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. 1997 Sep;71(9):6749–6756. doi: 10.1128/jvi.71.9.6749-6756.1997

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

P Isa 1, S López 1, L Segovia 1, C F Arias 1
PMCID: PMC191955  PMID: 9261399

Abstract

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.

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Selected References

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  1. 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]
  2. Burns J. W., Greenberg H. B., Shaw R. D., Estes M. K. Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11. J Virol. 1988 Jun;62(6):2164–2172. doi: 10.1128/jvi.62.6.2164-2172.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen D. Y., Estes M. K., Ramig R. F. Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope. J Virol. 1992 Jan;66(1):432–439. doi: 10.1128/jvi.66.1.432-439.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crawford S. E., Labbé M., Cohen J., Burroughs M. H., Zhou Y. J., Estes M. K. Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol. 1994 Sep;68(9):5945–5952. doi: 10.1128/jvi.68.9.5945-5952.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Fiore L., Greenberg H. B., Mackow E. R. The VP8 fragment of VP4 is the rhesus rotavirus hemagglutinin. Virology. 1991 Apr;181(2):553–563. doi: 10.1016/0042-6822(91)90888-i. [DOI] [PubMed] [Google Scholar]
  9. Fuentes-Pananá E. M., López S., Gorziglia M., Arias C. F. Mapping the hemagglutination domain of rotaviruses. J Virol. 1995 Apr;69(4):2629–2632. doi: 10.1128/jvi.69.4.2629-2632.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fukudome K., Yoshie O., Konno T. Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption. Virology. 1989 Sep;172(1):196–205. doi: 10.1016/0042-6822(89)90121-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Geourjon C., Deléage G. SOPM: a self-optimized method for protein secondary structure prediction. Protein Eng. 1994 Feb;7(2):157–164. doi: 10.1093/protein/7.2.157. [DOI] [PubMed] [Google Scholar]
  13. Giammarioli A. M., Mackow E. R., Fiore L., Greenberg H. B., Ruggeri F. M. Production and characterization of murine IgA monoclonal antibodies to the surface antigens of rhesus rotavirus. Virology. 1996 Nov 1;225(1):97–110. doi: 10.1006/viro.1996.0578. [DOI] [PubMed] [Google Scholar]
  14. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  15. Grodberg J., Davis K. L., Sykowski A. J. Alanine scanning mutagenesis of human erythropoietin identifies four amino acids which are critical for biological activity. Eur J Biochem. 1993 Dec 1;218(2):597–601. doi: 10.1111/j.1432-1033.1993.tb18413.x. [DOI] [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. Keljo D. J., Smith A. K. Characterization of binding of simian rotavirus SA-11 to cultured epithelial cells. J Pediatr Gastroenterol Nutr. 1988 Mar-Apr;7(2):249–256. doi: 10.1097/00005176-198803000-00015. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lim K., Ho J. X., Keeling K., Gilliland G. L., Ji X., Rüker F., Carter D. C. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. Protein Sci. 1994 Dec;3(12):2233–2244. doi: 10.1002/pro.5560031209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lizano M., López S., Arias C. F. The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus. J Virol. 1991 Mar;65(3):1383–1391. doi: 10.1128/jvi.65.3.1383-1391.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ludert J. E., Feng N., Yu J. H., Broome R. L., Hoshino Y., Greenberg H. B. Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo. J Virol. 1996 Jan;70(1):487–493. doi: 10.1128/jvi.70.1.487-493.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. López S., Arias C. F. Protein NS26 is highly conserved among porcine rotavirus strains. Nucleic Acids Res. 1993 Feb 25;21(4):1042–1042. doi: 10.1093/nar/21.4.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. López S., Espinosa R., Greenberg H. B., Arias C. F. Mapping the subgroup epitopes of rotavirus protein VP6. Virology. 1994 Oct;204(1):153–162. doi: 10.1006/viro.1994.1519. [DOI] [PubMed] [Google Scholar]
  24. López S., López I., Romero P., Méndez E., Soberón X., Arias C. F. Rotavirus YM gene 4: analysis of its deduced amino acid sequence and prediction of the secondary structure of the VP4 protein. J Virol. 1991 Jul;65(7):3738–3745. doi: 10.1128/jvi.65.7.3738-3745.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mackow E. R., Barnett J. W., Chan H., Greenberg H. B. The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant. J Virol. 1989 Apr;63(4):1661–1668. doi: 10.1128/jvi.63.4.1661-1668.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Mackow E. R., Yamanaka M. Y., Dang M. N., Greenberg H. B. DNA amplification-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites. Proc Natl Acad Sci U S A. 1990 Jan;87(2):518–522. doi: 10.1073/pnas.87.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Markwell M. A., Svennerholm L., Paulson J. C. Specific gangliosides function as host cell receptors for Sendai virus. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5406–5410. doi: 10.1073/pnas.78.9.5406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Merino E., Osuna J., Bolívar F., Soberón X. A general, PCR-based method for single or combinatorial oligonucleotide-directed mutagenesis on pUC/M13 vectors. Biotechniques. 1992 Apr;12(4):508–510. [PubMed] [Google Scholar]
  30. 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]
  31. Méndez E., Arias C. F., López S. Interactions between the two surface proteins of rotavirus may alter the receptor-binding specificity of the virus. J Virol. 1996 Feb;70(2):1218–1222. doi: 10.1128/jvi.70.2.1218-1222.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Patton J. T., Hua J., Mansell E. A. Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4. J Virol. 1993 Aug;67(8):4848–4855. doi: 10.1128/jvi.67.8.4848-4855.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paul R. W., Lee P. W. Glycophorin is the reovirus receptor on human erythrocytes. Virology. 1987 Jul;159(1):94–101. doi: 10.1016/0042-6822(87)90351-5. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rose G. D., Geselowitz A. R., Lesser G. J., Lee R. H., Zehfus M. H. Hydrophobicity of amino acid residues in globular proteins. Science. 1985 Aug 30;229(4716):834–838. doi: 10.1126/science.4023714. [DOI] [PubMed] [Google Scholar]
  37. Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
  38. Rost B., Sander C. Conservation and prediction of solvent accessibility in protein families. Proteins. 1994 Nov;20(3):216–226. doi: 10.1002/prot.340200303. [DOI] [PubMed] [Google Scholar]
  39. Rost B., Valencia A. Pitfalls of protein sequence analysis. Curr Opin Biotechnol. 1996 Aug;7(4):457–461. doi: 10.1016/s0958-1669(96)80124-8. [DOI] [PubMed] [Google Scholar]
  40. Sauter N. K., Hanson J. E., Glick G. D., Brown J. H., Crowther R. L., Park S. J., Skehel J. J., Wiley D. C. Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography. Biochemistry. 1992 Oct 13;31(40):9609–9621. doi: 10.1021/bi00155a013. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Shaw R. D., Vo P. T., Offit P. A., Coulson B. S., Greenberg H. B. Antigenic mapping of the surface proteins of rhesus rotavirus. Virology. 1986 Dec;155(2):434–451. doi: 10.1016/0042-6822(86)90205-9. [DOI] [PubMed] [Google Scholar]
  43. Stehle T., Yan Y., Benjamin T. L., Harrison S. C. Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature. 1994 May 12;369(6476):160–163. doi: 10.1038/369160a0. [DOI] [PubMed] [Google Scholar]
  44. Stultz C. M., White J. V., Smith T. F. Structural analysis based on state-space modeling. Protein Sci. 1993 Mar;2(3):305–314. doi: 10.1002/pro.5560020302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Taniguchi K., Urasawa T., Urasawa S. Species specificity and interspecies relatedness in VP4 genotypes demonstrated by VP4 sequence analysis of equine, feline, and canine rotavirus strains. Virology. 1994 May 1;200(2):390–400. doi: 10.1006/viro.1994.1203. [DOI] [PubMed] [Google Scholar]
  46. Vlasak R., Luytjes W., Spaan W., Palese P. Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4526–4529. doi: 10.1073/pnas.85.12.4526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weis W., Brown J. H., Cusack S., Paulson J. C., Skehel J. J., Wiley D. C. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988 Jun 2;333(6172):426–431. doi: 10.1038/333426a0. [DOI] [PubMed] [Google Scholar]
  48. White J. V., Stultz C. M., Smith T. F. Protein classification by stochastic modeling and optimal filtering of amino-acid sequences. Math Biosci. 1994 Jan;119(1):35–75. doi: 10.1016/0025-5564(94)90004-3. [DOI] [PubMed] [Google Scholar]
  49. Williams W. V., Kieber-Emmons T., Weiner D. B., Rubin D. H., Greene M. I. Contact residues and predicted structure of the reovirus type 3-receptor interaction. J Biol Chem. 1991 May 15;266(14):9241–9250. [PubMed] [Google Scholar]
  50. Yolken R. H., Willoughby R., Wee S. B., Miskuff R., Vonderfecht S. Sialic acid glycoproteins inhibit in vitro and in vivo replication of rotaviruses. J Clin Invest. 1987 Jan;79(1):148–154. doi: 10.1172/JCI112775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou Y. J., Burns J. W., Morita Y., Tanaka T., Estes M. K. Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure. J Virol. 1994 Jun;68(6):3955–3964. doi: 10.1128/jvi.68.6.3955-3964.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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