1887

Abstract

VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitutethe intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-78-8-1949
1997-08-01
2022-05-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/78/8/9266993.html?itemId=/content/journal/jgv/10.1099/0022-1317-78-8-1949&mimeType=html&fmt=ahah

References

  1. Au K. -S., Chan W. -K., Burns J. W., Estes M. K. 1989; Receptor activity of rotavirus non-structural glycoprotein NS28. Journal of Virology 63:4553–4562
    [Google Scholar]
  2. Au K. -S., Mattion N. M., Estes M. K. 1993; A subviral particle binding domain on the rotavirus non-structural glycoprotein NS28. Virology 194:665–673
    [Google Scholar]
  3. Clapp L. L., Patton J. T. 1991; Rotavirus morphogenesis: domains in the major inner capsid protein essential for binding to single-shelled particles and for trimerization. Virology 180:697–708
    [Google Scholar]
  4. Crawford S. E., Labbé M., Cohen J., Burroughs M. H., Zhou Y. -J., Estes M. K. 1994; Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. Journal of Virology 68:5945–5952
    [Google Scholar]
  5. Davison J. A., Moss B. 1990; New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. Nucleic Acids Research 18:4285–4286
    [Google Scholar]
  6. González S. A., Affranchino J. L. 1995a; Assembly of doublelayered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6. Journal of General Virology 76: 2357–2360
    [Google Scholar]
  7. González S. A., Affranchino J. L. 1995b; Mutational analysis ofthe conserved cysteine residues in the simian immunodeficiency virus matrix protein. Virology 210:501–507
    [Google Scholar]
  8. González S. A., Affranchino J. L., Gelderblom H. G., Burny A. 1993; Assembly of the matrix protein of simian immunodeficiency virus into virus-like particles. Virology 194:548–556
    [Google Scholar]
  9. Gorziglia M., Hoshino Y., Nishikawa K., Maloy W. L., Jones R. W., Kapikian A. Z., Chanock R. M. 1988; Comparative sequence analysis of the genomic segment 6 of four rotaviruses each with a different subgroup specificity. Journal of General Virology 69: 1659–1669
    [Google Scholar]
  10. Greenberg H. B., McAuliffe V., Valdesuso J., Wyatt R., Flores J., Kalica A., Hoshino Y., Singh N. 1983; Serological analysis of the subgroup protein of rotavirus using monoclonal antibodies. Infection and Immunity 39: 91–99
    [Google Scholar]
  11. Labbá M., Charpilienne A., Crawford S. E., Estes M. K., Cohen J. 1991; Expression of rotavirus VP2 produces empty core-like particles. Journal of Virology 65:2946–2952
    [Google Scholar]
  12. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  13. Mansell E. A., Ramig R. F., Patton J. T. 1994; Temperature- sensitive lesions in the capsid proteins of the rotavirus mutants tsF and tsG that affect virion assembly. Virology 204:69–81
    [Google Scholar]
  14. Mattion N. M., Cohen J., Estes M. K. 1994; The rotavirus proteins. In Viral infections of the Gastrointestinal Tract pp 169–249 Kapikian A. Z. Edited by New York: Marcel Dekker;
    [Google Scholar]
  15. Meyer J. C., Bergmann C. C., Bellamy A. R. 1989; Interaction of rotavirus cores with the non-structural glycoprotein NS28. Virology 171:98–107
    [Google Scholar]
  16. Poruchynsky M. S., Maas D. R., Atkinson P. H. 1991; Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER. Journal of Cell Biology 114:651–661
    [Google Scholar]
  17. Prasad B. V. V., Chiu W. 1994; Structure of rotavirus. Current Topics in Microbiology and Immunology 185:9–29
    [Google Scholar]
  18. Prasad B. V. V., Wang G. J., Clerx J. P. M., Chiu W. 1988; Threedimensional structure of rotavirus. Journal of Molecular Biology 199:269–275
    [Google Scholar]
  19. Shaw A. L., Rothnagel R., Chen D., Ramig R. F., Chiu W., Prasad B. V. V. 1993; Three dimensional visualization of the rotavirus hemagglutinin structure. Cell 74:693–701
    [Google Scholar]
  20. Shen S., Burke B., Desselberger U. 1994; Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability. Journal of Virology 68:1682–1688
    [Google Scholar]
  21. Tosser G., Labbé M., Brémont M., Cohen J. 1992; Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses. Journal of Virology 66:5825–5831
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-78-8-1949
Loading
/content/journal/jgv/10.1099/0022-1317-78-8-1949
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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