Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Virology logoLink to Journal of Virology
. 1995 Oct;69(10):6376–6388. doi: 10.1128/jvi.69.10.6376-6388.1995

Vaccinia virus morphogenesis is blocked by temperature-sensitive mutations in the F10 gene, which encodes protein kinase 2.

S Wang 1, S Shuman 1
PMCID: PMC189537  PMID: 7666539

Abstract

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts28, ts54, ts61, and ts15) composing a single complementation group have been mapped by marker rescue to the F10 open reading frame located within the genomic HindIII F DNA fragment. Sequencing of the F10 gene from wild-type and mutant viruses revealed single-amino-acid substitutions in the F10 polypeptide responsible for thermolabile growth. Although the ts mutants displayed normal patterns of viral protein synthesis, electron microscopy revealed a profound morphogenetic defect at the nonpermissive temperature (40 degrees C). Virion assembly was arrested at an early stage, with scant formation of membrane crescents and no progression to normal spherical immature particles. The F10 gene encodes a 52-kDa serine/threonine protein kinase (S. Lin and S. S. Broyles, Proc. Natl. Acad. Sci. USA 91:7653-7657, 1994). We expressed a His-tagged version of the wild-type, ts54, and ts61 F10 polypeptides in bacteria and purified these proteins by sequential nickel affinity and phosphocellulose chromatography steps. The wild-type F10 protein kinase activity was characterized in detail by using casein as a phosphate acceptor. Whereas the wild-type and ts61 kinases displayed similar thermal inactivation profiles, the ts54 kinase was thermosensitive in vitro. These findings suggest that protein phosphorylation plays an essential role at an early stage of virion assembly.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. Baldick C. J., Jr, Moss B. Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an Mr 62,000 polypeptide. Virology. 1987 Jan;156(1):138–145. doi: 10.1016/0042-6822(87)90444-2. [DOI] [PubMed] [Google Scholar]
  2. Carpenter M. S., DeLange A. M. Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression. Virology. 1992 May;188(1):233–244. doi: 10.1016/0042-6822(92)90753-c. [DOI] [PubMed] [Google Scholar]
  3. Christen L., Higman M. A., Niles E. G. Phenotypic characterization of three temperature-sensitive mutations in the vaccinia virus early gene transcription initiation factor. J Gen Virol. 1992 Dec;73(Pt 12):3155–3167. doi: 10.1099/0022-1317-73-12-3155. [DOI] [PubMed] [Google Scholar]
  4. Condit R. C., Motyczka A. Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology. 1981 Aug;113(1):224–241. doi: 10.1016/0042-6822(81)90150-1. [DOI] [PubMed] [Google Scholar]
  5. Condit R. C., Motyczka A., Spizz G. Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus. Virology. 1983 Jul 30;128(2):429–443. doi: 10.1016/0042-6822(83)90268-4. [DOI] [PubMed] [Google Scholar]
  6. Condit R. C., Niles E. G. Orthopoxvirus genetics. Curr Top Microbiol Immunol. 1990;163:1–39. doi: 10.1007/978-3-642-75605-4_1. [DOI] [PubMed] [Google Scholar]
  7. Dales S., Milovanovitch V., Pogo B. G., Weintraub S. B., Huima T., Wilton S., McFadden G. Biogenesis of vaccinia: isolation of conditional lethal mutants and electron microscopic characterization of their phenotypically expressed defects. Virology. 1978 Feb;84(2):403–428. doi: 10.1016/0042-6822(78)90258-1. [DOI] [PubMed] [Google Scholar]
  8. Davison A. J., Moss B. Structure of vaccinia virus late promoters. J Mol Biol. 1989 Dec 20;210(4):771–784. doi: 10.1016/0022-2836(89)90108-3. [DOI] [PubMed] [Google Scholar]
  9. Drillien R., Spehner D., Kirn A. Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants. Virology. 1982 Jun;119(2):372–381. doi: 10.1016/0042-6822(82)90096-4. [DOI] [PubMed] [Google Scholar]
  10. Dyster L. M., Niles E. G. Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins. Virology. 1991 Jun;182(2):455–467. doi: 10.1016/0042-6822(91)90586-z. [DOI] [PubMed] [Google Scholar]
  11. Ensinger M. J. Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J Virol. 1982 Sep;43(3):778–790. doi: 10.1128/jvi.43.3.778-790.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fathi Z., Condit R. C. Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component. Virology. 1991 Mar;181(1):273–276. doi: 10.1016/0042-6822(91)90492-t. [DOI] [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. Guan K. L., Broyles S. S., Dixon J. E. A Tyr/Ser protein phosphatase encoded by vaccinia virus. Nature. 1991 Mar 28;350(6316):359–362. doi: 10.1038/350359a0. [DOI] [PubMed] [Google Scholar]
  15. Hooda-Dhingra U., Thompson C. L., Condit R. C. Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. J Virol. 1989 Feb;63(2):714–729. doi: 10.1128/jvi.63.2.714-729.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson G. P., Goebel S. J., Paoletti E. An update on the vaccinia virus genome. Virology. 1993 Oct;196(2):381–401. doi: 10.1006/viro.1993.1494. [DOI] [PubMed] [Google Scholar]
