1887

Abstract

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354–389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.013805-0
2009-12-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/12/2912.html?itemId=/content/journal/jgv/10.1099/vir.0.013805-0&mimeType=html&fmt=ahah

References

  1. Bartholomeusz A. I., Wright P. J. 1993; Synthesis of dengue virus RNA in vitro : initiation and the involvement of proteins NS3 and NS5. Arch Virol 128:111–121 [CrossRef]
    [Google Scholar]
  2. Clark D. C., Lobigs M., Lee E., Howard M. J., Clark K., Blitvich B. J., Hall R. A. 2007; In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein. J Gen Virol 88:1175–1183 [CrossRef]
    [Google Scholar]
  3. Coia G., Parker M. D., Speight G., Byrne M. E., Westaway E. G. 1988; Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J Gen Virol 69:1–21 [CrossRef]
    [Google Scholar]
  4. Guyatt K. J., Westaway E. G., Khromykh A. A. 2001; Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. J Virol Methods 92:37–44 [CrossRef]
    [Google Scholar]
  5. Hall R. A., Kay B. H., Burgess G. W., Clancy P., Fanning I. D. 1990; Epitope analysis of the envelope and non-structural glycoproteins of Murray Valley encephalitis virus. J Gen Virol 71:2923–2930 [CrossRef]
    [Google Scholar]
  6. Heinz F. X. 1986; Epitope mapping of flavivirus glycoproteins. Adv Virus Res 31:103–168
    [Google Scholar]
  7. Hobson-Peters J., Toye P., Sánchez M. D., Bossart K. N., Wang L. F., Clark D. C., Cheah W. Y., Hall R. A. 2008; A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection. J Gen Virol 89:3063–3072 [CrossRef]
    [Google Scholar]
  8. Lanciotti R. S., Roehrig J. T., Deubel V., Smith J., Parker M., Steele K., Crise B., Volpe K. E., Crabtree M. B. other authors 1999; Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286:2333–2337 [CrossRef]
    [Google Scholar]
  9. Mackenzie J. M., Kenney M. T., Westaway E. G. 2007; West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. J Gen Virol 88:1163–1168 [CrossRef]
    [Google Scholar]
  10. Malet H., Egloff M. P., Selisko B., Butcher R. E., Wright P. J., Roberts M., Gruez A., Sulzenbacher G., Vonrhein C. other authors 2007; Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5. J Biol Chem 282:10678–10689 [CrossRef]
    [Google Scholar]
  11. Malet H., Massé N., Selisko B., Romette J. L., Alvarez K., Guillemot J. C., Tolou H., Yap T. L., Vasudevan S. other authors 2008; The flavivirus polymerase as a target for drug discovery. Antiviral Res 80:23–35 [CrossRef]
    [Google Scholar]
  12. Nomaguchi M., Teramoto T., Yu L., Markoff L., Padmanabhan R. 2004; Requirements for West Nile virus (−)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli . J Biol Chem 279:12141–12151 [CrossRef]
    [Google Scholar]
  13. Roosendaal J., Westaway E. G., Khromykh A. A., Mackenzie J. M. 2006; Regulated cleavages at the West Nile virus NS4A–2K–NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein. J Virol 80:4623–4632 [CrossRef]
    [Google Scholar]
  14. Selisko B., Dutartre H., Guillemot J. C., Debarnot C., Benarroch D., Khromykh A. A., Despres P., Egloff M. P., Canard B. 2006; Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases. Virology 351:145–158 [CrossRef]
    [Google Scholar]
  15. Tan B. H., Fu J., Sugrue R. J., Yap E. H., Chan Y. C., Tan Y. H. 1996; Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology 216:317–325 [CrossRef]
    [Google Scholar]
  16. Westaway E. G., Mackenzie J. M., Kenney M. T., Jones M. K., Khromykh A. A. 1997; Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J Virol 71:6650–6661
    [Google Scholar]
  17. Westaway E. G., Mackenzie J. M., Khromykh A. A. 2002; Replication and gene function in Kunjin virus. Curr Top Microbiol Immunol 267:323–351
    [Google Scholar]
  18. Wilson K. M., Gerometta M., Rylatt D. P., Bundesen P. G., McPhee D. A., Hillyard C. J., Kemp B. E. 1991; Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate. J Immunol Methods 138:111–119 [CrossRef]
    [Google Scholar]
  19. Wong S. J., Boyle R. H., Demarest V. L., Woodmansee A. N., Kramer L. D., Li H., Drebot M., Koski R. A., Fikrig E. other authors 2003; Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St Louis encephalitis virus infections and from flavivirus vaccination. J Clin Microbiol 41:4217–4223 [CrossRef]
    [Google Scholar]
  20. Zhou Y., Ray D., Zhao Y., Dong H., Ren S., Li Z., Guo Y., Bernard K. A., Shi P. Y., Li H. 2007; Structure and function of flavivirus NS5 methyltransferase. J Virol 81:3891–3903 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.013805-0
Loading
/content/journal/jgv/10.1099/vir.0.013805-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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