1887

Journal of General Virology header logo

Volume 96, Issue 5

Last 20 aa of the West Nile virus NS1′ protein are responsible for its retention in cells and the formation of unique heat-stable dimers Free

Abstract

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1′, a C-terminal extended product of NS1 generated as the result of a −1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1′, resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1′, or a combination of both, remains undetermined. Here, we showed that WNV NS1′ formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1′-expressing plasmid showed that stability of NS1′ dimers was linked to the penultimate 10 aa. To examine the role of NS1′ heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1′ to create a WNV producing a truncated version of NS1′ lacking the last 20 aa, but not affecting the PRF. NS1′ protein produced by this mutant virus was secreted more efficiently than WT NS1′, indicating that the sequence of the last 20 aa of NS1′ was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNV, suggesting that full-length NS1′ was not essential for virus replication and for virulence in mice.

  • Received:
  • Accepted:
  • Published Online:
© 2015 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.000053
2015-05-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/5/1042.html?itemId=/content/journal/jgv/10.1099/vir.0.000053&mimeType=html&fmt=ahah

References

  1. Alcon-LePoder S., Sivard P., Drouet M. T., Talarmin A., Rice C., Flamand M. 2006; Secretion of flaviviral non-structural protein NS1: from diagnosis to pathogenesis. Novartis Found Symp 277:233–247 [View Article][PubMed]
     [Google Scholar]
  2. 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]
  3. Avirutnan P., Punyadee N., Noisakran S., Komoltri C., Thiemmeca S., Auethavornanan K., Jairungsri A., Kanlaya R., Tangthawornchaikul N. et al. 2006; Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis 193:1078–1088 [View Article][PubMed]
     [Google Scholar]
  4. Avirutnan P., Fuchs A., Hauhart R. E., Somnuke P., Youn S., Diamond M. S., Atkinson J. P. 2010; Antagonism of the complement component C4 by flavivirus nonstructural protein NS1. J Exp Med 207:793–806 [View Article][PubMed]
     [Google Scholar]
  5. Beckham J. D., Tyler K. 2009; Clinical manifestations of neurological disease. In West Nile Encephalitis Virus Infection pp. 69–95 Edited by Diamond M. S. New York: Springer; [View Article]
     [Google Scholar]
  6. Blitvich B. J., Mackenzie J. S., Coelen R. J., Howard M. J., Hall R. A. 1995; A novel complex formed between the flavivirus E and NS1 proteins: analysis of its structure and function. Arch Virol 140:145–156 [View Article][PubMed]
     [Google Scholar]
  7. Chu P. W., Westaway E. G. 1992; Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA. Arch Virol 125:177–191 [View Article][PubMed]
     [Google Scholar]
  8. Chung K. M., Diamond M. S. 2008; Defining the levels of secreted non-structural protein NS1 after West Nile virus infection in cell culture and mice. J Med Virol 80:547–556 [View Article][PubMed]
     [Google Scholar]
  9. Chung K. M., Liszewski M. K., Nybakken G., Davis A. E., Townsend R. R., Fremont D. H., Atkinson J. P., Diamond M. S. 2006; West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H. Proc Natl Acad Sci U S A 103:19111–19116 [View Article][PubMed]
     [Google Scholar]
  10. Crook K. R., Miller-Kittrell M., Morrison C. R., Scholle F. 2014; Modulation of innate immune signaling by the secreted form of the West Nile virus NS1 glycoprotein. Virology 458-459:172–182 [View Article][PubMed]
     [Google Scholar]
  11. Doherty R. L., Carley J. G., MacKerras M. J., Marks E. N. 1963; Studies of arthropod-borne virus infections in Queensland. III. Isolation and characterization of virus strains from wild-caught mosquitoes in North Queensland. Aust J Exp Biol Med Sci 41:17–39 [View Article][PubMed]
     [Google Scholar]
  12. Falconar A. K., Young P. R. 1990; Immunoaffinity purification of native dimer forms of the flavivirus non-structural glycoprotein, NS1. J Virol Methods 30:323–332 [View Article][PubMed]
     [Google Scholar]
  13. Falgout B., Markoff L. 1995; Evidence that flavivirus NS1–NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum. J Virol 69:7232–7243[PubMed]
     [Google Scholar]
  14. Fan W. F., Mason P. W. 1990; Membrane association and secretion of the Japanese encephalitis virus NS1 protein from cells expressing NS1 cDNA. Virology 177:470–476 [View Article][PubMed]
     [Google Scholar]
  15. Firth A. E., Atkins J. F. 2009; A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1′ may derive from ribosomal frameshifting. Virol J 6:14–19 [View Article][PubMed]
     [Google Scholar]
  16. Flamand M., Megret F., Mathieu M., Lepault J., Rey F. A., Deubel V. 1999; Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol 73:6104–6110[PubMed]
     [Google Scholar]
  17. French E. L. 1952; Murray Valley encephalitis isolation and characterization of the aetiological agent. Med J Aust 1:100–103[PubMed]
     [Google Scholar]
  18. 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[PubMed]
     [Google Scholar]
  19. Hanna J. N., Ritchie S. A., Phillips D. A., Shield J., Bailey M. C., Mackenzie J. S., Poidinger M., McCall B. J., Mills P. J. 1996; An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 165:256–260[PubMed]
     [Google Scholar]
  20. 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]
  21. Khromykh A. A., Sedlak P. L., Guyatt K. J., Hall R. A., Westaway E. G. 1999; Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. J Virol 73:10272–10280[PubMed]
     [Google Scholar]
  22. Khromykh A. A., Sedlak P. L., Westaway E. G. 2000; cis- and trans-acting elements in flavivirus RNA replication. J Virol 74:3253–3263 [View Article][PubMed]
     [Google Scholar]
  23. Kurosu T., Chaichana P., Yamate M., Anantapreecha S., Ikuta K. 2007; Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1. Biochem Biophys Res Commun 362:1051–1056 [View Article][PubMed]
     [Google Scholar]
  24. Lanciotti R. S., Roehrig J. T., Deubel V., Smith J., Parker M., Steele K., Crise B., Volpe K. E., Crabtree M. B. et al. 1999; Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286:2333–2337 [View Article][PubMed]
     [Google Scholar]
  25. Leung J. Y., Pijlman G. P., Kondratieva N., Hyde J., Mackenzie J. M., Khromykh A. A. 2008; Role of nonstructural protein NS2A in flavivirus assembly. J Virol 82:4731–4741 [View Article][PubMed]
     [Google Scholar]
  26. Li K., Zhang S., Kronqvist M., Wallin M., Ekström M., Derse D., Garoff H. 2008; Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env. J Virol 82:7135–7143 [View Article][PubMed]
     [Google Scholar]
  27. Lin Y. L., Chen L. K., Liao C. L., Yeh C. T., Ma S. H., Chen J. L., Huang Y. L., Chen S. S., Chiang H. Y. 1998; DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice. J Virol 72:191–200[PubMed]
     [Google Scholar]
  28. Lindenbach B. D., Rice C. M. 1997; trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71:9608–9617[PubMed]
     [Google Scholar]
  29. Lindenbach B. D., Rice C. M. 2003; Molecular biology of flaviviruses. Adv Virus Res 59:23–61 [View Article][PubMed]
     [Google Scholar]
  30. Macdonald J., Tonry J., Hall R. A., Williams B., Palacios G., Ashok M. S., Jabado O., Clark D., Tesh R. B. et al. 2005; NS1 protein secretion during the acute phase of West Nile virus infection. J Virol 79:13924–13933 [View Article][PubMed]
     [Google Scholar]
  31. Mason P. W. 1989; Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169:354–364 [View Article][PubMed]
     [Google Scholar]
  32. Mason P. W., McAda P. C., Dalrymple J. M., Fournier M. J., Mason T. L. 1987; Expression of Japanese encephalitis virus antigens in Escherichia coli . Virology 158:361–372 [View Article][PubMed]
     [Google Scholar]
  33. Melian E. B., Hinzman E., Nagasaki T., Firth A. E., Wills N. M., Nouwens A. S., Blitvich B. J., Leung J., Funk A. et al. 2010; NS1′ of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness. J Virol 84:1641–1647 [View Article][PubMed]
     [Google Scholar]
  34. Melian E. B., Hall-Mendelin S., Du F., Owens N., Bosco-Lauth A. M., Nagasaki T., Rudd S., Brault A. C., Bowen R. A. et al. 2014; Programmed ribosomal frameshift alters expression of West Nile virus genes and facilitates virus replication in birds and mosquitoes. PLoS Pathog 10:e1004447 [View Article][PubMed]
     [Google Scholar]
  35. Muller D. A., Young P. R. 2013; The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res 98:192–208 [View Article][PubMed]
     [Google Scholar]
  36. Nowak T., Färber P. M., Wengler G., Wengler G. 1989; Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis. Virology 169:365–376 [View Article][PubMed]
     [Google Scholar]
  37. Petersen L. 2009; Global epidemiology of West Nile virus. In West Nile Encephalitis Virus Infection pp. 1–23 Edited by Diamond M. S. New York: Springer; [View Article]
     [Google Scholar]
  38. Pryor M. J., Wright P. J. 1993; The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specified by dengue virus. Virology 194:769–780 [View Article][PubMed]
     [Google Scholar]
  39. Pryor M. J., Wright P. J. 1994; Glycosylation mutants of dengue virus NS1 protein. J Gen Virol 75:1183–1187 [View Article][PubMed]
     [Google Scholar]
  40. Schlesinger J. J. 2006; Flavivirus nonstructural protein NS1: complementary surprises. Proc Natl Acad Sci U S A 103:18879–18880 [View Article][PubMed]
     [Google Scholar]
  41. Takamatsu Y., Okamoto K., Dinh D. T., Yu F., Hayasaka D., Uchida L., Nabeshima T., Buerano C. C., Morita K. 2014; NS1′ protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs. J Gen Virol 95:373–383 [View Article][PubMed]
     [Google Scholar]
  42. 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[PubMed]
     [Google Scholar]
  43. Westaway E. G., Mackenzie J. M., Khromykh A. A. 2002; Replication and gene function in Kunjin virus. Curr Top Microbiol Immunol 267:323–351[PubMed]
     [Google Scholar]
  44. Westaway E. G., Mackenzie J. M., Khromykh A. A. 2003; Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59:99–140 [View Article][PubMed]
     [Google Scholar]
  45. Wilson J. R., de Sessions P. F., Leon M. A., Scholle F. 2008; West Nile virus nonstructural protein 1 inhibits TLR3 signal transduction. J Virol 82:8262–8271 [View Article][PubMed]
     [Google Scholar]
  46. Winkler G., Randolph V. B., Cleaves G. R., Ryan T. E., Stollar V. 1988; Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162:187–196 [View Article][PubMed]
     [Google Scholar]
  47. Winkler G., Maxwell S. E., Ruemmler C., Stollar V. 1989; Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 171:302–305 [View Article][PubMed]
     [Google Scholar]
  48. Ye Q., Li X. F., Zhao H., Li S. H., Deng Y. Q., Cao R. Y., Song K. Y., Wang H. J., Hua R. H. et al. 2012; A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1′ formation and contributes to attenuation. J Gen Virol 93:1959–1964 [View Article][PubMed]
     [Google Scholar]
  49. Youn S., Li T., McCune B. T., Edeling M. A., Fremont D. H., Cristea I. M., Diamond M. S. 2012; Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus. J Virol 86:7360–7371 [View Article][PubMed]
     [Google Scholar]
  50. Youn S., Ambrose R. L., Mackenzie J. M., Diamond M. S. 2013; Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis. Virol J 10:339 [View Article][PubMed]
     [Google Scholar]
  51. Young L. B., Melian E. B., Khromykh A. A. 2013; NS1′ colocalizes with NS1 and can substitute for NS1 in West Nile virus replication. J Virol 87:9384–9390 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.000053
Loading
Last 20 aa of the West Nile virus NS1′ protein are responsible for its retention in cells and the formation of unique heat-stable dimers
J. Gen. Virol. 96, 1042 (2015); https://doi.org/10.1099/vir.0.000053
/content/journal/jgv/10.1099/vir.0.000053
/content/journal/jgv/10.1099/vir.0.000053
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