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. 1992 Oct;66(10):5805–5814. doi: 10.1128/jvi.66.10.5805-5814.1992

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

E F Meurs 1, Y Watanabe 1, S Kadereit 1, G N Barber 1, M G Katze 1, K Chong 1, B R Williams 1, A G Hovanessian 1
PMCID: PMC241456  PMID: 1382142

Abstract

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.

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Selected References

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

  1. Belkowski L. S., Sen G. C. Inhibition of vesicular stomatitis viral mRNA synthesis by interferons. J Virol. 1987 Mar;61(3):653–660. doi: 10.1128/jvi.61.3.653-660.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black T. L., Safer B., Hovanessian A., Katze M. G. The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J Virol. 1989 May;63(5):2244–2251. doi: 10.1128/jvi.63.5.2244-2251.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chebath J., Benech P., Revel M., Vigneron M. Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection. Nature. 1987 Dec 10;330(6148):587–588. doi: 10.1038/330587a0. [DOI] [PubMed] [Google Scholar]
  4. Chong K. L., Feng L., Schappert K., Meurs E., Donahue T. F., Friesen J. D., Hovanessian A. G., Williams B. R. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. doi: 10.1002/j.1460-2075.1992.tb05200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cigan A. M., Pabich E. K., Feng L., Donahue T. F. Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2784–2788. doi: 10.1073/pnas.86.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coccia E. M., Romeo G., Nissim A., Marziali G., Albertini R., Affabris E., Battistini A., Fiorucci G., Orsatti R., Rossi G. B. A full-length murine 2-5A synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication. Virology. 1990 Nov;179(1):228–233. doi: 10.1016/0042-6822(90)90292-y. [DOI] [PubMed] [Google Scholar]
  7. Dubois M. F., Galabru J., Lebon P., Safer B., Hovanessian A. G. Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress. J Biol Chem. 1989 Jul 25;264(21):12165–12171. [PubMed] [Google Scholar]
  8. Dubois M. F., Hovanessian A. G. Modified subcellular localization of interferon-induced p68 kinase during encephalomyocarditis virus infection. Virology. 1990 Dec;179(2):591–598. doi: 10.1016/0042-6822(90)90126-c. [DOI] [PubMed] [Google Scholar]
  9. Enoch T., Zinn K., Maniatis T. Activation of the human beta-interferon gene requires an interferon-inducible factor. Mol Cell Biol. 1986 Mar;6(3):801–810. doi: 10.1128/mcb.6.3.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feng G. S., Chong K., Kumar A., Williams B. R. Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5447–5451. doi: 10.1073/pnas.89.12.5447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galabru J., Hovanessian A. G. Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA. Cell. 1985 Dec;43(3 Pt 2):685–694. doi: 10.1016/0092-8674(85)90241-7. [DOI] [PubMed] [Google Scholar]
  12. Galabru J., Hovanessian A. Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem. 1987 Nov 15;262(32):15538–15544. [PubMed] [Google Scholar]
  13. Galabru J., Katze M. G., Robert N., Hovanessian A. G. The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur J Biochem. 1989 Jan 2;178(3):581–589. doi: 10.1111/j.1432-1033.1989.tb14485.x. [DOI] [PubMed] [Google Scholar]
  14. Hershey J. W. Protein phosphorylation controls translation rates. J Biol Chem. 1989 Dec 15;264(35):20823–20826. [PubMed] [Google Scholar]
  15. Hovanessian A. G., Galabru J. The double-stranded RNA-dependent protein kinase is also activated by heparin. Eur J Biochem. 1987 Sep 15;167(3):467–473. doi: 10.1111/j.1432-1033.1987.tb13360.x. [DOI] [PubMed] [Google Scholar]
  16. Hovanessian A. G. Interferon-induced and double-stranded RNA-activated enzymes: a specific protein kinase and 2',5'-oligoadenylate synthetases. J Interferon Res. 1991 Aug;11(4):199–205. doi: 10.1089/jir.1991.11.199. [DOI] [PubMed] [Google Scholar]
  17. Hovanessian A. G. The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res. 1989 Dec;9(6):641–647. doi: 10.1089/jir.1989.9.641. [DOI] [PubMed] [Google Scholar]
  18. Imani F., Jacobs B. L. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7887–7891. doi: 10.1073/pnas.85.21.7887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katze M. G., DeCorato D., Safer B., Galabru J., Hovanessian A. G. Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J. 1987 Mar;6(3):689–697. doi: 10.1002/j.1460-2075.1987.tb04809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katze M. G., Krug R. M. Translational control in influenza virus-infected cells. Enzyme. 1990;44(1-4):265–277. doi: 10.1159/000468764. [DOI] [PubMed] [Google Scholar]
