Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Nov 7;92(23):10516–10520. doi: 10.1073/pnas.92.23.10516

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

J Chou 1, J J Chen 1, M Gross 1, B Roizman 1
PMCID: PMC40642  PMID: 7479831

Abstract

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.

Full text

PDF
10520

Images in this article

10517Fig. 2
on p.10517
10518Fig. 3
on p.10518
10519Fig. 4
on p.10519

Selected References

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

  1. Chou J., Kern E. R., Whitley R. J., Roizman B. Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture. Science. 1990 Nov 30;250(4985):1262–1266. doi: 10.1126/science.2173860. [DOI] [PubMed] [Google Scholar]
  2. Chou J., Poon A. P., Johnson J., Roizman B. Differential response of human cells to deletions and stop codons in the gamma(1)34.5 gene of herpes simplex virus. J Virol. 1994 Dec;68(12):8304–8311. doi: 10.1128/jvi.68.12.8304-8311.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chou J., Roizman B. Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5247–5251. doi: 10.1073/pnas.91.12.5247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou J., Roizman B. The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3266–3270. doi: 10.1073/pnas.89.8.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chou J., Roizman B. The herpes simplex virus 1 gene for ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+. J Virol. 1990 Mar;64(3):1014–1020. doi: 10.1128/jvi.64.3.1014-1020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou J., Roizman B. The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component. J Virol. 1986 Feb;57(2):629–637. doi: 10.1128/jvi.57.2.629-637.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ejercito P. M., Kieff E. D., Roizman B. Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol. 1968 May;2(3):357–364. doi: 10.1099/0022-1317-2-3-357. [DOI] [PubMed] [Google Scholar]
  8. Fornace A. J., Jr, Jackman J., Hollander M. C., Hoffman-Liebermann B., Liebermann D. A. Genotoxic-stress-response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest. Ann N Y Acad Sci. 1992 Nov 21;663:139–153. doi: 10.1111/j.1749-6632.1992.tb38657.x. [DOI] [PubMed] [Google Scholar]
  9. Fornace A. J., Jr, Nebert D. W., Hollander M. C., Luethy J. D., Papathanasiou M., Fargnoli J., Holbrook N. J. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol. 1989 Oct;9(10):4196–4203. doi: 10.1128/mcb.9.10.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gross M., Kaplansky D. A. Identification of a Mr = 39,000 phosphoprotein in highly purified preparations of rabbit reticulocyte eIF-2 that is distinct from the Mr = 35,000 subunit phosphorylated by the hemin-controlled translational repressor. J Biol Chem. 1980 Jul 10;255(13):6270–6275. [PubMed] [Google Scholar]
  11. Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
  12. Liu F. Y., Roizman B. The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate. J Virol. 1991 Oct;65(10):5149–5156. doi: 10.1128/jvi.65.10.5149-5156.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lord K. A., Hoffman-Liebermann B., Liebermann D. A. Sequence of MyD116 cDNA: a novel myeloid differentiation primary response gene induced by IL6. Nucleic Acids Res. 1990 May 11;18(9):2823–2823. doi: 10.1093/nar/18.9.2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maurides P. A., Akkaraju G. R., Jagus R. Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting. Anal Biochem. 1989 Nov 15;183(1):144–151. doi: 10.1016/0003-2697(89)90182-6. [DOI] [PubMed] [Google Scholar]
  15. McGeoch D. J., Barnett B. C. Neurovirulence factor. Nature. 1991 Oct 17;353(6345):609–609. doi: 10.1038/353609b0. [DOI] [PubMed] [Google Scholar]
  16. McKie E. A., Hope R. G., Brown S. M., MacLean A. R. Characterization of the herpes simplex virus type 1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system. J Gen Virol. 1994 Apr;75(Pt 4):733–741. doi: 10.1099/0022-1317-75-4-733. [DOI] [PubMed] [Google Scholar]
  17. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pathak V. K., Schindler D., Hershey J. W. Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases. Mol Cell Biol. 1988 Feb;8(2):993–995. doi: 10.1128/mcb.8.2.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sarre T. F. The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells. Biosystems. 1989;22(4):311–325. doi: 10.1016/0303-2647(89)90053-1. [DOI] [PubMed] [Google Scholar]
  20. Scorsone K. A., Panniers R., Rowlands A. G., Henshaw E. C. Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis. J Biol Chem. 1987 Oct 25;262(30):14538–14543. [PubMed] [Google Scholar]
  21. Thomis D. C., Doohan J. P., Samuel C. E. Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. Virology. 1992 May;188(1):33–46. doi: 10.1016/0042-6822(92)90732-5. [DOI] [PubMed] [Google Scholar]
  22. Whitley R. J., Kern E. R., Chatterjee S., Chou J., Roizman B. Replication, establishment of latency, and induced reactivation of herpes simplex virus gamma 1 34.5 deletion mutants in rodent models. J Clin Invest. 1993 Jun;91(6):2837–2843. doi: 10.1172/JCI116527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zhan Q., Lord K. A., Alamo I., Jr, Hollander M. C., Carrier F., Ron D., Kohn K. W., Hoffman B., Liebermann D. A., Fornace A. J., Jr The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol. 1994 Apr;14(4):2361–2371. doi: 10.1128/mcb.14.4.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES