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. 1996 Sep;70(9):5832–5839. doi: 10.1128/jvi.70.9.5832-5839.1996

Trypsin activation pathway of rotavirus infectivity.

C F Arias 1, P Romero 1, V Alvarez 1, S López 1
PMCID: PMC190599  PMID: 8709201

Abstract

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

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Selected References

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  1. Almeida J. D., Hall T., Banatvala J. E., Totterdell B. M., Chrystie I. L. The effect of trypsin on the growth of rotavirus. J Gen Virol. 1978 Jul;40(1):213–218. doi: 10.1099/0022-1317-40-1-213. [DOI] [PubMed] [Google Scholar]
  2. Anthony I. D., Bullivant S., Dayal S., Bellamy A. R., Berriman J. A. Rotavirus spike structure and polypeptide composition. J Virol. 1991 Aug;65(8):4334–4340. doi: 10.1128/jvi.65.8.4334-4340.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arias C. F., Garcia G., Lopez S. Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide. J Virol. 1989 Dec;63(12):5393–5398. doi: 10.1128/jvi.63.12.5393-5398.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arias C. F., Lizano M., López S. Synthesis in Escherichia coli and immunological characterization of a polypeptide containing the cleavage sites associated with trypsin enhancement of rotavirus SA11 infectivity. J Gen Virol. 1987 Mar;68(Pt 3):633–642. doi: 10.1099/0022-1317-68-3-633. [DOI] [PubMed] [Google Scholar]
  5. Babiuk L. A., Mohammed K., Spence L., Fauvel M., Petro R. Rotavirus isolation and cultivation in the presence of trypsin. J Clin Microbiol. 1977 Dec;6(6):610–617. doi: 10.1128/jcm.6.6.610-617.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carr C. M., Kim P. S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993 May 21;73(4):823–832. doi: 10.1016/0092-8674(93)90260-w. [DOI] [PubMed] [Google Scholar]
  7. Clark S. M., Barnett B. B., Spendlove R. S. Production of high-titer bovine rotavirus with trypsin. J Clin Microbiol. 1979 Mar;9(3):413–417. doi: 10.1128/jcm.9.3.413-417.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark S. M., Roth J. R., Clark M. L., Barnett B. B., Spendlove R. S. Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. J Virol. 1981 Sep;39(3):816–822. doi: 10.1128/jvi.39.3.816-822.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Espejo R. T., López S., Arias C. Structural polypeptides of simian rotavirus SA11 and the effect of trypsin. J Virol. 1981 Jan;37(1):156–160. doi: 10.1128/jvi.37.1.156-160.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Espejo R., Martínez E., López S., Muñoz O. Different polypeptide composition of two human rotavirus types. Infect Immun. 1980 Apr;28(1):230–237. doi: 10.1128/iai.28.1.230-237.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Estes M. K., Cohen J. Rotavirus gene structure and function. Microbiol Rev. 1989 Dec;53(4):410–449. doi: 10.1128/mr.53.4.410-449.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Estes M. K., Graham D. Y., Mason B. B. Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J Virol. 1981 Sep;39(3):879–888. doi: 10.1128/jvi.39.3.879-888.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fernandes J., Tang D., Leone G., Lee P. W. Binding of reovirus to receptor leads to conformational changes in viral capsid proteins that are reversible upon virus detachment. J Biol Chem. 1994 Jun 24;269(25):17043–17047. [PubMed] [Google Scholar]
  14. Fricks C. E., Hogle J. M. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J Virol. 1990 May;64(5):1934–1945. doi: 10.1128/jvi.64.5.1934-1945.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fukuhara N., Yoshie O., Kitaoka S., Konno T. Role of VP3 in human rotavirus internalization after target cell attachment via VP7. J Virol. 1988 Jul;62(7):2209–2218. doi: 10.1128/jvi.62.7.2209-2218.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaljot K. T., Shaw R. D., Rubin D. H., Greenberg H. B. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis. J Virol. 1988 Apr;62(4):1136–1144. doi: 10.1128/jvi.62.4.1136-1144.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lamb R. A. Paramyxovirus fusion: a hypothesis for changes. Virology. 1993 Nov;197(1):1–11. doi: 10.1006/viro.1993.1561. [DOI] [PubMed] [Google Scholar]
  18. Liu M., Offit P. A., Estes M. K. Identification of the simian rotavirus SA11 genome segment 3 product. Virology. 1988 Mar;163(1):26–32. doi: 10.1016/0042-6822(88)90230-9. [DOI] [PubMed] [Google Scholar]
  19. López S., Arias C. F., Bell J. R., Strauss J. H., Espejo R. T. Primary structure of the cleavage site associated with trypsin enhancement of rotavirus SA11 infectivity. Virology. 1985 Jul 15;144(1):11–19. doi: 10.1016/0042-6822(85)90300-9. [DOI] [PubMed] [Google Scholar]
  20. López S., Arias C. F., Méndez E., Espejo R. T. Conservation in rotaviruses of the protein region containing the two sites associated with trypsin enhancement of infectivity. Virology. 1986 Oct 15;154(1):224–227. doi: 10.1016/0042-6822(86)90445-9. [DOI] [PubMed] [Google Scholar]
  21. Mackow E. R., Shaw R. D., Matsui S. M., Vo P. T., Dang M. N., Greenberg H. B. The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region. Proc Natl Acad Sci U S A. 1988 Feb;85(3):645–649. doi: 10.1073/pnas.85.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchell D. B., Both G. W. Complete nucleotide sequence of the simian rotavirus SA11 VP4 gene. Nucleic Acids Res. 1989 Mar 11;17(5):2122–2122. doi: 10.1093/nar/17.5.2122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Méndez E., Arias C. F., López S. Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture. J Virol. 1993 Sep;67(9):5253–5259. doi: 10.1128/jvi.67.9.5253-5259.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nandi P., Charpilienne A., Cohen J. Interaction of rotavirus particles with liposomes. J Virol. 1992 Jun;66(6):3363–3367. doi: 10.1128/jvi.66.6.3363-3367.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nishikawa K., Taniguchi K., Torres A., Hoshino Y., Green K., Kapikian A. Z., Chanock R. M., Gorziglia M. Comparative analysis of the VP3 gene of divergent strains of the rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus. J Virol. 1988 Nov;62(11):4022–4026. doi: 10.1128/jvi.62.11.4022-4026.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Padilla-Noriega L., Dunn S. J., López S., Greenberg H. B., Arias C. F. Identification of two independent neutralization domains on the VP4 trypsin cleavage products VP5* and VP8* of human rotavirus ST3. Virology. 1995 Jan 10;206(1):148–154. doi: 10.1016/s0042-6822(95)80029-8. [DOI] [PubMed] [Google Scholar]
  27. Prasad B. V., Burns J. W., Marietta E., Estes M. K., Chiu W. Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy. Nature. 1990 Feb 1;343(6257):476–479. doi: 10.1038/343476a0. [DOI] [PubMed] [Google Scholar]
  28. Rey F. A., Heinz F. X., Mandl C., Kunz C., Harrison S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature. 1995 May 25;375(6529):291–298. doi: 10.1038/375291a0. [DOI] [PubMed] [Google Scholar]
  29. Rossmann M. G. The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance. J Biol Chem. 1989 Sep 5;264(25):14587–14590. [PubMed] [Google Scholar]
  30. Ruiz M. C., Alonso-Torre S. R., Charpilienne A., Vasseur M., Michelangeli F., Cohen J., Alvarado F. Rotavirus interaction with isolated membrane vesicles. J Virol. 1994 Jun;68(6):4009–4016. doi: 10.1128/jvi.68.6.4009-4016.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shaw A. L., Rothnagel R., Chen D., Ramig R. F., Chiu W., Prasad B. V. Three-dimensional visualization of the rotavirus hemagglutinin structure. Cell. 1993 Aug 27;74(4):693–701. doi: 10.1016/0092-8674(93)90516-S. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Theil K. W., Bohl E. H., Agnes A. G. Cell culture propagation of porcine rotavirus (reovirus-like agent). Am J Vet Res. 1977 Nov;38(11):1765–1768. [PubMed] [Google Scholar]

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