1887

Abstract

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F subunit and a C-terminal, membrane-anchored F subunit. There is one cysteine in F and there are 15 cysteines in F, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F) and C241 (F). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4′-maleimidylstilbene 2,2′-disulfonic acid (AMS) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.068122-0
2014-12-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/12/2820.html?itemId=/content/journal/jgv/10.1099/vir.0.068122-0&mimeType=html&fmt=ahah

References

  1. Abell B. A., Brown D. T. 1993; Sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface. J Virol 67:5496–5501[PubMed]
    [Google Scholar]
  2. Abou-Jaoudé G., Sureau C. 2007; Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol–disulfide exchange. J Virol 81:13057–13066 [View Article][PubMed]
    [Google Scholar]
  3. Anthony R. P., Paredes A. M., Brown D. T. 1992; Disulfide bonds are essential for the stability of the Sindbis virus envelope. Virology 190:330–336 [View Article][PubMed]
    [Google Scholar]
  4. Baker K. A., Dutch R. E., Lamb R. A., Jardetzky T. S. 1999; Structural basis for paramyxovirus-mediated membrane fusion. Mol Cell 3:309–319 [View Article][PubMed]
    [Google Scholar]
  5. Barbouche R., Miquelis R., Jones I. M., Fenouillet E. 2003; Protein–disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion. J Biol Chem 278:3131–3136 [View Article][PubMed]
    [Google Scholar]
  6. Blissard G. W., Wenz J. R. 1992; Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66:6829–6835[PubMed]
    [Google Scholar]
  7. Carr C. M., Chaudhry C., Kim P. S. 1997; Influenza hemagglutinin is spring-loaded by a metastable native conformation. Proc Natl Acad Sci U S A 94:14306–14313 [View Article][PubMed]
    [Google Scholar]
  8. Day N. D., Branigan P. J., Liu C., Gutshall L. L., Luo J., Melero J. A., Sarisky R. T., Del Vecchio A. M. 2006; Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function. Virol J 3:34 [View Article][PubMed]
    [Google Scholar]
  9. Dutch R. E., Jardetzky T. S., Lamb R. A. 2000; Virus membrane fusion proteins: biological machines that undergo a metamorphosis. Biosci Rep 20:597–612 [View Article][PubMed]
    [Google Scholar]
  10. Federici B. A. 1997; Baculovirus pathogenesis. In The Baculoviruses pp. 33–59 Edited by Miller L. K. New York: Plenum Press; [View Article]
    [Google Scholar]
  11. Fenouillet E., Barbouche R., Jones I. M. 2007; Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus. Antioxid Redox Signal 9:1009–1034 [View Article][PubMed]
    [Google Scholar]
  12. Herniou E. A., Luque T., Chen X., Vlak J. M., Winstanley D., Cory J. S., O’Reilly D. R. 2001; Use of whole genome sequence data to infer baculovirus phylogeny. J Virol 75:8117–8126 [View Article][PubMed]
    [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. 1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59 [View Article][PubMed]
    [Google Scholar]
  14. IJkel W. F. J., Westenberg M., Goldbach R. W., Blissard G. W., Vlak J. M., Zuidema D. 2000; A novel baculovirus envelope fusion protein with a proprotein convertase cleavage site. Virology 275:30–41 [View Article][PubMed]
    [Google Scholar]
  15. Iwata S., Schmidt A. C., Titani K., Suzuki M., Kido H., Gotoh B., Hamaguchi M., Nagai Y. 1994; Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus. J Virol 68:3200–3206[PubMed]
    [Google Scholar]
  16. Jain S., McGinnes L. W., Morrison T. G. 2007; Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein. J Virol 81:2328–2339 [View Article][PubMed]
    [Google Scholar]
  17. Jain S., McGinnes L. W., Morrison T. G. 2009; Role of thiol/disulfide exchange in Newcastle disease virus entry. J Virol 83:241–249 [View Article][PubMed]
    [Google Scholar]
  18. Jehle J. A., Blissard G. W., Bonning B. C., Cory J. S., Herniou E. A., Rohrmann G. F., Theilmann D. A., Thiem S. M., Vlak J. M. 2006; On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151:1257–1266 [View Article][PubMed]
    [Google Scholar]
  19. Krey T., Thiel H.-J., Rümenapf T. 2005; Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry. J Virol 79:4191–4200 [View Article][PubMed]
    [Google Scholar]
  20. Lamb R. A., Jardetzky T. S. 2007; Structural basis of viral invasion: lessons from paramyxovirus F. Curr Opin Struct Biol 17:427–436 [View Article][PubMed]
    [Google Scholar]
  21. Li Z., Blissard G. W. 2010; Baculovirus GP64 disulfide bonds: the intermolecular disulfide bond of Autographa californica multicapsid nucleopolyhedrovirus GP64 is not essential for membrane fusion and virion budding. J Virol 84:8584–8595 [View Article][PubMed]
    [Google Scholar]
  22. Long G., Westenberg M., Wang H., Vlak J. M., Hu Z. 2006; Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein. J Gen Virol 87:839–846 [View Article][PubMed]
    [Google Scholar]
  23. Lung O., Westenberg M., Vlak J. M., Zuidema D., Blissard G. W. 2002; Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64. J Virol 76:5729–5736 [View Article][PubMed]
    [Google Scholar]
  24. Lung O. Y., Cruz-Alvarez M., Blissard G. W. 2003; Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, is a viral pathogenicity factor. J Virol 77:328–339 [View Article][PubMed]
    [Google Scholar]
  25. Maar D., Harmon B., Chu D., Schulz B., Aguilar H. C., Lee B., Negrete O. A. 2012; Cysteines in the stalk of the Nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation. J Virol 86:6632–6642 [View Article][PubMed]
    [Google Scholar]
  26. Markosyan R. M., Melikyan G. B., Cohen F. S. 2001; Evolution of intermediates of influenza virus hemagglutinin-mediated fusion revealed by kinetic measurements of pore formation. Biophys J 80:812–821 [View Article][PubMed]
    [Google Scholar]
  27. Markovic I., Pulyaeva H., Sokoloff A., Chernomordik L. V. 1998; Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates. J Cell Biol 143:1155–1166 [View Article][PubMed]
    [Google Scholar]
  28. McCaffrey K., Boo I., Tewierek K., Edmunds M. L., Poumbourios P., Drummer H. E. 2012; Role of conserved cysteine residues in hepatitis C virus glycoprotein e2 folding and function. J Virol 86:3961–3974 [View Article][PubMed]
    [Google Scholar]
  29. Pearson M. N., Groten C., Rohrmann G. F. 2000; Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J Virol 74:6126–6131 [View Article][PubMed]
    [Google Scholar]
  30. Rohrmann G. F., Karplus P. A. 2001; Relatedness of baculovirus and gypsy retrotransposon envelope proteins. BMC Evol Biol 1:1 [View Article][PubMed]
    [Google Scholar]
  31. Ryser H. J.-P., Levy E. M., Mandel R., DiSciullo G. J. 1994; Inhibition of human immunodeficiency virus infection by agents that interfere with thiol–disulfide interchange upon virus–receptor interaction. Proc Natl Acad Sci U S A 91:4559–4563 [View Article][PubMed]
    [Google Scholar]
  32. Schultz A. M., Henderson L. E., Oroszlan S. 1988; Fatty acylation of proteins. Annu Rev Cell Biol 4:611–647 [View Article][PubMed]
    [Google Scholar]
  33. Segal M. S., Bye J. M., Sambrook J. F., Gething M.-J. H. 1992; Disulfide bond formation during the folding of influenza virus hemagglutinin. J Cell Biol 118:227–244 [View Article][PubMed]
    [Google Scholar]
  34. Wang H., Deng F., Pijlman G. P., Chen X., Sun X., Vlak J. M., Hu Z. 2003; Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome. Virus Res 97:57–63 [View Article][PubMed]
    [Google Scholar]
  35. Wang M., Tan Y., Yin F., Deng F., Vlak J. M., Hu Z., Wang H. 2008a; The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F. J Virol 82:9800–9804 [View Article][PubMed]
    [Google Scholar]
  36. Wang M., Tan Y., Yin F., Deng F., Vlak J. M., Hu Z., Wang H. 2008b; The F protein of Helicoverpa armigera single nucleopolyhedrovirus can be substituted functionally with its homologue from Spodoptera exigua multiple nucleopolyhedrovirus. J Gen Virol 89:791–798 [View Article][PubMed]
    [Google Scholar]
  37. Wang M., Yin F., Shen S., Tan Y., Deng F., Vlak J. M., Hu Z., Wang H. 2010; Partial functional rescue of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus infectivity by replacement of F protein with GP64 from Autographa californica multicapsid nucleopolyhedrovirus. J Virol 84:11505–11514 [View Article][PubMed]
    [Google Scholar]
  38. Westenberg M., Wang H., IJkel W. F. J., Goldbach R. W., Vlak J. M., Zuidema D. 2002; Furin is involved in baculovirus envelope fusion protein activation. J Virol 76:178–184 [View Article][PubMed]
    [Google Scholar]
  39. Yin F., Wang M., Tan Y., Deng F., Vlak J. M., Hu Z., Wang H. 2008; A functional F analogue of Autographa californica nucleopolyhedrovirus GP64 from the Agrotis segetum granulovirus. J Virol 82:8922–8926 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.068122-0
Loading
/content/journal/jgv/10.1099/vir.0.068122-0
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