  17. Kane E. M., Shuman S. Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase. J Virol. 1992 Oct;66(10):5752–5762. doi: 10.1128/jvi.66.10.5752-5762.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kane E. M., Shuman S. Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component. J Virol. 1993 May;67(5):2689–2698. doi: 10.1128/jvi.67.5.2689-2698.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kao S. Y., Bauer W. R. Biosynthesis and phosphorylation of vaccinia virus structural protein VP11. Virology. 1987 Aug;159(2):399–407. doi: 10.1016/0042-6822(87)90479-x. [DOI] [PubMed] [Google Scholar]
  20. Li J., Pennington M. J., Broyles S. S. Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components. J Virol. 1994 Apr;68(4):2605–2614. doi: 10.1128/jvi.68.4.2605-2614.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin S., Broyles S. S. Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7653–7657. doi: 10.1073/pnas.91.16.7653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin S., Chen W., Broyles S. S. The vaccinia virus B1R gene product is a serine/threonine protein kinase. J Virol. 1992 May;66(5):2717–2723. doi: 10.1128/jvi.66.5.2717-2723.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McNulty-Kowalczyk A., Paoletti E. Mutations in ORF D13L and other genetic loci alter the rifampicin phenotype of vaccinia virus. Virology. 1993 Jun;194(2):638–646. doi: 10.1006/viro.1993.1303. [DOI] [PubMed] [Google Scholar]
  24. Miner J. N., Hruby D. E. Rifampicin prevents virosome localization of L65, an essential vaccinia virus polypeptide. Virology. 1989 May;170(1):227–237. doi: 10.1016/0042-6822(89)90370-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Paoletti E., Moss B. Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores. J Virol. 1972 Sep;10(3):417–424. doi: 10.1128/jvi.10.3.417-424.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rempel R. E., Traktman P. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins. J Virol. 1992 Jul;66(7):4413–4426. doi: 10.1128/jvi.66.7.4413-4426.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shuman S. Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10935–10939. doi: 10.1073/pnas.89.22.10935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Sodeik B., Griffiths G., Ericsson M., Moss B., Doms R. W. Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65. J Virol. 1994 Feb;68(2):1103–1114. doi: 10.1128/jvi.68.2.1103-1114.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Takahashi T., Oie M., Ichihashi Y. N-terminal amino acid sequences of vaccinia virus structural proteins. Virology. 1994 Aug 1;202(2):844–852. doi: 10.1006/viro.1994.1406. [DOI] [PubMed] [Google Scholar]
  32. Thompson C. L., Condit R. C. Marker rescue mapping of vaccinia virus temperature-sensitive mutants using overlapping cosmid clones representing the entire virus genome. Virology. 1986 Apr 15;150(1):10–20. doi: 10.1016/0042-6822(86)90261-8. [DOI] [PubMed] [Google Scholar]
  33. VanSlyke J. K., Franke C. A., Hruby D. E. Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins. J Gen Virol. 1991 Feb;72(Pt 2):411–416. doi: 10.1099/0022-1317-72-2-411. [DOI] [PubMed] [Google Scholar]
  34. Vanslyke J. K., Hruby D. E. Immunolocalization of vaccinia virus structural proteins during virion formation. Virology. 1994 Feb;198(2):624–635. doi: 10.1006/viro.1994.1074. [DOI] [PubMed] [Google Scholar]
  35. Vanslyke J. K., Whitehead S. S., Wilson E. M., Hruby D. E. The multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein: a degenerate conserved cleavage motif within core proteins. Virology. 1991 Aug;183(2):467–478. doi: 10.1016/0042-6822(91)90976-i. [DOI] [PubMed] [Google Scholar]
  36. Zhang Y. F., Moss B. Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor. J Virol. 1991 Nov;65(11):6101–6110. doi: 10.1128/jvi.65.11.6101-6110.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang Y., Ahn B. Y., Moss B. Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein. J Virol. 1994 Mar;68(3):1360–1370. doi: 10.1128/jvi.68.3.1360-1370.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y., Keck J. G., Moss B. Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus. J Virol. 1992 Nov;66(11):6470–6479. doi: 10.1128/jvi.66.11.6470-6479.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang Y., Moss B. Immature viral envelope formation is interrupted at the same stage by lac operator-mediated repression of the vaccinia virus D13L gene and by the drug rifampicin. Virology. 1992 Apr;187(2):643–653. doi: 10.1016/0042-6822(92)90467-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES