• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. Oct 1995; 69(10): 6376–6388.
PMCID: PMC189537

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

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 (2.7M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baldick CJ, 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. [PubMed]
  • Carpenter MS, DeLange AM. Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression. Virology. 1992 May;188(1):233–244. [PubMed]
  • Christen L, Higman MA, Niles EG. 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. [PubMed]
  • Condit RC, Motyczka A. Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology. 1981 Aug;113(1):224–241. [PubMed]
  • Condit RC, Motyczka A, Spizz G. Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus. Virology. 1983 Jul 30;128(2):429–443. [PubMed]
  • Condit RC, Niles EG. Orthopoxvirus genetics. Curr Top Microbiol Immunol. 1990;163:1–39. [PubMed]
  • Dales S, Milovanovitch V, Pogo BG, Weintraub SB, 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. [PubMed]
  • Davison AJ, Moss B. Structure of vaccinia virus late promoters. J Mol Biol. 1989 Dec 20;210(4):771–784. [PubMed]
  • Drillien R, Spehner D, Kirn A. Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants. Virology. 1982 Jun;119(2):372–381. [PubMed]
  • Dyster LM, Niles EG. Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins. Virology. 1991 Jun;182(2):455–467. [PubMed]
  • Ensinger MJ. Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J Virol. 1982 Sep;43(3):778–790. [PMC free article] [PubMed]
  • Fathi Z, Condit RC. Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component. Virology. 1991 Mar;181(1):273–276. [PubMed]
  • Goebel SJ, Johnson GP, Perkus ME, Davis SW, Winslow JP, Paoletti E. The complete DNA sequence of vaccinia virus. Virology. 1990 Nov;179(1):247–563. [PubMed]
  • Guan KL, Broyles SS, Dixon JE. A Tyr/Ser protein phosphatase encoded by vaccinia virus. Nature. 1991 Mar 28;350(6316):359–362. [PubMed]
  • Hooda-Dhingra U, Thompson CL, Condit RC. 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. [PMC free article] [PubMed]
  • Johnson GP, Goebel SJ, Paoletti E. An update on the vaccinia virus genome. Virology. 1993 Oct;196(2):381–401. [PubMed]
  • Kane EM, 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. [PMC free article] [PubMed]
  • Kane EM, 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. [PMC free article] [PubMed]
  • Kao SY, Bauer WR. Biosynthesis and phosphorylation of vaccinia virus structural protein VP11. Virology. 1987 Aug;159(2):399–407. [PubMed]
  • Li J, Pennington MJ, Broyles SS. 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. [PMC free article] [PubMed]
  • Lin S, Broyles SS. 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. [PMC free article] [PubMed]
  • Lin S, Chen W, Broyles SS. The vaccinia virus B1R gene product is a serine/threonine protein kinase. J Virol. 1992 May;66(5):2717–2723. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Miner JN, Hruby DE. Rifampicin prevents virosome localization of L65, an essential vaccinia virus polypeptide. Virology. 1989 May;170(1):227–237. [PubMed]
  • Morgan C. Vaccinia virus reexamined: development and release. Virology. 1976 Aug;73(1):43–58. [PubMed]
  • Paoletti E, Moss B. Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores. J Virol. 1972 Sep;10(3):417–424. [PMC free article] [PubMed]
  • Rempel RE, 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sodeik B, Doms RW, Ericsson M, Hiller G, Machamer CE, 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. [PMC free article] [PubMed]
  • Sodeik B, Griffiths G, Ericsson M, Moss B, Doms RW. Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65. J Virol. 1994 Feb;68(2):1103–1114. [PMC free article] [PubMed]
  • Takahashi T, Oie M, Ichihashi Y. N-terminal amino acid sequences of vaccinia virus structural proteins. Virology. 1994 Aug 1;202(2):844–852. [PubMed]
  • Thompson CL, Condit RC. 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. [PubMed]
  • VanSlyke JK, Franke CA, Hruby DE. 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. [PubMed]
  • Vanslyke JK, Hruby DE. Immunolocalization of vaccinia virus structural proteins during virion formation. Virology. 1994 Feb;198(2):624–635. [PubMed]
  • Vanslyke JK, Whitehead SS, Wilson EM, Hruby DE. 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. [PubMed]
  • Zhang Y, Ahn BY, 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. [PMC free article] [PubMed]
  • Zhang Y, Keck JG, 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. [PMC free article] [PubMed]
  • Zhang YF, 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. [PMC free article] [PubMed]
  • 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. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...