Epstein-Barr virus (EBV) is linked with a number of human malignancies. In vitro EBV is able to efficiently immortalise normal human B-lymphocytes [ 11.
Biochemical Society Transactions (1995) 23 3135 Interaction of the dsRNA-dependent protein kinme PKR with Epstein-Barr virus W RNAs. ANDROULLA ELIA, ANDREA SCHOFIELD and MICHAEL J. CLEMENS Division of Biochemistry, St George‘s Hospital Medical School, Cranmer Terrace, London. SW17 ORE, U.K. Epstein-Barr virus (EBV) is linked with a number of human malignancies. In vitro EBV is able to efficiently immortalise normal human B-lymphocytes [ 11. In the majority of the infected cells the virus enters into a latent state in which only a few viral genes are expressed. These genes include the six nuclear antigens (EBNAs 1-6).three membrane proteins and two small RNAs (EBER-I and -2).Since the coordinated action of these gene products is probably sufficient to promote cell proliferation and immortalisation it is important to understand the mechanisms by which their expression is regulated. The EBNAs are derived from mRNAs which arise from a single complex transcription unit that spans a large portion of the viral genome. The messages for each individual EBNA are probably generated by differential splicing of this unit. These mRNAs all share common 5’ exons and have unique 3’ exons. EBNA transcription is usually driven by one of two promoters, located in the BamHI C fragment of the short unique region 121, and in the BamHl W major internal repeat sequence of the genome 131. An additional promoter for EBNA-I only has been identified within the BamHI F region 141. The interferon induced protein kinase, PKR, is intimately involved in the regulation of protein synthesis in response to viral infections. This enzyme is dependent for its activity on doublestranded RNA (dsRNA) which can be produced by symmetrical transcription of viral genomes. Extensive loop structures in mRNAs may also activate PKR. The computer predicted secondary structure of EBV RNA transcribed from the W repeat sequence indicates a possible hairpin structure. This suggests that the RNA may have the ability to bind to PKR and regulate this protein kinase. Furthermore, the presence of repeats of Wencoded RNA at the 5’ ends of a number of viral mRNAs is reminiscent of the situation with HIV-I RNAs, where the TAR sequence is a common 5’ element. TAR has been shown to bind to PKR and regulate it 151. For these reasons we have investigated structural and functional interactions between the W RNAs and PKR, i n comparison with the behaviour of another EBV RNA, EBER- I . Cytoplasmic RNA was extracted from tissue culture cells and analysed by agarose gel electrophoresis and blotting 161. Small RNAs were synthesized by in vitro transcription from recombinant plasmids using T7 RNA polymerase 17,8].U.V. cross-linking of RNA-protein complexes was carried out as described previously 171. After digestion with RNAses T I and A the samples were subjected to SDS polyacrylamide gel electrophoresis and autoradiography. The formation of RNA-protein complexes was also assayed by retention of radioactivity on cellulose nitrate filters 181. Protein synthesis was measured in rabbit reticulocyte lysates by the incorporation of L-[14C] leucine into acid-insoluble material, in the presence or absence of the synthetic dsRNA, PO~Y(~).PO~Y(C). When total cytoplasmic RNA from EBV-positive cell lines was electrophoresed on a 1 % agarose gel and hybridized with a ”Plabelled W riboprobe a number of RNA species were detected. A clearly defined ladder of small RNAs with sizes estimated to be from 0.38 kb to 1.9kb could be seen. The size interval between these small RNAs is approximately 200 nt which is the combined size of the two exons W I and W2 that make up the W transcript. Each small RNA was labelled by in v i m transcription in the presence of [a3’P]UTP. Labelled small RNAs were incubated with
PKR and subjected to U.V. crosslinking. The W l W 2 RNAs, like EBER- 1, could be cross-linked to a protein that migrates to a similar position on the gel as PKR (69 kDa). The binding of labelled W1W2 RNA to PKR was competed out by unlabelled EBER-I and (WIW2), RNAs, as well as by W l W 2 itself. In contrast, unlabelled tRNA was a poor competitor for binding to PKR. The filter-binding assay was used to examine the competition for binding to PKR between tRNA and the W1W2 RNAs or EBER-I. Only a 1000-fold molar excess of tRNA showed any significant effect on labelled RNA binding. The effect of the W l W 2 RNA repeats on protein synthesis, compared with the effect of EBER-I, was investigated using the reticulocyte lysate system. I n the absence of poly(I).poly(C) the W l W 2 RNAs caused a shut-off of protein synthesis. In the presence of poly(I).poly(C) at a concentration sufficient fur activation of PKR the W1W2 repeat RNAs had no effect oil overall protein synthesis, suggesting that even at high concentrations they are unable to block the activation of PKR by dsRNA. In contrast, EBER-I inhibited the activation of PKR by dsRNA; thus rescue of protein synthesis from inhibition by poly(l).poly(C) was observed in the presence of this RNA. The main conclusions to be drawn from this work are that thc W l W 2 repeat RNAs bind to PKR in a specific manner. This binding is at the same site on PKR as for EBER-I. The functional role of the W l W 2 repeat RNAs has yet to be established but the reticulocyte lysate assays suggest that these RNAs are able to shut off protein synthesis. It is possible that Epstein-Barr virus encodes RNAs capable of both activating PKR (the W l W 2 RNAs) and blocking the activation of PKR (EBER-I), which may provide fine-tuning for the activity of the protein kinase at different stages in the life-cycle of the virus. This work is funded by a grant from The Leukaemia Research Fund. A.E. is supported by a studentship from the Medical Research Council. We are grateful to Drs P.J. Farrell, M.G. Katze, D.H. Levin and D.R. Gewert for gifts of materials. I . Sugden,B. (1989)Cell 57,5-7 2. Bodescot,M., Perricaudet,M. & Farrel1.P.J. (1987)J.Virol. 61, 3424-3430 3. Samp1e.J.. Hummel,M., Braun,D., Birkenbach,M. & Kieff,E. (1986)Proc. Natl. Acad.Sci. U .S.A. 83, 5096-5100 4. Schaefer, B.C., Woisetschlaeger ,M ., S trominger ,J .L. & Speck,S.H. (I99I ) Proc. Natl. Acad.Sci. U .S.A. 88, 6550-6554 5. Gunnery,S., Rice.A.P., Robertson,H.D. & Mathews,M. B. ( I 990) Proc. Natl. Acad.Sci. U .S.A. 87, 8687-8691 6.Clarke,P.A., Sharp,N.A., Arrand,J. R. & C1emens.M. J. ( 1990) Biochim.Biophys.Acta 1050, 167-173 7. C1arke.P.A.. Schwemmle,M., SchickingerJ., Hi1se.K. & Clemens,M.J. (1991)Nucleic Acids Res. 19,243-248 8. Sharp,T.V., Schwemmle,M., Jeffrey,I., Laing,K., Mellor,H., Proud,C.G., Hi1se.K. & Clemens,M.J. (1993)Nucleic Acids Res. 21, 4483-4490
