Báo cáo khoa học: Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1

doi:10.1111/j.1432-1033.2004.04099.x

Eur. J. Biochem. 271, 1895–1905 (2004) (cid:1) FEBS 2004

Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1

Androulla Elia, Jashmin Vyas*, Kenneth G. Laing† and Michael J. Clemens

Translational Control Group, Department of Basic Medical Sciences, St George’s Hospital Medical School, London, UK

dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 pre- vents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.

Keywords: double-stranded RNA; EBV; protein kinase R; RNA–protein interactions; translational control.

Epstein–Barr virus (EBV) is a potent mitogenic and anti- apoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phe- notype and that this may be related to inhibition of the pro- apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by

Epstein-Barr virus (EBV) is linked to a number of human malignancies, including Burkitt’s lymphoma, nasopharyn- geal carcinoma and lymphoproliferative diseases in immu- nocompromised patients. In vitro, EBV is able to efficiently immortalize normal human B lymphocytes [1–3]. In the majority of these infected cells, the virus enters into a latent state in which only a few viral genes are expressed. These viral genes encode six nuclear antigens [Epstein-Barr nuclear antigens (EBNAs) 1–6], three membrane proteins [latent membrane protein (LMP)-1, -2A and -2B] and two small RNAs (EBER-1 and -2) [3]. Several of these genes may contribute to the growth-promoting and transforming abilities of EBV.

The abundantly expressed RNA, EBER-1, is a highly structured molecule that binds to the interferon-induced

protein kinase R (PKR) [4], and blocks activation of this enzyme by double-stranded RNA (dsRNA) [5–8]. Although relatively high concentrations of EBER-1 are required to obtain this effect, it has been calculated that the levels achieved in EBV-infected cells lie within this concentration range [9] and thus the regulation of PKR by this small RNA is probably of physiological relevance. It has been shown that the expression of EBER-1 in EBV-negative B cells promotes a tumorigenic phenotype [10,11]. Consistent with the above in vitro effects of the RNA, inhibition of the pro-apoptotic effects of PKR may be responsible for this effect [12]. Recently, we have reported that EBER-1 can also stimulate expression of a cotransfected reporter gene independently of any effect on PKR [9]. Thus, the viral RNA must have at least one additional mode of action that may also contribute to its antiapoptotic and cell-transforming abilities.

In EBV-infected cells, EBER-1 has been shown to bind to L22, an abundant, 14.3 kDa ribosomal protein. The function of L22 remains unclear, although it is involved in the 3;21 chromosomal translocation that characterizes some forms of acute myeloid leukaemia [13]. L22 is relocated from the nucleolus to the nucleoplasm following EBV infection [14], but the significance of this has yet to be established. L22 is a component of an EBER-RNP particle in which the RNA is also associated with the La antigen [15,16]. A number of other cellular and viral RNAs, including telomerase RNA, can also bind to L22 [17–19].

As both PKR and L22 are ligands for EBER-1, we investigated the significance of the ability of L22 to bind RNA for the regulation of PKR activity by EBER-1. The data reported here suggest that L22 counteracts the inhib- itory effect of the viral RNA towards the protein kinase

Correspondence to M. J. Clemens, Department of Basic Medical Sciences, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. Fax: + 44 20 87252992, Tel.: + 44 20 87255762, E-mail: M.Clemens@sghms.ac.uk Abbreviations: dsRBD, double-stranded RNA-binding domain; dsRBM, double-stranded RNA-binding motif; dsRNA, double- stranded RNA; EBNA, Epstein-Barr nuclear antigen; EBV, Epstein- Barr virus; LMP, latent membrane protein; PKR, protein kinase R. Present addresses: *Centre for Cutaneous Research, Barts and The London, Queen Mary’s School of Medicine and Dentistry, 2 Newark St., London E1 2AT, UK; (cid:1)Department of Cellular and Molecular Medicine, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. (Received 3 December 2003, revised 18 March 2004, accepted 19 March 2004)

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in vitro and in vivo. Moreover, L22 also blocks the PKR- independent effect of EBER-1 in transfected cells. These results suggest that L22 may exert a protective effect against the transforming potential of EBER-1 and EBV in vivo.

Materials and methods

Materials

Recombinant baculovirus, encoding PKR K296R, was a gift from Dr G. Barber (University of Miami). Monoclonal antibody against PKR was a gift from Dr E. Meurs (Institut Pasteur, Paris) and antibody against L22 was a gift from Dr J. Steitz (Yale University). T7 RNA polymerase was purchased from Cambio, and poly(I).poly(C) was obtained from Sigma. Radiochemicals were from NEN.

Preparation of purified PKR

HeLa cells were grown in spinner culture in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco). Exponentially growing cells were treated with human interferon-a (1000 UÆmL)1 for 24 h) to induce PKR, which was purified from the ribosomal salt wash obtained from 20 L of cells [20,21]. The protein was subjected to chromatography on DEAE-cellulose (where it elutes in the flow-through fraction), followed by chroma- tography on a Mono-S FPLC column (Pharmacia). Proteins were eluted with a 50–450 mM KCl gradient and the PKR-containing fractions identified by Western blot- ting. All buffers contained the protease inhibitors aprotinin (1 lgÆmL)1), leupeptin (1 lgÆmL)1), pepstatin (1 lgÆmL)1) and phenylmethanesulfonyl fluoride (1 lM). The catalyti- cally inactive K296R mutant form of PKR was purified to near homogeneity from Sf9 insect cells infected with a recombinant baculovirus, as described previously [22] (Fig. 1A).

Preparation and purification of recombinant L22

promoter, has been described previously [5,6]. The plasmid was linearized with the restriction enzyme Sau3A and transcribed using T7 RNA polymerase [5]. For the produc- tion of radiolabelled transcript, unlabelled UTP was omit- ted from the reaction and replaced with 500 lCiÆmL)1 of [32P]UTP[aP].

Preparation of 32P-labelled poly(I).poly(C)

Recombinant L22 was expressed using the L22/pQE31 vector (a kind gift from Dr Jonny Wood, Glasgow). This construct gives high level expression of His-tagged L22 in Escherichia coli M15 after induction with 1 mM isopropyl thio-b-D-galactoside for 3.5 h at 37 (cid:2)C. Induced bacteria were lysed and sonicated and, after treatment with DNase I and RNase A, the clarified lysate was incubated with 1 mL of Ni-nitrilotriacetic acid beads (Qiagen). The L22 was eluted from the washed beads with 50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole (pH 8.0) and desalted using a PD-10 column. All buffers used during the purification contained protease inhibitors, as described above. The protein was further purified using a 100K Microcon spin column to remove larger products and then concentrated on a 3000K Centricon column. Bradford analysis [23] was used to measure the concentration of the essentially homogeneous protein (Fig. 1A).

Invitrosynthesis of EBER-1

The transcription plasmid pPAC-1, for the in vitro synthesis of EBER-1 under the direction of a T7 RNA polymerase

Thirty micrograms of poly(I).poly(C) (Sigma) was dephos- phorylated by incubation with 4 U of alkaline phosphatase (Roche) in 50 lL of 50 mM Tris/HCl, pH 9.0, containing 1 mM MgCl2, 0.1 mM ZnCl2 and 1 mM spermidine. After 30 min at 37 (cid:2)C, the enzyme was inactivated with 10 mM EDTA (pH 8.0) and heated to 68 (cid:2)C for 10 min. The RNA was extracted with phenol/chloroform, precipitated with isopropanol in the presence of carrier glycogen and labelled at the 5¢ end with 10 U of T4 polynucleotide kinase and

