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. 2000 Oct;6(10):1455–1467. doi: 10.1017/s1355838200001187

De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation.

D Chen 1, J T Patton 1
PMCID: PMC1370016  PMID: 11073221

Abstract

The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG.

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Selected References

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  1. Blumenthal T. Q beta replicase template specificity: different templates require different GTP concentrations for initiation. Proc Natl Acad Sci U S A. 1980 May;77(5):2601–2605. doi: 10.1073/pnas.77.5.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyle J. F., Holmes K. V. RNA-binding proteins of bovine rotavirus. J Virol. 1986 May;58(2):561–568. doi: 10.1128/jvi.58.2.561-568.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buck K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res. 1996;47:159–251. doi: 10.1016/S0065-3527(08)60736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen D., Luongo C. L., Nibert M. L., Patton J. T. Rotavirus open cores catalyze 5'-capping and methylation of exogenous RNA: evidence that VP3 is a methyltransferase. Virology. 1999 Dec 5;265(1):120–130. doi: 10.1006/viro.1999.0029. [DOI] [PubMed] [Google Scholar]
  5. Chen D., Patton J. T. Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis. J Virol. 1998 Sep;72(9):7387–7396. doi: 10.1128/jvi.72.9.7387-7396.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen D., Zeng C. Q., Wentz M. J., Gorziglia M., Estes M. K., Ramig R. F. Template-dependent, in vitro replication of rotavirus RNA. J Virol. 1994 Nov;68(11):7030–7039. doi: 10.1128/jvi.68.11.7030-7039.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gallegos C. O., Patton J. T. Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles. Virology. 1989 Oct;172(2):616–627. doi: 10.1016/0042-6822(89)90204-3. [DOI] [PubMed] [Google Scholar]
  8. Imai M., Akatani K., Ikegami N., Furuichi Y. Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments. J Virol. 1983 Jul;47(1):125–136. doi: 10.1128/jvi.47.1.125-136.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kao C. C., Sun J. H. Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers. J Virol. 1996 Oct;70(10):6826–6830. doi: 10.1128/jvi.70.10.6826-6830.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Labbé M., Baudoux P., Charpilienne A., Poncet D., Cohen J. Identification of the nucleic acid binding domain of the rotavirus VP2 protein. J Gen Virol. 1994 Dec;75(Pt 12):3423–3430. doi: 10.1099/0022-1317-75-12-3423. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lawton J. A., Zeng C. Q., Mukherjee S. K., Cohen J., Estes M. K., Prasad B. V. Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer. J Virol. 1997 Oct;71(10):7353–7360. doi: 10.1128/jvi.71.10.7353-7360.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Liu M., Mattion N. M., Estes M. K. Rotavirus VP3 expressed in insect cells possesses guanylyltransferase activity. Virology. 1992 May;188(1):77–84. doi: 10.1016/0042-6822(92)90736-9. [DOI] [PubMed] [Google Scholar]
  14. Mansell E. A., Patton J. T. Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity. J Virol. 1990 Oct;64(10):4988–4996. doi: 10.1128/jvi.64.10.4988-4996.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCrae M. A., McCorquodale J. G. Molecular biology of rotaviruses. V. Terminal structure of viral RNA species. Virology. 1983 Apr 15;126(1):204–212. doi: 10.1016/0042-6822(83)90472-5. [DOI] [PubMed] [Google Scholar]
  16. Midthun K., Greenberg H. B., Hoshino Y., Kapikian A. Z., Wyatt R. G., Chanock R. M. Reassortant rotaviruses as potential live rotavirus vaccine candidates. J Virol. 1985 Mar;53(3):949–954. doi: 10.1128/jvi.53.3.949-954.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mitchell D. B., Both G. W. Completion of the genomic sequence of the simian rotavirus SA11: nucleotide sequences of segments 1, 2, and 3. Virology. 1990 Jul;177(1):324–331. doi: 10.1016/0042-6822(90)90487-c. [DOI] [PubMed] [Google Scholar]
  18. Patton J. T., Chen D. RNA-binding and capping activities of proteins in rotavirus open cores. J Virol. 1999 Feb;73(2):1382–1391. doi: 10.1128/jvi.73.2.1382-1391.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Patton J. T., Chnaiderman J., Spencer E. Open reading frame in rotavirus mRNA specifically promotes synthesis of double-stranded RNA: template size also affects replication efficiency. Virology. 1999 Nov 10;264(1):167–180. doi: 10.1006/viro.1999.9989. [DOI] [PubMed] [Google Scholar]
  20. Patton J. T., Jones M. T., Kalbach A. N., He Y. W., Xiaobo J. Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. J Virol. 1997 Dec;71(12):9618–9626. doi: 10.1128/jvi.71.12.9618-9626.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Patton J. T. Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis. J Virol. 1996 Nov;70(11):7940–7947. doi: 10.1128/jvi.70.11.7940-7947.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Patton J. T., Wentz M., Xiaobo J., Ramig R. F. cis-Acting signals that promote genome replication in rotavirus mRNA. J Virol. 1996 Jun;70(6):3961–3971. doi: 10.1128/jvi.70.6.3961-3971.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pizarro J. L., Sandino A. M., Pizarro J. M., Fernández J., Spencer E. Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3. J Gen Virol. 1991 Feb;72(Pt 2):325–332. doi: 10.1099/0022-1317-72-2-325. [DOI] [PubMed] [Google Scholar]
  24. Plotch S. J., Krug R. M. Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J Virol. 1977 Jan;21(1):24–34. doi: 10.1128/jvi.21.1.24-34.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Prasad B. V., Rothnagel R., Zeng C. Q., Jakana J., Lawton J. A., Chiu W., Estes M. K. Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. Nature. 1996 Aug 1;382(6590):471–473. doi: 10.1038/382471a0. [DOI] [PubMed] [Google Scholar]
  26. Taraporewala Z., Chen D., Patton J. T. Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity. J Virol. 1999 Dec;73(12):9934–9943. doi: 10.1128/jvi.73.12.9934-9943.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Valenzuela S., Pizarro J., Sandino A. M., Vásquez M., Fernández J., Hernández O., Patton J., Spencer E. Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase. J Virol. 1991 Jul;65(7):3964–3967. doi: 10.1128/jvi.65.7.3964-3967.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wentz M. J., Patton J. T., Ramig R. F. The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis. J Virol. 1996 Nov;70(11):7833–7841. doi: 10.1128/jvi.70.11.7833-7841.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zeng C. Q., Wentz M. J., Cohen J., Estes M. K., Ramig R. F. Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol. 1996 May;70(5):2736–2742. doi: 10.1128/jvi.70.5.2736-2742.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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