Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Jul;71(7):5218–5226. doi: 10.1128/jvi.71.7.5218-5226.1997

Identification and analysis of three myristylated vaccinia virus late proteins.

K H Martin 1, D W Grosenbach 1, C A Franke 1, D E Hruby 1
PMCID: PMC191757  PMID: 9188589

Abstract

Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. 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]
  2. 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]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Bryant M., Ratner L. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A. 1990 Jan;87(2):523–527. doi: 10.1073/pnas.87.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Child S. J., Hruby D. E. Evidence for multiple species of vaccinia virus-encoded palmitylated proteins. Virology. 1992 Nov;191(1):262–271. doi: 10.1016/0042-6822(92)90188-u. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chung T. D., Wymer J. P., Kulka M., Smith C. C., Aurelian L. Myristylation and polylysine-mediated activation of the protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). Virology. 1990 Nov;179(1):168–178. doi: 10.1016/0042-6822(90)90286-z. [DOI] [PubMed] [Google Scholar]
  9. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Deichaite I., Casson L. P., Ling H. P., Resh M. D. In vitro synthesis of pp60v-src: myristylation in a cell-free system. Mol Cell Biol. 1988 Oct;8(10):4295–4301. doi: 10.1128/mcb.8.10.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duronio R. J., Rudnick D. A., Adams S. P., Towler D. A., Gordon J. I. Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli. J Biol Chem. 1991 Jun 5;266(16):10498–10504. [PubMed] [Google Scholar]
  13. 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]
  14. Engelstad M., Smith G. L. The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence. Virology. 1993 Jun;194(2):627–637. doi: 10.1006/viro.1993.1302. [DOI] [PubMed] [Google Scholar]
  15. Franke C. A., Reynolds P. L., Hruby D. E. Fatty acid acylation of vaccinia virus proteins. J Virol. 1989 Oct;63(10):4285–4291. doi: 10.1128/jvi.63.10.4285-4291.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Franke C. A., Wilson E. M., Hruby D. E. Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25. J Virol. 1990 Dec;64(12):5988–5996. doi: 10.1128/jvi.64.12.5988-5996.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gaedigk-Nitschko K., Ding M. X., Levy M. A., Schlesinger M. J. Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure. Virology. 1990 Mar;175(1):282–291. doi: 10.1016/0042-6822(90)90210-i. [DOI] [PubMed] [Google Scholar]
  18. Gheysen D., Jacobs E., de Foresta F., Thiriart C., Francotte M., Thines D., De Wilde M. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 1989 Oct 6;59(1):103–112. doi: 10.1016/0092-8674(89)90873-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Grosenbach D. W., Ulaeto D. O., Hruby D. E. Palmitylation of the vaccinia virus 37-kDa major envelope antigen. Identification of a conserved acceptor motif and biological relevance. J Biol Chem. 1997 Jan 17;272(3):1956–1964. doi: 10.1074/jbc.272.3.1956. [DOI] [PubMed] [Google Scholar]
  21. Göttlinger H. G., Sodroski J. G., Haseltine W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781–5785. doi: 10.1073/pnas.86.15.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heuckeroth R. O., Towler D. A., Adams S. P., Glaser L., Gordon J. I. 11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase. J Biol Chem. 1988 Feb 15;263(5):2127–2133. [PubMed] [Google Scholar]
  23. Hiller G., Weber K. Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment. J Virol. 1985 Sep;55(3):651–659. doi: 10.1128/jvi.55.3.651-659.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hruby D. E., Franke C. A. Viral acylproteins: greasing the wheels of assembly. Trends Microbiol. 1993 Apr;1(1):20–25. doi: 10.1016/0966-842x(93)90020-r. [DOI] [PubMed] [Google Scholar]
  26. Hruby D. E., Guarino L. A., Kates J. R. Vaccinia virus replication. I. Requirement for the host-cell nucleus. J Virol. 1979 Feb;29(2):705–715. doi: 10.1128/jvi.29.2.705-715.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnson D. R., Bhatnagar R. S., Knoll L. J., Gordon J. I. Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem. 1994;63:869–914. doi: 10.1146/annurev.bi.63.070194.004253. [DOI] [PubMed] [Google Scholar]
  28. Jones T. L., Simonds W. F., Merendino J. J., Jr, Brann M. R., Spiegel A. M. Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment. Proc Natl Acad Sci U S A. 1990 Jan;87(2):568–572. doi: 10.1073/pnas.87.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kamps M. P., Buss J. E., Sefton B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4625–4628. doi: 10.1073/pnas.82.14.4625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krauzewicz N., Streuli C. H., Stuart-Smith N., Jones M. D., Wallace S., Griffin B. E. Myristylated polyomavirus VP2: role in the life cycle of the virus. J Virol. 1990 Sep;64(9):4414–4420. doi: 10.1128/jvi.64.9.4414-4420.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kräusslich H. G., Hölscher C., Reuer Q., Harber J., Wimmer E. Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol. 1990 May;64(5):2433–2436. doi: 10.1128/jvi.64.5.2433-2436.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Moscufo N., Simons J., Chow M. Myristoylation is important at multiple stages in poliovirus assembly. J Virol. 1991 May;65(5):2372–2380. doi: 10.1128/jvi.65.5.2372-2380.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parkinson J. E., Smith G. L. Vaccinia virus gene A36R encodes a M(r) 43-50 K protein on the surface of extracellular enveloped virus. Virology. 1994 Oct;204(1):376–390. doi: 10.1006/viro.1994.1542. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Que Q., Li Y., Wang I. N., Lane L. C., Chaney W. G., Van Etten J. L. Protein glycosylation and myristylation in Chlorella virus PBCV-1 and its antigenic variants. Virology. 1994 Sep;203(2):320–327. doi: 10.1006/viro.1994.1490. [DOI] [PubMed] [Google Scholar]
  40. Ravanello M. P., Hruby D. E. Characterization of the vaccinia virus L1R myristylprotein as a component of the intracellular virion envelope. J Gen Virol. 1994 Jun;75(Pt 6):1479–1483. doi: 10.1099/0022-1317-75-6-1479. [DOI] [PubMed] [Google Scholar]
  41. Ravanello M. P., Hruby D. E. Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly. J Virol. 1994 Oct;68(10):6401–6410. doi: 10.1128/jvi.68.10.6401-6410.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rein A., McClure M. R., Rice N. R., Luftig R. B., Schultz A. M. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7246–7250. doi: 10.1073/pnas.83.19.7246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Resh M. D. Membrane interactions of pp60v-src: a model for myristylated tyrosine protein kinases. Oncogene. 1990 Oct;5(10):1437–1444. [PubMed] [Google Scholar]
  44. Schmelz M., Sodeik B., Ericsson M., Wolffe E. J., Shida H., Hiller G., Griffiths G. Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. J Virol. 1994 Jan;68(1):130–147. doi: 10.1128/jvi.68.1.130-147.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Stevenson F. T., Bursten S. L., Fanton C., Locksley R. M., Lovett D. H. The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7245–7249. doi: 10.1073/pnas.90.15.7245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Streuli C. H., Griffin B. E. Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature. 1987 Apr 9;326(6113):619–622. doi: 10.1038/326619a0. [DOI] [PubMed] [Google Scholar]
  49. Tooze J., Hollinshead M., Reis B., Radsak K., Kern H. Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes. Eur J Cell Biol. 1993 Feb;60(1):163–178. [PubMed] [Google Scholar]
  50. Towler D. A., Eubanks S. R., Towery D. S., Adams S. P., Glaser L. Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase. J Biol Chem. 1987 Jan 25;262(3):1030–1036. [PubMed] [Google Scholar]
  51. Ulaeto D., Grosenbach D., Hruby D. E. The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle. J Virol. 1996 Jun;70(6):3372–3377. doi: 10.1128/jvi.70.6.3372-3377.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. VanSlyke J. K., Hruby D. E. Posttranslational modification of vaccinia virus proteins. Curr Top Microbiol Immunol. 1990;163:185–206. doi: 10.1007/978-3-642-75605-4_7. [DOI] [PubMed] [Google Scholar]
  53. Wolffe E. J., Isaacs S. N., Moss B. Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination. J Virol. 1993 Aug;67(8):4732–4741. doi: 10.1128/jvi.67.8.4732-4741.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wolffe E. J., Vijaya S., Moss B. A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology. 1995 Aug 1;211(1):53–63. doi: 10.1006/viro.1995.1378. [DOI] [PubMed] [Google Scholar]

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

RESOURCES