Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Aug;67(8):4732–4741. doi: 10.1128/jvi.67.8.4732-4741.1993

Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination.

E J Wolffe 1, S N Isaacs 1, B Moss 1
PMCID: PMC237859  PMID: 8331727

Abstract

The structure, formation, and function of the virion membranes are among the least well understood aspects of vaccinia virus replication. In this study, we investigated the role of gp42, a glycoprotein component of the extracellular enveloped form of vaccinia virus (EEV) encoded by the B5R gene. The B5R gene was deleted by homologous recombination from vaccinia virus strains IHD-J and WR, which produce high and low levels of EEV, respectively. Isolation of recombinant viruses was facilitated by the insertion into the genome of a cassette containing the Escherichia coli gpt and lacZ genes flanked by the ends of the B5R gene to provide simultaneous antibiotic selection and color screening. Deletion mutant viruses of both strains formed tiny plaques, and those of the IHD-J mutant lacked the characteristic comet shape caused by release of EEV. Nevertheless, similar yields of intracellular infectious virus were obtained whether cells were infected with the B5R deletion mutants or their parental strains. In the case of IHD-J, however, this deletion severely reduced the amount of infectious extracellular virus. Metabolic labeling studies demonstrated that the low extracellular infectivity corresponded with a decrease in EEV particles in the medium. Electron microscopic examination revealed that mature intracellular naked virions (INV) were present in cells infected with mutant virus, but neither membrane-wrapped INV nor significant amounts of plasma membrane-associated virus were observed. Syncytium formation, which occurs in cells infected with wild-type WR and IHD-J virus after brief low-pH treatment, did not occur in cells infected with the B5R deletion mutants. By contrast, syncytium formation induced by antibody to the viral hemagglutinin occurred, suggesting that different mechanisms are involved. When assayed by intracranial injection into weanling mice, both IHD-J and WR mutant viruses were found to be significantly attenuated. These findings demonstrate that the 42-kDa glycoprotein of the EEV is required for efficient membrane enwrapment of INV, externalization of the virus, and transmission and that gp42 contributes to viral virulence in strains producing both low and high levels of EEV.

Full text

PDF

Images in this article

4735Image
on p.4735
4735Image
on p.4735
4738Image
on p.4738
4739Image
on p.4739

Selected References

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

  1. Appleyard G., Andrews C. Neutralizing activities of antisera to poxvirus soluble antigens. J Gen Virol. 1974 May;23(2):197–200. doi: 10.1099/0022-1317-23-2-197. [DOI] [PubMed] [Google Scholar]
  2. Appleyard G., Hapel A. J., Boulter E. A. An antigenic difference between intracellular and extracellular rabbitpox virus. J Gen Virol. 1971 Oct;13(1):9–17. doi: 10.1099/0022-1317-13-1-9. [DOI] [PubMed] [Google Scholar]
  3. Blasco R., Moss B. Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. J Virol. 1991 Nov;65(11):5910–5920. doi: 10.1128/jvi.65.11.5910-5920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blasco R., Moss B. Role of cell-associated enveloped vaccinia virus in cell-to-cell spread. J Virol. 1992 Jul;66(7):4170–4179. doi: 10.1128/jvi.66.7.4170-4179.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blasco R., Sisler J. R., Moss B. Dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene. J Virol. 1993 Jun;67(6):3319–3325. doi: 10.1128/jvi.67.6.3319-3325.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boulter E. A., Appleyard G. Differences between extracellular and intracellular forms of poxvirus and their implications. Prog Med Virol. 1973;16:86–108. [PubMed] [Google Scholar]
  7. Buller R. M., Chakrabarti S., Cooper J. A., Twardzik D. R., Moss B. Deletion of the vaccinia virus growth factor gene reduces virus virulence. J Virol. 1988 Mar;62(3):866–874. doi: 10.1128/jvi.62.3.866-874.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buller R. M., Smith G. L., Cremer K., Notkins A. L., Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. 1985 Oct 31-Nov 6Nature. 317(6040):813–815. doi: 10.1038/317813a0. [DOI] [PubMed] [Google Scholar]
  9. Doms R. W., Blumenthal R., Moss B. Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol. 1990 Oct;64(10):4884–4892. doi: 10.1128/jvi.64.10.4884-4892.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duncan S. A., Smith G. L. Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress. J Virol. 1992 Mar;66(3):1610–1621. doi: 10.1128/jvi.66.3.1610-1621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engelstad M., Howard S. T., Smith G. L. A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope. Virology. 1992 Jun;188(2):801–810. doi: 10.1016/0042-6822(92)90535-w. [DOI] [PubMed] [Google Scholar]
  12. Falkner F. G., Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol. 1988 Jun;62(6):1849–1854. doi: 10.1128/jvi.62.6.1849-1854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goebel S. J., Johnson G. P., Perkus M. E., Davis S. W., Winslow J. P., Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247-66, 517-63. doi: 10.1016/0042-6822(90)90294-2. [DOI] [PubMed] [Google Scholar]
  14. Gong S. C., Lai C. F., Esteban M. Vaccinia virus induces cell fusion at acid pH and this activity is mediated by the N-terminus of the 14-kDa virus envelope protein. Virology. 1990 Sep;178(1):81–91. doi: 10.1016/0042-6822(90)90381-z. [DOI] [PubMed] [Google Scholar]
  15. Hiller G., Eibl H., Weber K. Characterization of intracellular and extracellular vaccinia virus variants: N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine interferes with cytoplasmic virus dissemination and release. J Virol. 1981 Sep;39(3):903–913. doi: 10.1128/jvi.39.3.903-913.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirt P., Hiller G., Wittek R. Localization and fine structure of a vaccinia virus gene encoding an envelope antigen. J Virol. 1986 Jun;58(3):757–764. doi: 10.1128/jvi.58.3.757-764.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ichihashi Y., Dales S. Biogenesis of poxviruses: interrelationship between hemagglutinin production and polykaryocytosis. Virology. 1971 Dec;46(3):533–543. doi: 10.1016/0042-6822(71)90057-2. [DOI] [PubMed] [Google Scholar]
  18. Ichihashi Y., Matsumoto S., Dales S. Biogenesis of poxviruses: role of A-type inclusions and host cell membranes in virus dissemination. Virology. 1971 Dec;46(3):507–532. doi: 10.1016/0042-6822(71)90056-0. [DOI] [PubMed] [Google Scholar]
  19. Ichihashi Y., Oie M. Epitope mosaic on the surface proteins of orthopoxviruses. Virology. 1988 Mar;163(1):133–144. doi: 10.1016/0042-6822(88)90240-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Isaacs S. N., Wolffe E. J., Payne L. G., Moss B. Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope. J Virol. 1992 Dec;66(12):7217–7224. doi: 10.1128/jvi.66.12.7217-7224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee M. S., Roos J. M., McGuigan L. C., Smith K. A., Cormier N., Cohen L. K., Roberts B. E., Payne L. G. Molecular attenuation of vaccinia virus: mutant generation and animal characterization. J Virol. 1992 May;66(5):2617–2630. doi: 10.1128/jvi.66.5.2617-2630.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morgan C. Vaccinia virus reexamined: development and release. Virology. 1976 Aug;73(1):43–58. doi: 10.1016/0042-6822(76)90059-3. [DOI] [PubMed] [Google Scholar]
  23. Moses A. M., Howanitz J., van Gemert M., Miller M. Clofibrate-induced antidiuresis. J Clin Invest. 1973 Mar;52(3):535–542. doi: 10.1172/JCI107213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Payne L. G. Characterization of vaccinia virus glycoproteins by monoclonal antibody precipitation. Virology. 1992 Mar;187(1):251–260. doi: 10.1016/0042-6822(92)90313-e. [DOI] [PubMed] [Google Scholar]
  25. Payne L. G. Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus. J Virol. 1979 Jul;31(1):147–155. doi: 10.1128/jvi.31.1.147-155.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Payne L. G., Kristensson K. Extracellular release of enveloped vaccinia virus from mouse nasal epithelial cells in vivo. J Gen Virol. 1985 Mar;66(Pt 3):643–646. doi: 10.1099/0022-1317-66-3-643. [DOI] [PubMed] [Google Scholar]
  27. Payne L. G., Norrby E. Presence of haemagglutinin in the envelope of extracellular vaccinia virus particles. J Gen Virol. 1976 Jul;32(1):63–72. doi: 10.1099/0022-1317-32-1-63. [DOI] [PubMed] [Google Scholar]
  28. Payne L. G. Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J Gen Virol. 1980 Sep;50(1):89–100. doi: 10.1099/0022-1317-50-1-89. [DOI] [PubMed] [Google Scholar]
  29. Payne L. Polypeptide composition of extracellular enveloped vaccinia virus. J Virol. 1978 Jul;27(1):28–37. doi: 10.1128/jvi.27.1.28-37.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rodriguez J. F., Esteban M. Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein. J Virol. 1987 Nov;61(11):3550–3554. doi: 10.1128/jvi.61.11.3550-3554.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rodriguez J. F., Paez E., Esteban M. A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers. J Virol. 1987 Feb;61(2):395–404. doi: 10.1128/jvi.61.2.395-404.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmutz C., Payne L. G., Gubser J., Wittek R. A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release. J Virol. 1991 Jul;65(7):3435–3442. doi: 10.1128/jvi.65.7.3435-3442.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Seki M., Oie M., Ichihashi Y., Shida H. Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants. Virology. 1990 Apr;175(2):372–384. doi: 10.1016/0042-6822(90)90422-n. [DOI] [PubMed] [Google Scholar]
  34. Shida H. Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology. 1986 Apr 30;150(2):451–462. doi: 10.1016/0042-6822(86)90309-0. [DOI] [PubMed] [Google Scholar]
  35. Smith G. L., Chan Y. S., Howard S. T. Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat. J Gen Virol. 1991 Jun;72(Pt 6):1349–1376. doi: 10.1099/0022-1317-72-6-1349. [DOI] [PubMed] [Google Scholar]
  36. Sodeik B., Doms R. W., Ericsson M., Hiller G., Machamer C. E., van 't Hof W., van Meer G., Moss B., Griffiths G. Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks. J Cell Biol. 1993 May;121(3):521–541. doi: 10.1083/jcb.121.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Takahashi-Nishimaki F., Funahashi S., Miki K., Hashizume S., Sugimoto M. Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins. Virology. 1991 Mar;181(1):158–164. doi: 10.1016/0042-6822(91)90480-y. [DOI] [PubMed] [Google Scholar]
  38. Turner G. S., Squires E. J. Inactivated smallpox vaccine: immunogenicity of inactivated intracellular and extracellular vaccinia virus. J Gen Virol. 1971 Oct;13(1):19–25. doi: 10.1099/0022-1317-13-1-19. [DOI] [PubMed] [Google Scholar]

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

RESOURCES