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. 1997 Oct;71(10):7353–7360. doi: 10.1128/jvi.71.10.7353-7360.1997

Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.

J A Lawton 1, C Q Zeng 1, S K Mukherjee 1, J Cohen 1, M K Estes 1, B V Prasad 1
PMCID: PMC192080  PMID: 9311813

Abstract

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.

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Selected References

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  1. Brüssow H., Bruttin A., Marc-Martin S. Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine. J Virol. 1990 Aug;64(8):3635–3642. doi: 10.1128/jvi.64.8.3635-3642.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cohen J. Ribonucleic acid polymerase activity associated with purified calf rotavirus. J Gen Virol. 1977 Sep;36(3):395–402. doi: 10.1099/0022-1317-36-3-395. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Crowther R. A. Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs. Philos Trans R Soc Lond B Biol Sci. 1971 May 27;261(837):221–230. doi: 10.1098/rstb.1971.0054. [DOI] [PubMed] [Google Scholar]
  5. Dubochet J., Adrian M., Chang J. J., Homo J. C., Lepault J., McDowall A. W., Schultz P. Cryo-electron microscopy of vitrified specimens. Q Rev Biophys. 1988 May;21(2):129–228. doi: 10.1017/s0033583500004297. [DOI] [PubMed] [Google Scholar]
  6. Fuller S. D. The T=4 envelope of Sindbis virus is organized by interactions with a complementary T=3 capsid. Cell. 1987 Mar 27;48(6):923–934. doi: 10.1016/0092-8674(87)90701-x. [DOI] [PubMed] [Google Scholar]
  7. Labbé M., Baudoux P., Charpilienne A., Poncet D., Cohen J. Identification of the nucleic acid binding domain of the rotavirus VP2 protein. J Gen Virol. 1994 Dec;75(Pt 12):3423–3430. doi: 10.1099/0022-1317-75-12-3423. [DOI] [PubMed] [Google Scholar]
  8. Labbé M., Charpilienne A., Crawford S. E., Estes M. K., Cohen J. Expression of rotavirus VP2 produces empty corelike particles. J Virol. 1991 Jun;65(6):2946–2952. doi: 10.1128/jvi.65.6.2946-2952.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Lawton J. A., Estes M. K., Prasad B. V. Three-dimensional visualization of mRNA release from actively transcribing rotavirus particles. Nat Struct Biol. 1997 Feb;4(2):118–121. doi: 10.1038/nsb0297-118. [DOI] [PubMed] [Google Scholar]
  11. Lawton J. A., Venkataram Prasad B. V. Automated software package for icosahedral virus reconstruction. J Struct Biol. 1996 Jan-Feb;116(1):209–215. doi: 10.1006/jsbi.1996.0032. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Mitchell D. B., Both G. W. Completion of the genomic sequence of the simian rotavirus SA11: nucleotide sequences of segments 1, 2, and 3. Virology. 1990 Jul;177(1):324–331. doi: 10.1016/0042-6822(90)90487-c. [DOI] [PubMed] [Google Scholar]
  14. Olson N. H., Baker T. S. Magnification calibration and the determination of spherical virus diameters using cryo-microscopy. Ultramicroscopy. 1989 Jul-Aug;30(3):281–297. doi: 10.1016/0304-3991(89)90057-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Prasad B. V., Rothnagel R., Zeng C. Q., Jakana J., Lawton J. A., Chiu W., Estes M. K. Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. Nature. 1996 Aug 1;382(6590):471–473. doi: 10.1038/382471a0. [DOI] [PubMed] [Google Scholar]
  17. Prasad B. V., Wang G. J., Clerx J. P., Chiu W. Three-dimensional structure of rotavirus. J Mol Biol. 1988 Jan 20;199(2):269–275. doi: 10.1016/0022-2836(88)90313-0. [DOI] [PubMed] [Google Scholar]
  18. Prasad B. V., Yamaguchi S., Roy P. Three-dimensional structure of single-shelled bluetongue virus. J Virol. 1992 Apr;66(4):2135–2142. doi: 10.1128/jvi.66.4.2135-2142.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaw A. L., Rothnagel R., Zeng C. Q., Lawton J. A., Ramig R. F., Estes M. K., Prasad B. V. Rotavirus structure: interactions between the structural proteins. Arch Virol Suppl. 1996;12:21–27. doi: 10.1007/978-3-7091-6553-9_3. [DOI] [PubMed] [Google Scholar]
  20. Shaw A. L., Samal S. K., Subramanian K., Prasad B. V. The structure of aquareovirus shows how the different geometries of the two layers of the capsid are reconciled to provide symmetrical interactions and stabilization. Structure. 1996 Aug 15;4(8):957–967. doi: 10.1016/s0969-2126(96)00102-5. [DOI] [PubMed] [Google Scholar]
  21. Yeager M., Dryden K. A., Olson N. H., Greenberg H. B., Baker T. S. Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J Cell Biol. 1990 Jun;110(6):2133–2144. doi: 10.1083/jcb.110.6.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zeng C. Q., Labbé M., Cohen J., Prasad B. V., Chen D., Ramig R. F., Estes M. K. Characterization of rotavirus VP2 particles. Virology. 1994 May 15;201(1):55–65. doi: 10.1006/viro.1994.1265. [DOI] [PubMed] [Google Scholar]
  23. Zeng C. Q., Wentz M. J., Cohen J., Estes M. K., Ramig R. F. Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol. 1996 May;70(5):2736–2742. doi: 10.1128/jvi.70.5.2736-2742.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou Z. H., Prasad B. V., Jakana J., Rixon F. J., Chiu W. Protein subunit structures in the herpes simplex virus A-capsid determined from 400 kV spot-scan electron cryomicroscopy. J Mol Biol. 1994 Sep 30;242(4):456–469. doi: 10.1006/jmbi.1994.1594. [DOI] [PubMed] [Google Scholar]

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