Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1991 Apr;65(4):2088–2092. doi: 10.1128/jvi.65.4.2088-2092.1991

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

D C Ansardi 1, D C Porter 1, C D Morrow 1
PMCID: PMC240068  PMID: 1848318

Abstract

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Full text

PDF
2088

Images in this article

2090Image
on p.2090
2090Image
on p.2090
2091Image
on p.2091
2091Image
on p.2091

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnold E., Luo M., Vriend G., Rossmann M. G., Palmenberg A. C., Parks G. D., Nicklin M. J., Wimmer E. Implications of the picornavirus capsid structure for polyprotein processing. Proc Natl Acad Sci U S A. 1987 Jan;84(1):21–25. doi: 10.1073/pnas.84.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chakrabarti S., Brechling K., Moss B. Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol Cell Biol. 1985 Dec;5(12):3403–3409. doi: 10.1128/mcb.5.12.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chow M., Newman J. F., Filman D., Hogle J. M., Rowlands D. J., Brown F. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. 1987 Jun 11;327(6122):482–486. doi: 10.1038/327482a0. [DOI] [PubMed] [Google Scholar]
  4. Compton S. R., Nelsen B., Kirkegaard K. Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions. J Virol. 1990 Sep;64(9):4067–4075. doi: 10.1128/jvi.64.9.4067-4075.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Etchison D., Milburn S. C., Edery I., Sonenberg N., Hershey J. W. Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem. 1982 Dec 25;257(24):14806–14810. [PubMed] [Google Scholar]
  6. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  7. Hanecak R., Semler B. L., Anderson C. W., Wimmer E. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3973–3977. doi: 10.1073/pnas.79.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanecak R., Semler B. L., Ariga H., Anderson C. W., Wimmer E. Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase. Cell. 1984 Jul;37(3):1063–1073. doi: 10.1016/0092-8674(84)90441-0. [DOI] [PubMed] [Google Scholar]
  9. Jacobson M. F., Asso J., Baltimore D. Further evidence on the formation of poliovirus proteins. J Mol Biol. 1970 May 14;49(3):657–669. doi: 10.1016/0022-2836(70)90289-5. [DOI] [PubMed] [Google Scholar]
  10. Jacobson M. F., Baltimore D. Morphogenesis of poliovirus. I. Association of the viral RNA with coat protein. J Mol Biol. 1968 Apr 28;33(2):369–378. doi: 10.1016/0022-2836(68)90195-2. [DOI] [PubMed] [Google Scholar]
  11. Jewell J. E., Ball L. A., Rueckert R. Limited expression of poliovirus by vaccinia virus recombinants due to inhibition of the vector by proteinase 2A. J Virol. 1990 Mar;64(3):1388–1393. doi: 10.1128/jvi.64.3.1388-1393.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kräusslich H. G., Nicklin M. J., Toyoda H., Etchison D., Wimmer E. Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220. J Virol. 1987 Sep;61(9):2711–2718. doi: 10.1128/jvi.61.9.2711-2718.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marc D., Masson G., Girard M., van der Werf S. Lack of myristoylation of poliovirus capsid polypeptide VP0 prevents the formation of virions or results in the assembly of noninfectious virus particles. J Virol. 1990 Sep;64(9):4099–4107. doi: 10.1128/jvi.64.9.4099-4107.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morrow C. D., Warren B., Lentz M. R. Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6050–6054. doi: 10.1073/pnas.84.17.6050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Onodera S., Cardamone J. J., Jr, Phillips B. A. Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids. J Virol. 1986 May;58(2):610–618. doi: 10.1128/jvi.58.2.610-618.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Phillips B. A. In vitro assembly of poliovirus. II. Evidence for the self-assembly of 14 S particles into empty capsids. Virology. 1971 May;44(2):307–316. doi: 10.1016/0042-6822(71)90262-5. [DOI] [PubMed] [Google Scholar]
  17. Phillips B. A. In vitro assembly of polioviruses. I. Kinetics of the assembly of empty capsids and the role of extracts from infected cells. Virology. 1969 Dec;39(4):811–821. doi: 10.1016/0042-6822(69)90018-x. [DOI] [PubMed] [Google Scholar]
  18. Phillips B. A., Summers D. F., Maizel J. V., Jr In vitro assembly of poliovirus-related particles. Virology. 1968 Jun;35(2):216–226. doi: 10.1016/0042-6822(68)90262-6. [DOI] [PubMed] [Google Scholar]
  19. Putnak J. R., Phillips B. A. Picornaviral structure and assembly. Microbiol Rev. 1981 Jun;45(2):287–315. doi: 10.1128/mr.45.2.287-315.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Richards O. C., Ivanoff L. A., Bienkowska-Szewczyk K., Butt B., Petteway S. R., Jr, Rothstein M. A., Ehrenfeld E. Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA. Virology. 1987 Dec;161(2):348–356. doi: 10.1016/0042-6822(87)90127-9. [DOI] [PubMed] [Google Scholar]
  21. Semler B. L., Dorner A. J., Wimmer E. Production of infectious poliovirus from cloned cDNA is dramatically increased by SV40 transcription and replication signals. Nucleic Acids Res. 1984 Jun 25;12(12):5123–5141. doi: 10.1093/nar/12.12.5123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sonenberg N. Regulation of translation by poliovirus. Adv Virus Res. 1987;33:175–204. doi: 10.1016/s0065-3527(08)60318-8. [DOI] [PubMed] [Google Scholar]
  23. Toyoda H., Nicklin M. J., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell. 1986 Jun 6;45(5):761–770. doi: 10.1016/0092-8674(86)90790-7. [DOI] [PubMed] [Google Scholar]
  24. Turner P. C., Young D. C., Flanegan J. B., Moyer R. W. Interference with vaccinia virus growth caused by insertion of the coding sequence for poliovirus protease 2A. Virology. 1989 Dec;173(2):509–521. doi: 10.1016/0042-6822(89)90563-1. [DOI] [PubMed] [Google Scholar]
  25. Urakawa T., Ferguson M., Minor P. D., Cooper J., Sullivan M., Almond J. W., Bishop D. H. Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J Gen Virol. 1989 Jun;70(Pt 6):1453–1463. doi: 10.1099/0022-1317-70-6-1453. [DOI] [PubMed] [Google Scholar]
  26. Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]
  27. Ypma-Wong M. F., Semler B. L. Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins. J Virol. 1987 Oct;61(10):3181–3189. doi: 10.1128/jvi.61.10.3181-3189.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES