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. 1996 Nov 15;24(22):4471–4478. doi: 10.1093/nar/24.22.4471

Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

A Elia 1, K G Laing 1, A Schofield 1, V J Tilleray 1, M J Clemens 1
PMCID: PMC146269  PMID: 8948637

Abstract

During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription.

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Selected References

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  1. Alfieri C., Birkenbach M., Kieff E. Early events in Epstein-Barr virus infection of human B lymphocytes. Virology. 1991 Apr;181(2):595–608. doi: 10.1016/0042-6822(91)90893-g. [DOI] [PubMed] [Google Scholar]
  2. Allday M. J., Crawford D. H., Griffin B. E. Epstein-Barr virus latent gene expression during the initiation of B cell immortalization. J Gen Virol. 1989 Jul;70(Pt 7):1755–1764. doi: 10.1099/0022-1317-70-7-1755. [DOI] [PubMed] [Google Scholar]
  3. Barber G. N., Tomita J., Garfinkel M. S., Meurs E., Hovanessian A., Katze M. G. Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase. Virology. 1992 Dec;191(2):670–679. doi: 10.1016/0042-6822(92)90242-h. [DOI] [PubMed] [Google Scholar]
  4. Bass B. L., Hurst S. R., Singer J. D. Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs. Curr Biol. 1994 Apr 1;4(4):301–314. doi: 10.1016/s0960-9822(00)00069-5. [DOI] [PubMed] [Google Scholar]
  5. Bischoff J. R., Samuel C. E. Mechanism of interferon action. Activation of the human P1/eIF-2 alpha protein kinase by individual reovirus s-class mRNAs: s1 mRNA is a potent activator relative to s4 mRNA. Virology. 1989 Sep;172(1):106–115. doi: 10.1016/0042-6822(89)90112-8. [DOI] [PubMed] [Google Scholar]
  6. Bodescot M., Chambraud B., Farrell P., Perricaudet M. Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide. EMBO J. 1984 Aug;3(8):1913–1917. doi: 10.1002/j.1460-2075.1984.tb02067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bodescot M., Perricaudet M. Epstein-Barr virus mRNAs produced by alternative splicing. Nucleic Acids Res. 1986 Sep 11;14(17):7103–7114. doi: 10.1093/nar/14.17.7103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bodescot M., Perricaudet M., Farrell P. J. A promoter for the highly spliced EBNA family of RNAs of Epstein-Barr virus. J Virol. 1987 Nov;61(11):3424–3430. doi: 10.1128/jvi.61.11.3424-3430.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clarke P. A., Schwemmle M., Schickinger J., Hilse K., Clemens M. J. Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI. Nucleic Acids Res. 1991 Jan 25;19(2):243–248. doi: 10.1093/nar/19.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke P. A., Sharp N. A., Arrand J. R., Clemens M. J. Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state. Biochim Biophys Acta. 1990 Aug 27;1050(1-3):167–173. doi: 10.1016/0167-4781(90)90161-t. [DOI] [PubMed] [Google Scholar]
  11. Clarke P. A., Sharp N. A., Clemens M. J. Translational control by the Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates. Eur J Biochem. 1990 Nov 13;193(3):635–641. doi: 10.1111/j.1432-1033.1990.tb19381.x. [DOI] [PubMed] [Google Scholar]
  12. Farrell P. J., Balkow K., Hunt T., Jackson R. J., Trachsel H. Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis. Cell. 1977 May;11(1):187–200. doi: 10.1016/0092-8674(77)90330-0. [DOI] [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. Graeble M. A., Churcher M. J., Lowe A. D., Gait M. J., Karn J. Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6184–6188. doi: 10.1073/pnas.90.13.6184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green S. R., Mathews M. B. Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. Genes Dev. 1992 Dec;6(12B):2478–2490. doi: 10.1101/gad.6.12b.2478. [DOI] [PubMed] [Google Scholar]
