Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 87, Issue 9

Characterization of norovirus 3D RNA-dependent RNA polymerase activity and initiation of RNA synthesis No Access

Abstract

Norovirus (NV) 3D is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3D activity and initiation of RNA synthesis have been examined . Recombinant NV 3D, as well as a 3D active-site mutant were expressed in and purified. NV 3D was able to synthesize RNA and displayed flexibility with respect to the use of Mg or Mn as a cofactor. NV 3D yielded two different products when incubated with synthetic RNA : (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining initiation of RNA synthesis. NV 3D displays properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.81802-0
2006-09-01
2025-04-22
Loading full text...

Full text loading...

References

  1. Arnold J. J., Ghosh S. K., Cameron C. E. 1999; Poliovirus RNA-dependent RNA polymerase (3Dpol). Divalent cation modulation of primer, template, and nucleotide selection. J Biol Chem 274:37060–37069 [CrossRef]
     [Google Scholar]
  2. Asanaka M., Atmar R. L., Ruvolo V., Crawford S. E., Neill F. H., Estes M. K. 2005; Replication and packaging of Norwalk virus RNA in cultured mammalian cells. Proc Natl Acad Sci U S A 102:10327–10332 [CrossRef]
     [Google Scholar]
  3. Atmar R. L., Estes M. K. 2001; Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin Microbiol Rev 14:15–37 [CrossRef]
     [Google Scholar]
  4. Behrens S. E., Tomei L., De Francesco R. 1996; Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15:12–22
     [Google Scholar]
  5. Belliot G., Sosnovtsev S. V., Chang K. O., Babu V., Uche U., Arnold J. J., Cameron C. E., Green K. Y. 2005; Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. J Virol 79:2393–2403 [CrossRef]
     [Google Scholar]
  6. Crotty S., Gohara D., Gilligan D. K., Karelsky S., Cameron C. E., Andino R. 2003; Manganese-dependent polioviruses caused by mutations within the viral polymerase. J Virol 77:5378–5388 [CrossRef]
     [Google Scholar]
  7. Fukushi S., Kojima S., Takai R., Hoshino F. B., Oka T., Takeda N., Katayama K., Kageyama T. 2004; Poly(A)- and primer-independent RNA polymerase of norovirus. J Virol 78:3889–3896 [CrossRef]
     [Google Scholar]
  8. Green K. Y., Chanock R. M., Kapikian A. Z. 2001; Human caliciviruses. In Fields Virology , 4th edn. Edited by Knipe D. M., Howley P. M., Griffin D. E., Lamb R. A., Martin M. A., Roizman B, Straus S. E. Lippincott Williams & Wilkins;
     [Google Scholar]
  9. Kao C. C., Sun J. H. 1996; Initiation of minus-strand RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase: use of oligoribonucleotide primers. J Virol 70:6826–6830
     [Google Scholar]
  10. Kao C. C., Del Vecchio A. M., Zhong W. 1999; De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase. Virology 253:1–7 [CrossRef]
     [Google Scholar]
  11. Luo G., Hamatake R. K., Mathis D. M., Racela J., Rigat K. L., Lemm J., Colonno R. J. 2000; De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol 74:851–863 [CrossRef]
     [Google Scholar]
  12. Parashar U. D., Monroe S. S. 2001; ‘Norwalk-like viruses' as a cause of foodborne disease outbreaks. Rev Med Virol 11:243–252 [CrossRef]
     [Google Scholar]
  13. Ranjith-Kumar C. T., Gajewski J., Gutshall L., Maley D., Sarisky R. T., Kao C. C. 2001; Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis. J Virol 75:8615–8623 [CrossRef]
     [Google Scholar]
  14. Rohayem J., Diestelkoetter P., Weigle B., Oehmichen A., Schmitz M., Mehlhorn J., Conrad K., Rieber E. P. 2000; Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res 60:1815–1817
     [Google Scholar]
  15. Rohayem J., Munch J., Rethwilm A. 2005; Evidence of recombination in the norovirus capsid gene. J Virol 79:4977–4990 [CrossRef]
     [Google Scholar]
  16. Sambrook J., Russel D. W. 2001 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  17. Sun J. H., Adkins S., Faurote G., Kao C. C. 1996; Initiation of (–)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides. Virology 226:1–12 [CrossRef]
     [Google Scholar]
  18. Temme A., Traub O., Willecke K. 1998; Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes. Cell Tissue Res 294:345–350 [CrossRef]
     [Google Scholar]
  19. Vazquez A. L., Alonso J. M., Parra F. 2000; Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase. J Virol 74:3888–3891 [CrossRef]
     [Google Scholar]
  20. Wobus C. E., Karst S. M., Thackray L. B. & 7 other authors 2004; Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2:e432 [CrossRef]
     [Google Scholar]
  21. Zhong W., Uss A. S., Ferrari E., Lau J. Y., Hong Z. 2000; De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74:2017–2022 [CrossRef]
     [Google Scholar]
/content/journal/jgv/10.1099/vir.0.81802-0
Loading
Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis
J. Gen. Virol. 87, 2621 (2006); https://doi.org/10.1099/vir.0.81802-0
/content/journal/jgv/10.1099/vir.0.81802-0
/content/journal/jgv/10.1099/vir.0.81802-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error