D. C., Emini E. A., Wimmer E. Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C. J Virol. 1986 Feb;57(2):638–646. doi: 10.1128/jvi.57.2.638-646.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pincus S. E., Rohl H., Wimmer E. Guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements. Virology. 1987 Mar;157(1):83–88. doi: 10.1016/0042-6822(87)90316-3. [DOI] [PubMed] [Google Scholar]
  31. Pincus S. E., Wimmer E. Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. J Virol. 1986 Nov;60(2):793–796. doi: 10.1128/jvi.60.2.793-796.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rodríguez P. L., Carrasco L. Poliovirus protein 2C contains two regions involved in RNA binding activity. J Biol Chem. 1995 Apr 28;270(17):10105–10112. doi: 10.1074/jbc.270.17.10105. [DOI] [PubMed] [Google Scholar]
  33. Rodríguez P. L., Carrasco L. Poliovirus protein 2C has ATPase and GTPase activities. J Biol Chem. 1993 Apr 15;268(11):8105–8110. [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schlegel A., Giddings T. H., Jr, Ladinsky M. S., Kirkegaard K. Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol. 1996 Oct;70(10):6576–6588. doi: 10.1128/jvi.70.10.6576-6588.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takeda N., Kuhn R. J., Yang C. F., Takegami T., Wimmer E. Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. J Virol. 1986 Oct;60(1):43–53. doi: 10.1128/jvi.60.1.43-53.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tershak D. R. Inhibition of poliovirus polymerase by guanidine in vitro. J Virol. 1982 Jan;41(1):313–318. doi: 10.1128/jvi.41.1.313-318.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tolskaya E. A., Romanova L. I., Kolesnikova M. S., Gmyl A. P., Gorbalenya A. E., Agol V. I. Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization. J Mol Biol. 1994 Mar 11;236(5):1310–1323. doi: 10.1016/0022-2836(94)90060-4. [DOI] [PubMed] [Google Scholar]
  39. Tuschall D. M., Hiebert E., Flanegan J. B. Poliovirus RNA-dependent RNA polymerase synthesizes full-length copies of poliovirion RNA, cellular mRNA, and several plant virus RNAs in vitro. J Virol. 1982 Oct;44(1):209–216. doi: 10.1128/jvi.44.1.209-216.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Van Dyke T. A., Flanegan J. B. Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. J Virol. 1980 Sep;35(3):732–740. doi: 10.1128/jvi.35.3.732-740.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Dyke T. A., Rickles R. J., Flanegan J. B. Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase. J Biol Chem. 1982 Apr 25;257(8):4610–4617. [PubMed] [Google Scholar]
  42. Villa-Komaroff L., McDowell M., Baltimore D., Lodish H. F. Translation of reovirus mRNA, poliovirus RNA and bacteriophage Qbeta RNA in cell-free extracts of mammalian cells. Methods Enzymol. 1974;30:709–723. doi: 10.1016/0076-6879(74)30068-7. [DOI] [PubMed] [Google Scholar]
  43. Young D. C., Tuschall D. M., Flanegan J. B. Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro. J Virol. 1985 May;54(2):256–264. doi: 10.1128/jvi.54.2.256-264.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES