1887

Abstract

Infectious entry of the nonenveloped rotavirus virion requires proteolysis of the spike protein VP4 to mediate conformational changes associated with membrane penetration. We sequenced and characterized an isolate that was cultured in the absence of trypsin and found that it is more resistant to proteolysis than WT virus. A substitution mutation abrogates one of the defined trypsin-cleavage sites, suggesting that blocking proteolysis at this site reduces the overall kinetics of proteolysis. Kinetic analysis of the membrane penetration-associated conformational change indicated that the ‘fold-back’ of the mutant spike protein is slower than that of WT. Despite these apparent biochemical defects, the mutant virus replicates in an identical manner to the WT virus. These findings enhance an understanding of VP4 functions and establish new strategies to interrogate rotavirus cell entry.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.050674-0
2013-06-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/94/6/1296.html?itemId=/content/journal/jgv/10.1099/vir.0.050674-0&mimeType=html&fmt=ahah

References

  1. Arias C. F., Romero P., Alvarez V., López S. 1996; Trypsin activation pathway of rotavirus infectivity. J Virol 70:5832–5839[PubMed]
    [Google Scholar]
  2. Arnold, M., Patton, J. T. & McDonald, S. M. (2009). Culturing, storage, and quantification of rotaviruses. Cur Prot Microbiol Chapter 15, 15C.3
  3. Costello D. A., Lee D. W., Drewes J., Vasquez K. A., Kisler K., Wiesner U., Pollack L., Whittaker G. R., Daniel S. 2012; Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics. Anal Chem 84:8480–8489 [View Article][PubMed]
    [Google Scholar]
  4. Crawford S. E., Mukherjee S. K., Estes M. K., Lawton J. A., Shaw A. L., Ramig R. F., Prasad B. V. 2001; Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol 75:6052–6061 [View Article][PubMed]
    [Google Scholar]
  5. Dormitzer P. R., Greenberg H. B., Harrison S. C. 2001; Proteolysis of monomeric recombinant rotavirus VP4 yields an oligomeric VP5* core. J Virol 75:7339–7350 [View Article][PubMed]
    [Google Scholar]
  6. Dormitzer P. R., Nason E. B., Venkataram Prasad B. V., Harrison S. C. 2004; Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature 430:1053–1058 [View Article][PubMed]
    [Google Scholar]
  7. Estes M. K., Graham D. Y., Mason B. B. 1981; Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J Virol 39:879–888[PubMed]
    [Google Scholar]
  8. Floyd D. L., Ragains J. R., Skehel J. J., Harrison S. C., van Oijen A. M. 2008; Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci U S A 105:15382–15387 [View Article][PubMed]
    [Google Scholar]
  9. Gilbert J. M., Greenberg H. B. 1998; Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without. J Virol 72:5323–5327[PubMed]
    [Google Scholar]
  10. Greenberg H. B., Estes M. K. 2009; Rotaviruses: from pathogenesis to vaccination. Gastroenterology 136:1939–1951 [View Article][PubMed]
    [Google Scholar]
  11. Kim I. S., Trask S. D., Babyonyshev M., Dormitzer P. R., Harrison S. C. 2010; Effect of mutations in VP5 hydrophobic loops on rotavirus cell entry. J Virol 84:6200–6207 [View Article][PubMed]
    [Google Scholar]
  12. López S., Arias C. F., Bell J. R., Strauss J. H., Espejo R. T. 1985; Primary structure of the cleavage site associated with trypsin enhancement of rotavirus SA11 infectivity. Virology 144:11–19 [View Article][PubMed]
    [Google Scholar]
  13. Mrukowicz J. Z., Wetzel J. D., Goral M. I., Fogo A. B., Wright P. F., Dermody T. S. 1998; Viruses and cells with mutations affecting viral entry are selected during persistent rotavirus infections of MA104 cells. J Virol 72:3088–3097[PubMed]
    [Google Scholar]
  14. Padilla-Noriega L., Werner-Eckert R., Mackow E. R., Gorziglia M., Larralde G., Taniguchi K., Greenberg H. B. 1993; Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies. J Clin Microbiol 31:622–628[PubMed]
    [Google Scholar]
  15. Parashar U. D., Burton A., Lanata C., Boschi-Pinto C., Shibuya K., Steele D., Birmingham M., Glass R. I. 2009; Global mortality associated with rotavirus disease among children in 2004. J Infect Dis 200:Suppl 1S9–S15 [View Article][PubMed]
    [Google Scholar]
  16. Settembre E. C., Chen J. Z., Dormitzer P. R., Grigorieff N., Harrison S. C. 2011; Atomic model of an infectious rotavirus particle. EMBO J 30:408–416 [View Article][PubMed]
    [Google Scholar]
  17. Trask S. D., Kim I. S., Harrison S. C., Dormitzer P. R. 2010; A rotavirus spike protein conformational intermediate binds lipid bilayers. J Virol 84:1764–1770 [View Article][PubMed]
    [Google Scholar]
  18. Trask S. D., McDonald S. M., Patton J. T. 2012; Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol 10:165–177 [View Article][PubMed]
    [Google Scholar]
  19. Wolf M., Vo P. T., Greenberg H. B. 2011; Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes. J Virol 85:2492–2503 [View Article][PubMed]
    [Google Scholar]
  20. Yoder J. D., Trask S. D., Vo T. P., Binka M., Feng N., Harrison S. C., Greenberg H. B., Dormitzer P. R. 2009; VP5* rearranges when rotavirus uncoats. J Virol 83:11372–11377 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.050674-0
Loading
/content/journal/jgv/10.1099/vir.0.050674-0
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