  21. Katze M. G., Wambach M., Wong M. L., Garfinkel M., Meurs E., Chong K., Williams B. R., Hovanessian A. G., Barber G. N. Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system. Mol Cell Biol. 1991 Nov;11(11):5497–5505. doi: 10.1128/mcb.11.11.5497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krust B., Galabru J., Hovanessian A. G. Further characterization of the protein kinase activity mediated by interferon in mouse and human cells. J Biol Chem. 1984 Jul 10;259(13):8494–8498. [PubMed] [Google Scholar]
  23. Laurent A. G., Krust B., Galabru J., Svab J., Hovanessian A. G. Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4341–4345. doi: 10.1073/pnas.82.13.4341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989 Jul 28;58(2):227–229. doi: 10.1016/0092-8674(89)90833-7. [DOI] [PubMed] [Google Scholar]
  25. Lenardo M. J., Fan C. M., Maniatis T., Baltimore D. The involvement of NF-kappa B in beta-interferon gene regulation reveals its role as widely inducible mediator of signal transduction. Cell. 1989 Apr 21;57(2):287–294. doi: 10.1016/0092-8674(89)90966-5. [DOI] [PubMed] [Google Scholar]
  26. Maheshwari R. K., Jay F. T., Friedman R. M. Selective inhibition of glycoprotein and membrane protein of vesicular stomatitis virus from interferon-treated cells. Science. 1980 Feb 1;207(4430):540–541. doi: 10.1126/science.6243416. [DOI] [PubMed] [Google Scholar]
  27. Marcus P. I., Sekellick M. J. Interferon action III. The rate of primary transcription of vesicular stomatitis virus is inhibited by interferon action. J Gen Virol. 1978 Mar;38(3):391–408. doi: 10.1099/0022-1317-38-3-391. [DOI] [PubMed] [Google Scholar]
  28. Mellits K. H., Kostura M., Mathews M. B. Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function. Cell. 1990 Jun 1;61(5):843–852. doi: 10.1016/0092-8674(90)90194-j. [DOI] [PubMed] [Google Scholar]
  29. Meurs E., Chong K., Galabru J., Thomas N. S., Kerr I. M., Williams B. R., Hovanessian A. G. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. doi: 10.1016/0092-8674(90)90374-n. [DOI] [PubMed] [Google Scholar]
  30. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  31. Ohno S., Konno Y., Akita Y., Yano A., Suzuki K. A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. J Biol Chem. 1990 Apr 15;265(11):6296–6300. [PubMed] [Google Scholar]
  32. Rice A. P., Duncan R., Hershey J. W., Kerr I. M. Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells. J Virol. 1985 Jun;54(3):894–898. doi: 10.1128/jvi.54.3.894-898.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ringold G. M., Dieckmann B., Vannice J. L., Trahey M., McCormick F. Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3964–3968. doi: 10.1073/pnas.81.13.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Roy S., Agy M., Hovanessian A. G., Sonenberg N., Katze M. G. The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase. J Virol. 1991 Feb;65(2):632–640. doi: 10.1128/jvi.65.2.632-640.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roy S., Katze M. G., Parkin N. T., Edery I., Hovanessian A. G., Sonenberg N. Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product. Science. 1990 Mar 9;247(4947):1216–1219. doi: 10.1126/science.2180064. [DOI] [PubMed] [Google Scholar]
  36. Rysiecki G., Gewert D. R., Williams B. R. Constitutive expression of a 2',5'-oligoadenylate synthetase cDNA results in increased antiviral activity and growth suppression. J Interferon Res. 1989 Dec;9(6):649–657. doi: 10.1089/jir.1989.9.649. [DOI] [PubMed] [Google Scholar]
  37. Singh V. K., Maheshwari R. K., Damewood G. P., 4th, Stephensen C. B., Oliver C., Friedman R. M. Interferon alters intracellular transport of vesicular stomatitis virus glycoprotein. J Biol Regul Homeost Agents. 1988 Apr-Jun;2(2):53–62. [PubMed] [Google Scholar]
  38. Ulker N., Samuel C. E. Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. II. Effect on viral macromolecular synthesis. J Biol Chem. 1985 Apr 10;260(7):4324–4330. [PubMed] [Google Scholar]
  39. Visvanathan K. V., Goodbourn S. Double-stranded RNA activates binding of NF-kappa B to an inducible element in the human beta-interferon promoter. EMBO J. 1989 Apr;8(4):1129–1138. doi: 10.1002/j.1460-2075.1989.tb03483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wathelet M. G., Clauss I. M., Content J., Huez G. A. Regulation of two interferon-inducible human genes by interferon, poly(rI).poly(rC) and viruses. Eur J Biochem. 1988 Jun 1;174(2):323–329. doi: 10.1111/j.1432-1033.1988.tb14101.x. [DOI] [PubMed] [Google Scholar]
  41. Whitaker-Dowling P., Youngner J. S. Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase. Virology. 1984 Aug;137(1):171–181. doi: 10.1016/0042-6822(84)90020-5. [DOI] [PubMed] [Google Scholar]

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