Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence Free

Abstract

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV) and a weakly virulent Australian subtype (WNV). Five amino acid differences occur in WNV compared with WNV (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV prM to the same level as that of WNV, and enhanced the secretion of WNV prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

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2012-09-01
2024-03-28
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References

  1. Adams S. C., Broom A. K., Sammels L. M., Hartnett A. C., Howard M. J., Coelen R. J., Mackenzie J. S., Hall R. A. 1995; Glycosylation and antigenic variation among Kunjin virus isolates. Virology 206:49–56 [View Article][PubMed]
    [Google Scholar]
  2. Arnold K., Bordoli L., Kopp J., Schwede T. 2006; The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201 [View Article][PubMed]
    [Google Scholar]
  3. Audsley M., Edmonds J., Liu W., Mokhonov V., Mokhonova E., Melian E. B., Prow N., Hall R. A., Khromykh A. A. 2011; Virulence determinants between New York 99 and Kunjin strains of West Nile virus. Virology 414:63–73 [View Article][PubMed]
    [Google Scholar]
  4. Beasley D. W., Whiteman M. C., Zhang S., Huang C. Y., Schneider B. S., Smith D. R., Gromowski G. D., Higgs S., Kinney R. M., Barrett A. D. 2005; Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. J Virol 79:8339–8347 [View Article][PubMed]
    [Google Scholar]
  5. Bosshart H., Humphrey J., Deignan E., Davidson J., Drazba J., Yuan L., Oorschot V., Peters P. J., Bonifacino J. S. 1994; The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system. J Cell Biol 126:1157–1172 [View Article][PubMed]
    [Google Scholar]
  6. Brault A. C., Huang C. Y., Langevin S. A., Kinney R. M., Bowen R. A., Ramey W. N., Panella N. A., Holmes E. C., Powers A. M., Miller B. R. 2007; A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 39:1162–1166 [View Article][PubMed]
    [Google Scholar]
  7. Chang G. J., Davis B. S., Hunt A. R., Holmes D. A., Kuno G. 2001; Flavivirus DNA vaccines: current status and potential. Ann N Y Acad Sci 951:272–285 [View Article][PubMed]
    [Google Scholar]
  8. Ellgaard L., Helenius A. 2003; Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4:181–191 [View Article][PubMed]
    [Google Scholar]
  9. Goto A., Yoshii K., Obara M., Ueki T., Mizutani T., Kariwa H., Takashima I. 2005; Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion. Vaccine 23:3043–3052 [View Article][PubMed]
    [Google Scholar]
  10. Hall R. A., Broom A. K., Smith D. W., Mackenzie J. S. 2002; The ecology and epidemiology of Kunjin virus. Curr Top Microbiol Immunol 267:253–269 [View Article][PubMed]
    [Google Scholar]
  11. Hanna S. L., Pierson T. C., Sanchez M. D., Ahmed A. A., Murtadha M. M., Doms R. W. 2005; N-linked glycosylation of West Nile virus envelope proteins influences particle assembly and infectivity. J Virol 79:13262–13274 [View Article][PubMed]
    [Google Scholar]
  12. Hunt A. R., Hall R. A., Kerst A. J., Nasci R. S., Savage H. M., Panella N. A., Gottfried K. L., Burkhalter K. L., Roehrig J. T. 2002; Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay. J Clin Microbiol 40:2023–2030 [View Article][PubMed]
    [Google Scholar]
  13. Keelapang P., Sriburi R., Supasa S., Panyadee N., Songjaeng A., Jairungsri A., Puttikhunt C., Kasinrerk W., Malasit P., Sittisombut N. 2004; Alterations of pr-M cleavage and virus export in pr-M junction chimeric dengue viruses. J Virol 78:2367–2381 [View Article][PubMed]
    [Google Scholar]
  14. Khromykh A. A., Kenney M. T., Westaway E. G. 1998; trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. J Virol 72:7270–7279[PubMed]
    [Google Scholar]
  15. Kim J. H., Johannes L., Goud B., Antony C., Lingwood C. A., Daneman R., Grinstein S. 1998; Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release. Proc Natl Acad Sci U S A 95:2997–3002 [View Article][PubMed]
    [Google Scholar]
  16. Konishi E., Mason P. W. 1993; Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol 67:1672–1675[PubMed]
    [Google Scholar]
  17. Lanciotti R. S., Ebel G. D., Deubel V., Kerst A. J., Murri S., Meyer R., Bowen M., McKinney N., Morrill W. E. other authors 2002; Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 298:96–105 [View Article][PubMed]
    [Google Scholar]
  18. Li L., Lok S. M., Yu I. M., Zhang Y., Kuhn R. J., Chen J., Rossmann M. G. 2008; The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319:1830–1834 [View Article][PubMed]
    [Google Scholar]
  19. Lin Y. J., Wu S. C. 2005; Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus. J Virol 79:8535–8544 [View Article][PubMed]
    [Google Scholar]
  20. Lin Y.-J., Peng J.-G., Wu S.-C. 2010; Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM) protein. J Biomed Sci 17:39 [View Article][PubMed]
    [Google Scholar]
  21. Liu W. J., Chen H. B., Khromykh A. A. 2003; Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication. J Virol 77:7804–7813 [View Article][PubMed]
    [Google Scholar]
  22. Lorenz I. C., Allison S. L., Heinz F. X., Helenius A. 2002; Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 76:5480–5491 [View Article][PubMed]
    [Google Scholar]
  23. Molloy S. S., Bresnahan P. A., Leppla S. H., Klimpel K. R., Thomas G. 1992; Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402[PubMed]
    [Google Scholar]
  24. Molloy S. S., Anderson E. D., Jean F., Thomas G. 1999; Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9:28–35 [View Article][PubMed]
    [Google Scholar]
  25. Mukherjee S., Lin T. Y., Dowd K. A., Manhart C. J., Pierson T. C. 2011; The infectivity of prM-containing partially mature West Nile virus does not require the activity of cellular furin-like proteases. J Virol 85:12067–12072 [View Article][PubMed]
    [Google Scholar]
  26. Prow N. A., May F. J., Westlake D. J., Hurrelbrink R. J., Biron R. M., Leung J. Y., McMinn P. C., Clark D. C., Mackenzie J. S. other authors 2011; Determinants of attenuation in the envelope protein of the flavivirus Alfuy. J Gen Virol 92:2286–2296 [View Article][PubMed]
    [Google Scholar]
  27. Pryor M. J., Azzola L., Wright P. J., Davidson A. D. 2004; Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly. J Gen Virol 85:3627–3636 [View Article][PubMed]
    [Google Scholar]
  28. Reed L. J., Muench H. 1938; A simple method for estimating fifty per cent end points.. Am J Hygiene 27:493–497 [View Article][PubMed]
    [Google Scholar]
  29. Scherret J. H., Mackenzie J. S., Khromykh A. A., Hall R. A. 2001a; Biological significance of glycosylation of the envelope protein of Kunjin virus. Ann N Y Acad Sci 951:361–363 [View Article][PubMed]
    [Google Scholar]
  30. Scherret J. H., Poidinger M., Mackenzie J. S., Broom A. K., Deubel V., Lipkin W. I., Briese T., Gould E. A., Hall R. A. 2001b; The relationships between West Nile and Kunjin viruses. Emerg Infect Dis 7:697–705[PubMed] [CrossRef]
    [Google Scholar]
  31. Setoh Y. X., Hobson-Peters J., Prow N. A., Young P. R., Hall R. A. 2011; Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen. J Virol Methods 175:20–27 [View Article][PubMed]
    [Google Scholar]
  32. Sousa M., Parodi A. J. 1995; The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. EMBO J 14:4196–4203[PubMed]
    [Google Scholar]
  33. Stadler K., Allison S. L., Schalich J., Heinz F. X. 1997; Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71:8475–8481[PubMed]
    [Google Scholar]
  34. Tan T. T., Bhuvanakantham R., Li J., Howe J., Ng M. L. 2009; Tyrosine 78 of premembrane protein is essential for assembly of West Nile virus. J Gen Virol 90:1081–1092 [View Article][PubMed]
    [Google Scholar]
  35. Vey M., Schäfer W., Berghöfer S., Klenk H. D., Garten W. 1994; Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol 127:1829–1842 [View Article][PubMed]
    [Google Scholar]
  36. Voorhees P., Deignan E., van Donselaar E., Humphrey J., Marks M. S., Peters P. J., Bonifacino J. S. 1995; An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J 14:4961–4975[PubMed]
    [Google Scholar]
  37. Yoshii K., Konno A., Goto A., Nio J., Obara M., Ueki T., Hayasaka D., Mizutani T., Kariwa H., Takashima I. 2004; Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion. J Gen Virol 85:3049–3058 [View Article][PubMed]
    [Google Scholar]
  38. Yoshii K., Igarashi M., Ichii O., Yokozawa K., Ito K., Kariwa H., Takashima I. 2012; A conserved region in the prM protein is a critical determinant in the assembly of flavivirus particles. J Gen Virol 93:27–38 [View Article][PubMed]
    [Google Scholar]
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