Fig. 1. Competition between protein kinase R (PKR) and L22 for binding to EBER-1. (A) Recombinant PKR (K296R) and L22 were purified as described in the Materials and methods. The purities of the two proteins were determined by SDS gel electrophoresis, followed by staining with Coomassie Blue. Molecular mass markers are indicated to the left of each panel. (B) Purified L22 (132 ng) was incubated with 2 · 105 counts per minute of [32P]UTP[aP]-labelled EBER-1 in the absence of PKR (lane 2) or in the presence of increasing amounts of purified PKR K296R (1- and 10-fold molar excess over L22; lanes 3 and 4). Conversely, PKR (450 ng) was incubated with the labelled EBER-1 in the absence of L22 (lane 5) or in the presence of increasing amounts of L22 (1- and 10-fold molar excess over PKR; lanes 6 and 7). The samples were UV cross-linked, as described in the Materials and methods, separated on a 15% SDS gel and analysed by autoradio- graphy. Molecular mass markers are shown in lane 1, and the positions of the cross-linked [PKR.EBER-1] and [L22.EBER-1] complexes in lanes 2–7 are indicated. (Note that the quantity, by weight, of L22 used in lanes 2–4, was less than that of the PKR used in lanes 5–7, in order to give approximately equimolar amounts of the two proteins in the two sets of incubations.)

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50 lCi of [32P]ATP[cP] in the presence of 70 mM Tris/HCl, pH 7.6, 10 mM MgCl2 and 5 mM dithiothreitol. The labelled RNA was extracted and precipitated, as described above, and dissolved in 20 lL of 50 mM KCl to maintain the double-stranded structure.

EDTA, 30% (v/v) glycerol and 2 lM [32P]ATP[cP] (2.5–5 lCi). The incubation mixtres also included poly(I). poly(C), EBER-1 and L22 as indicated in the figure legends. After 20 min at 30 (cid:2)C, proteins were denatured with 2· SDS sample buffer and separated by SDS-PAGE (15% gels). The dried gels were analysed by autoradiogra- phy and quantified using a STORM phosphoimager.

UV cross-linking assays for EBER-1–protein interactions

Transient transfection of murine embryonic fibroblasts

105

cells)

Fibroblasts from wild-type and PKR knockout cells were transiently transfected with plasmids (each at 1 lg of DNA per (pLEXIII), L22 encoding EBER-1 (pFLAGCMV2-L22) and PKR (pCMV-PKR), in various combinations. FuGENE 6 reagent was used for the transfections, as described previously [28]. Plasmid pcDNA3 was used as a control. At the same time the cells were transfected with an expression vector for the reporter enzyme b-galactosidase (pRSVLacZII) [9]. The cells were harvested 48 h later and the enzyme activity in extracts was assayed using a luminescence-based method (Tropix Prod- ucts, Bedford, MA, USA). The data were calculated as relative light units per unit of protein in the extracts.

UV cross-linking of radiolabelled EBER-1 to purified L22 or PKR was carried out as described previously [6]. For convenience, the K296R mutant form of PKR was used in such experiments, as this protein can be easily produced in pure form. Although this mutant has lost all protein kinase activity, it retains the normal RNA-binding characteristics of wild-type PKR [24]. Radioactive RNA (2 · 105 counts per minute) was incubated with the proteins for 15 min at 30 (cid:2)C in the presence of 100 mM KCl, 10 mM Tris/HCl, pH 7.5, in a final volume of 25 lL. RNA was cross-linked to protein by irradiation at 254 nm for 5 min at 4 (cid:2)C. The samples were then adjusted to 0.5% (v/v) in N-lauroyl- sarcosine. Each sample was treated with 20 U of RNase T1 and 20 lg of RNase A, for 1 h at 37 (cid:2)C, and analysed by SDS gel electrophoresis. Cross-linked complexes were identified by autoradiography.

Results

Poly(I).poly(C)-binding assays

Competition between L22 and PKR for EBER-1

In view of the fact that EBER-1 is able to interact with both L22 and PKR, it was of interest to determine whether the two proteins compete with each other for binding to the small RNA. We investigated this using UV-induced cross- linking of the radiolabelled RNA to L22 and PKR when the two proteins were added together, in different ratios. The results in Fig. 1B (lane 2) show that EBER-1 can be cross- linked to L22, as expected. However, in the presence of increasing amounts of PKR, the intensity of EBER-1 cross- linking to a fixed amount of L22 was reduced by up to 45%; this was coincident with the appearance of a labelled band of (cid:1) 69 kDa, corresponding to PKR itself (Fig. 1B; lanes 3 and 4). Conversely, EBER-1 could be cross-linked to PKR, as shown previously [6] (Fig. 1B; lane 5), but as the amount of L22 was increased, the intensity of this cross-linking strongly diminished and a labelled band appeared at 14 kDa, corresponding to L22 (Fig. 1B, lanes 6 and 7). These data indicate that PKR and L22 do compete with each other for EBER-1 and suggest that the two proteins bind to the same or an overlapping site on the small RNA molecule.

The binding of poly(I).poly(C) to L22 or PKR was assayed using filter-binding assays. This assay is based on the retention of radioactive RNA on cellulose nitrate filters 32P-labelled poly(I).poly(C) when bound to protein. (1 · 105 counts per minute) was incubated with various amounts of L22 or PKR K296R, in 25 lL reaction in the presence of 10 mM Tris/HCl, pH 7.6, volumes, 100 mM KCl and 0.8 mM Mg acetate for 15 min, as described previously [7]. The complexes were rapidly filtered through cellulose nitrate filters on a microtitre plate and thoroughly washed with the above buffer. The extent of complex formation was quantified by scintillation counting of the excised filters. For competition assays, increasing amounts of nonradioactive poly(I).poly(C) were included to yield up to a 100-fold molar excess over the labelled form. The abilities of proteins to bind to double-stranded RNA (dsRNA) were also assessed using immobilized poly(I). poly(C). Ten microlitres of poly(I).poly(C)-agarose beads were incubated with varying amounts of PKR, L22 and EBER-1 using conditions described in Kumar et al. [25]. After 2 h of incubation at room temperature, the beads were washed thoroughly with 1 mL of binding buffer and SDS sample buffer was added. Samples were separated on a 15% SDS gel and Western blotting analysis was carried out using anti-PKR or anti-L22 primary antibodies and an alkaline phosphatase-tagged secondary antibody. The blots were developed using Nitro Blue tetrazolium substrate, as previously described [26], and quantified by densitometry.

Measurement of PKR activation

We have previously shown that EBER-1 competes with the synthetic dsRNA ligand poly(I).poly(C) for interaction with the dsRNA-binding domain (dsRBD) of PKR [7]. However, ribosomal protein L22 does not possess a canonical dsRBD and would not be expected to interact with dsRNA. Figure 2A shows that, indeed, even very high concentrations of poly(I).poly(C) did not inhibit the binding of EBER-1 to L22 in vitro, as determined using the UV cross-linking assay. Consistent with this finding, in a filter- binding assay L22 showed very poor binding of labelled poly(I).poly(C), in contrast to PKR (Fig. 2B). Moreover, the binding to L22 that did occur was nonspecific because it could not be competed by excess unlabelled poly(I).poly(C)

Activation of PKR was assessed by autophosphorylation of the protein kinase in the presence of [32P]ATP[cP] [27]. Incubation mixtures (10 lL) contained 10 mM Tris/HCl, pH 7.5, 100 mM KCl, 5 mM MnCl2, 5 mM MgCl2, 0.1 mM

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addition of L22 alone had a small inhibitory effect on the binding of PKR to poly(I).poly(C) (Fig. 3B,C; lanes 4 and 5). However, L22 partially relieved the inhibitory effect of EBER-1 on the binding of PKR to poly(I).poly(C)-agarose (Fig. 3B,C; lane 6 vs. lane 7), suggesting that L22 sequesters the EBER-1 and makes it unavailable for competition with the immobilized dsRNA.

(Fig. 2C), unlike the binding to PKR (Fig. 2D). In addition, L22 bound poorly to immobilized poly(I).poly(C) (attached to agarose beads) (Fig. 3A). These data confirm that although L22 can bind EBER-1, the ribosomal protein is a weak dsRNA-binding protein. It is therefore probable that L22 recognizes a feature of EBER-1 that is distinct from the double-stranded hairpin structures present in this molecule.

Interaction between L22 and PKR

Although L22 has very low dsRNA-binding activity and, on its own, interacts poorly with immobilized poly(I).poly(C) (Fig. 3A), nevertheless, in the presence of PKR the ribosomal protein could be coprecipitated with the protein kinase on poly(I).poly(C)-agarose beads (Fig. 3B; bottom panel, lanes 4 and 5), suggesting a possible direct interaction between the two proteins. However this PKR-dependent association of L22 with the poly(I).poly(C)-agarose could not be seen in the additional presence of EBER-1 (Fig. 3B; bottom panel, lane 7), whereas there was still some binding of PKR to the immobilized dsRNA under these conditions.

As L22 and PKR compete for EBER-1 binding (Fig. 1B), we also investigated whether L22 can interfere with the ability of EBER-1 to inhibit the binding of dsRNA to PKR. To do this we examined the extent of capture of PKR on poly(I).poly(C)-agarose. Figure 3B shows that PKR bound to poly(I).poly(C)-agarose beads (lane 1) and that the addition of soluble poly(I).poly(C) prevented such binding (Fig. 3B,C; lanes 2 and 3). Similarly, the addition of soluble EBER-1 to the assay also partially prevented the binding of PKR to the beads, although the small RNA was a less efficient competitor than poly(I).poly(C) itself (Fig. 3B,C; lane 6). This is consistent with the lower affinity of PKR for EBER-1, relative to that for poly(I).poly(C) [7]. The

Fig. 2. Comparison of the binding of protein kinase R (PKR) and L22 to double-stranded RNA (dsRNA). (A) Absence of competition between EBER-1 and dsRNA for binding to L22. A total of 180 ng of L22 was incubated with 2 · 105 counts per minute of labelled EBER-1 in the presence of increasing concentrations of unlabelled poly(I).poly(C), and RNA–protein complexes were then UV cross-linked and analysed as described in the legend to Fig. 1. (B) Relative abilities of PKR and L22 to bind dsRNA. End-labelled poly(I).poly(C) (105 counts per minute) was incubated with the indicated amounts of PKR K296R (squares) or L22 (triangles) and the extent of complex formation was measured by filter binding, as described in the Materials and methods. Incubations were performed in triplicate and the data represent the mean ± SEM values. [Note that the molecular mass of PKR is more than four times higher than that of L22, so that although much larger amounts of the latter (in molar terms) were used, the level of dsRNA binding was still considerably less.] (C) and (D) Filter-binding assays were carried out using 105 counts per minute of end-labelled poly(I).poly(C) in the presence of increasing concentrations of unlabelled poly(I).poly(C); the dsRNA was incubated with 13 ng of L22 (C) or 18 ng of PKR (D), and binding of the labelled poly(I).poly(C) was assessed as described in (B).

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level seen with poly(I).poly(C) alone (Fig. 4C; compare lane 2 with lanes 5 and 6). These results indicate that L22 can indirectly regulate PKR activity through its ability to sequester EBER-1. Interestingly, in the same experiment, L22 itself was also phosphorylated in a poly(I).poly(C)- dependent manner, suggesting that L22 can serve as a substrate for PKR, at least in vitro. Further evidence of this is presented in Fig. 5A (lower panel, lanes 7 and 8). The latter figure also illustrates that L22 alone had no effect either on basal PKR activity (upper panel, lane 1 vs. lanes 3–5) or on the activation of the protein kinase by poly(I).poly(C) (lane 2 vs. lanes 6–8). Figure 5B provides confirmation that the protein which is phosphorylated when recombinant L22 is added to assays containing active PKR is indeed the ribosomal protein itself. Following a kinase assay, gel-separated proteins were transferred to a poly(vinylidene difluoride) (PVDF) membrane and subjec- ted to immunoblotting analysis using a monoclonal anti- body to L22. The PVDF membrane was also exposed to X-ray film. Alignment of the autoradiograph with the blot indicated that the major radioactive band at (cid:1) 14 kDa had an identical mobility to that of the immunoreactive L22.

Effects of EBER-1 and L22 on reporter gene expression

We have previously shown that transient transfection of an EBER-1 expression vector into mammalian cells stimulates the expression of a cotransfected reporter gene [9]. Further- more, this effect could be observed in embryonic fibroblasts from both wild-type and PKR knockout mice, suggesting at least one additional, PKR-independent, mode of action of the small viral RNA. We therefore examined whether L22 is able to interfere with the effect of EBER-1 in intact cells, both in the presence and absence of PKR. We also investigated the effect of enhancing PKR expression by transient transfection [28], and the modulation of this effect by EBER-1 and L22.

