1887

Abstract

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001346
2019-11-04
2019-11-15
Loading full text...

Full text loading...

References

  1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW et al. The global distribution and burden of dengue. Nature 2013;496: 504– 507 [CrossRef]
    [Google Scholar]
  2. Lai S, Huang Z, Zhou H, Anders KL, Perkins TA et al. The changing epidemiology of dengue in China, 1990-2014: a descriptive analysis of 25 years of nationwide surveillance data. BMC Med 2015;13: 100 [CrossRef]
    [Google Scholar]
  3. Wang C, Yang W, Fan J, Wang F, Jiang B et al. Spatial and temporal patterns of dengue in guangdong province of China. Asia Pac J Public Health 2015;27: NP844– NP853 [CrossRef]
    [Google Scholar]
  4. Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery. Antiviral Res 2009;81: 6– 15 [CrossRef]
    [Google Scholar]
  5. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 1990;44: 649– 688 [CrossRef]
    [Google Scholar]
  6. Mackenzie JM, Westaway EG. Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 2001;75: 10787– 10799 [CrossRef]
    [Google Scholar]
  7. Zhang Y, Corver J, Chipman PR, Zhang W, Pletnev SV et al. Structures of immature flavivirus particles. EMBO J 2003;22: 2604– 2613 [CrossRef]
    [Google Scholar]
  8. Zhang Y, Kaufmann B, Chipman PR, Kuhn RJ, Rossmann MG. Structure of immature West Nile virus. J Virol 2007;81: 6141– 6145 [CrossRef]
    [Google Scholar]
  9. Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002;108: 717– 725 [CrossRef]
    [Google Scholar]
  10. Stadler K, Allison SL, Schalich J, Heinz FX. Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 1997;71: 8475– 8481
    [Google Scholar]
  11. Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 2005;3: 13– 22 [CrossRef]
    [Google Scholar]
  12. Amberg SM, Nestorowicz A, McCourt DW, Rice CM. NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J Virol 1994;68: 3794– 3802
    [Google Scholar]
  13. Amberg SM, Rice CM. Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 1999;73: 8083– 8094
    [Google Scholar]
  14. Lobigs M. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc Natl Acad Sci USA 1993;90: 6218– 6222 [CrossRef]
    [Google Scholar]
  15. Lobigs M, Lee E, Ng ML, Pavy M, Lobigs P. A flavivirus signal peptide balances the catalytic activity of two proteases and thereby facilitates virus morphogenesis. Virology 2010;401: 80– 89 [CrossRef]
    [Google Scholar]
  16. Stocks CE, Lobigs M. Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and PRM. J Virol 1998;72: 2141– 2149
    [Google Scholar]
  17. Junglen S, Kopp A, Kurth A, Pauli G, Ellerbrok H et al. A new flavivirus and a new vector: characterization of a novel flavivirus isolated from uranotaenia mosquitoes from a tropical rain forest. J Virol 2009;83: 4462– 4468 [CrossRef]
    [Google Scholar]
  18. O'Neil KT, DeGrado WF. A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science 1990;250: 646– 651 [CrossRef]
    [Google Scholar]
  19. Rana J, Slon Campos JL, Leccese G, Francolini M, Bestagno M et al. Role of capsid anchor in the morphogenesis of Zika virus. J Virol 2018;92: [CrossRef]
    [Google Scholar]
  20. Pierson TC, Sánchez MD, Puffer BA, Ahmed AA, Geiss BJ et al. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection. Virology 2006;346: 53– 65 [CrossRef]
    [Google Scholar]
  21. VanBlargan LA, Davis KA, Dowd KA, Akey DL, Smith JL et al. Context-Dependent cleavage of the capsid protein by the West Nile virus protease modulates the efficiency of virus assembly. J Virol 2015;89: 8632– 8642 [CrossRef]
    [Google Scholar]
  22. Rana J, Slon Campos JL, Leccese G, Francolini M, Bestagno M et al. Role of capsid anchor in the morphogenesis of Zika virus. J Virol 2018;92: [CrossRef]
    [Google Scholar]
  23. Davis CW, Mattei LM, Nguyen HY, Ansarah-Sobrinho C, Doms RW et al. The location of asparagine-linked glycans on West Nile virions controls their interactions with CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin). J Biol Chem 2006;281: 37183– 37194 [CrossRef]
    [Google Scholar]
  24. Goto A, Yoshii K, Obara M, Ueki T, Mizutani T et al. Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion. Vaccine 2005;23: 3043– 3052 [CrossRef]
    [Google Scholar]
  25. Khromykh AA, Westaway EG. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 1997;71: 1497– 1505
    [Google Scholar]
  26. Scholle F, Girard YA, Zhao Q, Higgs S, Mason PW. Trans-Packaged West Nile virus-like particles: infectious properties in vitro and in infected mosquito vectors. J Virol 2004;78: 11605– 11614 [CrossRef]
    [Google Scholar]
  27. Whitby K, Pierson TC, Geiss B, Lane K, Engle M et al. Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo. J Virol 2005;79: 8698– 8706 [CrossRef]
    [Google Scholar]
  28. Yoshii K, Goto A, Kawakami K, Kariwa H, Takashima I. Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus. J Gen Virol 2008;89: 200– 211 [CrossRef]
    [Google Scholar]
  29. Paetzel M, Dalbey RE, Strynadka NC. Crystal structure of a bacterial signal peptidase in complex with a beta-lactam inhibitor. Nature 1998;396: 186– 190 [CrossRef]
    [Google Scholar]
  30. von Heijne G. Life and death of a signal peptide. Nature 1998;396: 111– 113 [CrossRef]
    [Google Scholar]
  31. Carrère-Kremer S, Montpellier C, Lorenzo L, Brulin B, Cocquerel L et al. Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions. J Biol Chem 2004;279: 41384– 41392 [CrossRef]
    [Google Scholar]
  32. Keelapang P, Sriburi R, Supasa S, Panyadee N, Songjaeng A et al. Alterations of pr-M cleavage and virus export in pr-M junction chimeric dengue viruses. J Virol 2004;78: 2367– 2381 [CrossRef]
    [Google Scholar]
  33. Heinz FX, Allison SL. Flavivirus structure and membrane fusion. Adv Virus Res 2003;59: 63– 97
    [Google Scholar]
  34. Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH et al. Conformational changes of the flavivirus E glycoprotein. Structure 2004;12: 1607– 1618 [CrossRef]
    [Google Scholar]
  35. Shapiro D, Brandt WE, Russell PK. Change involving a viral membrane glycoprotein during morphogenesis of group B arboviruses. Virology 1972;50: 906– 911 [CrossRef]
    [Google Scholar]
  36. Wengler G, Wengler G. Cell-Associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J Virol 1989;63: 2521– 2526
    [Google Scholar]
  37. Heinz FX, Stiasny K, Püschner-Auer G, Holzmann H, Allison SL et al. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 1994;198: 109– 117 [CrossRef]
    [Google Scholar]
  38. Junjhon J, Edwards TJ, Utaipat U, Bowman VD, Holdaway HA et al. Influence of pr-M cleavage on the heterogeneity of extracellular dengue virus particles. J Virol 2010;84: 8353– 8358 [CrossRef]
    [Google Scholar]
  39. Nelson S, Jost CA, Xu Q, Ess J, Martin JE et al. Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog 2008;4: e1000060 [CrossRef]
    [Google Scholar]
  40. Randolph VB, Winkler G, Stollar V. Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 1990;174: 450– 458 [CrossRef]
    [Google Scholar]
  41. Bennett KM, Gorham RD, Gusti V, Trinh L, Morikis D et al. Hybrid flagellin as a T cell independent vaccine scaffold. BMC Biotechnol 2015;15: 71 [CrossRef]
    [Google Scholar]
  42. Poggianella M, Slon Campos JL, Chan KR, Tan HC, Bestagno M et al. Dengue E protein domain III-based DNA immunisation induces strong antibody responses to all four viral serotypes. PLoS Negl Trop Dis 2015;9: e0003947 [CrossRef]
    [Google Scholar]
  43. Petris G, Bestagno M, Arnoldi F, Burrone OR. New tags for recombinant protein detection and O-glycosylation reporters. PLoS One 2014;9: e96700 [CrossRef]
    [Google Scholar]
  44. Mossenta M, Marchese S, Poggianella M, Slon Campos JL, Burrone OR. Role of N-glycosylation on Zika virus E protein secretion, viral assembly and infectivity. Biochem Biophys Res Commun 2017;492: 579– 586 [CrossRef]
    [Google Scholar]
  45. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989; p 1546
    [Google Scholar]
  46. Drozdetskiy A, Cole C, Procter J, Barton GJ. JPred4: a protein secondary structure prediction server. Nucleic Acids Res 2015;43: W389– W394 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001346
Loading
/content/journal/jgv/10.1099/jgv.0.001346
Loading

Data & Media loading...

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