  16. Gunnery S., Green S. R., Mathews M. B. Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11557–11561. doi: 10.1073/pnas.89.23.11557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gunnery S., Rice A. P., Robertson H. D., Mathews M. B. Tat-responsive region RNA of human immunodeficiency virus 1 can prevent activation of the double-stranded-RNA-activated protein kinase. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8687–8691. doi: 10.1073/pnas.87.22.8687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gurevich V. V., Pokrovskaya I. D., Obukhova T. A., Zozulya S. A. Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal Biochem. 1991 Jun;195(2):207–213. doi: 10.1016/0003-2697(91)90318-n. [DOI] [PubMed] [Google Scholar]
  19. Henry G. L., McCormack S. J., Thomis D. C., Samuel C. E. Mechanism of interferon action. Translational control and the RNA-dependent protein kinase (PKR): antagonists of PKR enhance the translational activity of mRNAs that include a 161 nucleotide region from reovirus S1 mRNA. J Biol Regul Homeost Agents. 1994 Jan-Mar;8(1):15–24. [PubMed] [Google Scholar]
  20. Hunter T., Hunt T., Jackson R. J., Robertson H. D. The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975 Jan 25;250(2):409–417. [PubMed] [Google Scholar]
  21. Kao S. Y., Calman A. F., Luciw P. A., Peterlin B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987 Dec 3;330(6147):489–493. doi: 10.1038/330489a0. [DOI] [PubMed] [Google Scholar]
  22. Kostura M., Mathews M. B. Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI. Mol Cell Biol. 1989 Apr;9(4):1576–1586. doi: 10.1128/mcb.9.4.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
  25. Kure S., Tada K., Wada J., Yoshie O. Inhibition of Epstein-Barr virus infection in vitro by recombinant human interferons alpha and gamma. Virus Res. 1986 Sep;5(4):377–390. doi: 10.1016/0168-1702(86)90030-4. [DOI] [PubMed] [Google Scholar]
  26. Lemay G. Transcriptional and translational events during reovirus infection. Biochem Cell Biol. 1988 Aug;66(8):803–812. doi: 10.1139/o88-092. [DOI] [PubMed] [Google Scholar]
  27. Maitra R. K., McMillan N. A., Desai S., McSwiggen J., Hovanessian A. G., Sen G., Williams B. R., Silverman R. H. HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes. Virology. 1994 Nov 1;204(2):823–827. doi: 10.1006/viro.1994.1601. [DOI] [PubMed] [Google Scholar]
  28. Manche L., Green S. R., Schmedt C., Mathews M. B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol. 1992 Nov;12(11):5238–5248. doi: 10.1128/mcb.12.11.5238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mathews M. B., Shenk T. Adenovirus virus-associated RNA and translation control. J Virol. 1991 Nov;65(11):5657–5662. doi: 10.1128/jvi.65.11.5657-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McBryant S. J., Kassavetis G. A., Gottesfeld J. M. Repression of vertebrate RNA polymerase III transcription by DNA binding proteins located upstream from the transcription start site. J Mol Biol. 1995 Jul 14;250(3):315–326. doi: 10.1006/jmbi.1995.0379. [DOI] [PubMed] [Google Scholar]
  31. Mellits K. H., Pe'ery T., Manche L., Robertson H. D., Mathews M. B. Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector. Nucleic Acids Res. 1990 Sep 25;18(18):5401–5406. doi: 10.1093/nar/18.18.5401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Muesing M. A., Smith D. H., Cabradilla C. D., Benton C. V., Lasky L. A., Capon D. J. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature. 1985 Feb 7;313(6002):450–458. doi: 10.1038/313450a0. [DOI] [PubMed] [Google Scholar]
  33. Müller F., Clarkson S. G. Nucleotide sequence of genes coding for tRNAPhe and tRNATyr from a repeating unit of X. laevis DNA. Cell. 1980 Feb;19(2):345–353. doi: 10.1016/0092-8674(80)90509-7. [DOI] [PubMed] [Google Scholar]
  34. Nilsson T., Sjöblom A., Masucci M. G., Rymo L. Viral and cellular factors influence the activity of the Epstein-Barr virus BCR2 and BWR1 promoters in cells of different phenotype. Virology. 1993 Apr;193(2):774–785. doi: 10.1006/viro.1993.1186. [DOI] [PubMed] [Google Scholar]
  35. O'Malley R. P., Duncan R. F., Hershey J. W., Mathews M. B. Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells. Virology. 1989 Jan;168(1):112–118. doi: 10.1016/0042-6822(89)90409-1. [DOI] [PubMed] [Google Scholar]
  36. O'Neill R. E., Racaniello V. R. Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. J Virol. 1989 Dec;63(12):5069–5075. doi: 10.1128/jvi.63.12.5069-5075.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Price N., Proud C. The guanine nucleotide-exchange factor, eIF-2B. Biochimie. 1994;76(8):748–760. doi: 10.1016/0300-9084(94)90079-5. [DOI] [PubMed] [Google Scholar]
  38. Robertson H. D., Manche L., Mathews M. B. Paradoxical interactions between human delta hepatitis agent RNA and the cellular protein kinase PKR. J Virol. 1996 Aug;70(8):5611–5617. doi: 10.1128/jvi.70.8.5611-5617.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rogers R. P., Strominger J. L., Speck S. H. Epstein-Barr virus in B lymphocytes: viral gene expression and function in latency. Adv Cancer Res. 1992;58:1–26. doi: 10.1016/s0065-230x(08)60288-2. [DOI] [PubMed] [Google Scholar]
  40. Rogers R. P., Woisetschlaeger M., Speck S. H. Alternative splicing dictates translational start in Epstein-Barr virus transcripts. EMBO J. 1990 Jul;9(7):2273–2277. doi: 10.1002/j.1460-2075.1990.tb07398.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Sample J., Brooks L., Sample C., Young L., Rowe M., Gregory C., Rickinson A., Kieff E. Restricted Epstein-Barr virus protein expression in Burkitt lymphoma is due to a different Epstein-Barr nuclear antigen 1 transcriptional initiation site. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6343–6347. doi: 10.1073/pnas.88.14.6343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Samuel C. E. The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. J Biol Chem. 1993 Apr 15;268(11):7603–7606. [PubMed] [Google Scholar]
  44. Schaefer B. C., Strominger J. L., Speck S. H. Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10565–10569. doi: 10.1073/pnas.92.23.10565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schaefer B. C., Woisetschlaeger M., Strominger J. L., Speck S. H. Exclusive expression of Epstein-Barr virus nuclear antigen 1 in Burkitt lymphoma arises from a third promoter, distinct from the promoters used in latently infected lymphocytes. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6550–6554. doi: 10.1073/pnas.88.15.6550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schmedt C., Green S. R., Manche L., Taylor D. R., Ma Y., Mathews M. B. Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR). J Mol Biol. 1995 May 26;249(1):29–44. doi: 10.1006/jmbi.1995.0278. [DOI] [PubMed] [Google Scholar]
  47. SenGupta D. N., Silverman R. H. Activation of interferon-regulated, dsRNA-dependent enzymes by human immunodeficiency virus-1 leader RNA. Nucleic Acids Res. 1989 Feb 11;17(3):969–978. doi: 10.1093/nar/17.3.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sharp T. V., Schwemmle M., Jeffrey I., Laing K., Mellor H., Proud C. G., Hilse K., Clemens M. J. Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA. Nucleic Acids Res. 1993 Sep 25;21(19):4483–4490. doi: 10.1093/nar/21.19.4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sharp T. V., Xiao Q., Jeffrey I., Gewert D. R., Clemens M. J. Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR. Eur J Biochem. 1993 Jun 15;214(3):945–948. doi: 10.1111/j.1432-1033.1993.tb17998.x. [DOI] [PubMed] [Google Scholar]
  50. Tsai C. N., Liu S. T., Chang Y. S. Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene. DNA Cell Biol. 1995 Sep;14(9):767–776. doi: 10.1089/dna.1995.14.767. [DOI] [PubMed] [Google Scholar]
  51. Woisetschlaeger M., Strominger J. L., Speck S. H. Mutually exclusive use of viral promoters in Epstein-Barr virus latently infected lymphocytes. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6498–6502. doi: 10.1073/pnas.86.17.6498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Woisetschlaeger M., Yandava C. N., Furmanski L. A., Strominger J. L., Speck S. H. Promoter switching in Epstein-Barr virus during the initial stages of infection of B lymphocytes. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1725–1729. doi: 10.1073/pnas.87.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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