Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Dec;71(12):9618–9626. doi: 10.1128/jvi.71.12.9618-9626.1997

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

J T Patton 1, M T Jones 1, A N Kalbach 1, Y W He 1, J Xiaobo 1
PMCID: PMC230270  PMID: 9371626

Abstract

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Altenburg B. C., Graham D. Y., Estes M. K. Ultrastructural study of rotavirus replication in cultured cells. J Gen Virol. 1980 Jan;46(1):75–85. doi: 10.1099/0022-1317-46-1-75. [DOI] [PubMed] [Google Scholar]
  2. Bican P., Cohen J., Charpilienne A., Scherrer R. Purification and characterization of bovine rotavirus cores. J Virol. 1982 Sep;43(3):1113–1117. doi: 10.1128/jvi.43.3.1113-1117.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blacklow N. R., Greenberg H. B. Viral gastroenteritis. N Engl J Med. 1991 Jul 25;325(4):252–264. doi: 10.1056/NEJM199107253250406. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Clapp L. L., Patton J. T. Rotavirus morphogenesis: domains in the major inner capsid protein essential for binding to single-shelled particles and for trimerization. Virology. 1991 Feb;180(2):697–708. doi: 10.1016/0042-6822(91)90083-n. [DOI] [PubMed] [Google Scholar]
  6. Cohen J., Charpilienne A., Chilmonczyk S., Estes M. K. Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus. Virology. 1989 Jul;171(1):131–140. doi: 10.1016/0042-6822(89)90519-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen J. Ribonucleic acid polymerase activity associated with purified calf rotavirus. J Gen Virol. 1977 Sep;36(3):395–402. doi: 10.1099/0022-1317-36-3-395. [DOI] [PubMed] [Google Scholar]
  8. Eventoff W., Rossmann M. G., Taylor S. S., Torff H. J., Meyer H., Keil W., Kiltz H. H. Structural adaptations of lactate dehydrogenase isozymes. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2677–2681. doi: 10.1073/pnas.74.7.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukuhara N., Nishikawa K., Gorziglia M., Kapikian A. Z. Nucleotide sequence of gene segment 1 of a porcine rotavirus strain. Virology. 1989 Dec;173(2):743–749. doi: 10.1016/0042-6822(89)90590-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gombold J. L., Estes M. K., Ramig R. F. Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments. Virology. 1985 May;143(1):309–320. doi: 10.1016/0042-6822(85)90118-7. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kapikian A. Z. Overview of viral gastroenteritis. Arch Virol Suppl. 1996;12:7–19. doi: 10.1007/978-3-7091-6553-9_2. [DOI] [PubMed] [Google Scholar]
  14. Kattoura M. D., Chen X., Patton J. T. The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase. Virology. 1994 Aug 1;202(2):803–813. doi: 10.1006/viro.1994.1402. [DOI] [PubMed] [Google Scholar]
  15. Kattoura M. D., Clapp L. L., Patton J. T. The rotavirus nonstructural protein, NS35, possesses RNA-binding activity in vitro and in vivo. Virology. 1992 Dec;191(2):698–708. doi: 10.1016/0042-6822(92)90245-k. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Labbé M., Charpilienne A., Crawford S. E., Estes M. K., Cohen J. Expression of rotavirus VP2 produces empty corelike particles. J Virol. 1991 Jun;65(6):2946–2952. doi: 10.1128/jvi.65.6.2946-2952.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Lawton J. A., Estes M. K., Prasad B. V. Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. Nat Struct Biol. 1997 Feb;4(2):118–121. doi: 10.1038/nsb0297-118. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Liu M., Offit P. A., Estes M. K. Identification of the simian rotavirus SA11 genome segment 3 product. Virology. 1988 Mar;163(1):26–32. doi: 10.1016/0042-6822(88)90230-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Mansell E. A., Ramig R. F., Patton J. T. Temperature-sensitive lesions in the capsid proteins of the rotavirus mutants tsF and tsG that affect virion assembly. Virology. 1994 Oct;204(1):69–81. doi: 10.1006/viro.1994.1511. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Patton J. T., Gallegos C. O. Rotavirus RNA replication: single-stranded RNA extends from the replicase particle. J Gen Virol. 1990 May;71(Pt 5):1087–1094. doi: 10.1099/0022-1317-71-5-1087. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Petrie B. L., Greenberg H. B., Graham D. Y., Estes M. K. Ultrastructural localization of rotavirus antigens using colloidal gold. Virus Res. 1984;1(2):133–152. doi: 10.1016/0168-1702(84)90069-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Prasad B. V., Wang G. J., Clerx J. P., Chiu W. Three-dimensional structure of rotavirus. J Mol Biol. 1988 Jan 20;199(2):269–275. doi: 10.1016/0022-2836(88)90313-0. [DOI] [PubMed] [Google Scholar]
  33. Ramig R. F., Petrie B. L. Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis. J Virol. 1984 Mar;49(3):665–673. doi: 10.1128/jvi.49.3.665-673.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sandino A. M., Jashes M., Faúndez G., Spencer E. Role of the inner protein capsid on in vitro human rotavirus transcription. J Virol. 1986 Nov;60(2):797–802. doi: 10.1128/jvi.60.2.797-802.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Shaw A. L., Rothnagel R., Zeng C. Q., Lawton J. A., Ramig R. F., Estes M. K., Prasad B. V. Rotavirus structure: interactions between the structural proteins. Arch Virol Suppl. 1996;12:21–27. doi: 10.1007/978-3-7091-6553-9_3. [DOI] [PubMed] [Google Scholar]
  37. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Vásquez M., Sandino A. M., Pizarro J. M., Fernández J., Valenzuela S., Spencer E. Function of rotavirus VP3 polypeptide in viral morphogenesis. J Gen Virol. 1993 May;74(Pt 5):937–941. doi: 10.1099/0022-1317-74-5-937. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wentz M. J., Zeng C. Q., Patton J. T., Estes M. K., Ramig R. F. Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro. Arch Virol Suppl. 1996;12:59–67. doi: 10.1007/978-3-7091-6553-9_7. [DOI] [PubMed] [Google Scholar]
  42. Yeager M., Dryden K. A., Olson N. H., Greenberg H. B., Baker T. S. Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J Cell Biol. 1990 Jun;110(6):2133–2144. doi: 10.1083/jcb.110.6.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]

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

RESOURCES