This suggests that L22 which is bound to EBER-1 is unable to form a complex with PKR. Thus, L22 and EBER-1 may have mutually antagonistic effects on each other’s ability to associate with the protein kinase.

Figure 6A shows that EBER-1 enhanced reporter gene expression in wild-type cells by 2.9-fold and that this effect was inhibited, by 67%, by coexpression of L22. Expression of the ribosomal protein alone had no effect. As reported previously [9], EBER-1 also enhanced b-galactosidase activity in the PKR–/– cells, although to a smaller extent relative to the pcDNA-transfected control (Fig. 6B) (note, however, that PKR–/– cells show a much higher basal level of expression of the reporter gene than do PKR+/+ cells). Again, the effect of EBER-1 was abrogated by coexpression of L22.

Effect of L22 on PKR activity

As reported previously [7], EBER-1 inhibited the activation of PKR by poly(I).poly(C) and had virtually no ability itself to activate the kinase (Fig. 4A,B). As L22 interfered with the binding of EBER-1 to PKR (Fig. 1B), and at least partially restored the ability of PKR to bind to poly(I). poly(C) in the presence of EBER-1 (Fig. 3B,C), we investi- gated whether the ribosomal protein could rescue the ability of PKR to undergo autophosphorylation and activation in the presence of EBER-1. The experiment illustrated in Fig. 4C shows that in the presence of relatively low concentrations of both poly(I).poly(C) and EBER-1, L22 was able to restore the autophosphorylation of PKR to the

As reported in previous studies [9], cotransfection of PKR was very inhibitory to reporter gene activity in both PKR+/+ and PKR–/– cells. However, in the absence of exogenous L22, additional transfection of EBER-1 increased b-galactosidase activity in the presence of exogen- ous PKR by 77.8% in PKR+/+ cells and 44.5% in PKR–/– cells (Fig. 6C). In the presence of transfected L22, this stimulation was strongly inhibited (Fig. 6C). These results are consistent with our in vitro findings that L22 prevents the inhibition of PKR by EBER-1. Nevertheless, it is clear that EBER-1 can also stimulate reporter gene expression in the complete absence of the protein kinase and that this effect too is markedly inhibited by L22, presumably because of sequestration of the RNA by the ribosomal protein.

Fig. 3. Interactions of L22 and protein kinase R (PKR) with immobi- lized double-stranded RNA (dsRNA). (A) L22 alone binds poorly to dsRNA. Poly(I).poly(C)-agarose beads were incubated for 2 h at room temperature with increasing amounts of purified L22 (up to 200 ng). After extensive washing of the beads, the association of the protein with the immobilized poly(I).poly(C) was determined by SDS gel electrophoresis followed by immunoblotting (lanes 1–4). The highest amount of input L22 is shown in the lane designated (cid:3)load(cid:4). (B) L22 associates with poly(I).poly(C)-agarose only in the presence of PKR. Beads were incubated, as described in (A), with the indicated combi- nations of PKR (200 ng), L22 (200 or 400 ng), soluble poly(I).poly(C) (15 or 150 lg) or EBER-1 (100 lg). The association of PKR and L22 with the immobilized poly(I).poly(C) was determined by immuno- blotting, as described in (A). (C) The extent of inhibition of the binding of PKR to the poly(I).poly(C)-agarose beads, by the various combi- nations of soluble poly(I).poly(C), EBER-1 and L22, was quantified by densitometry of the PKR blot shown in (B).

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Fig. 4. Effects of EBER-1 and L22 on the activation of protein kinase R (PKR) by double- stranded RNA (dsRNA). (A) and (B) Wild- type PKR was incubated with [32P]ATP[cP] in the absence or presence of the indicated con- centrations of poly(I).poly(C) and EBER-1 for 20 min at 30 (cid:2)C, as described in the Materials and methods. The samples were analysed for PKR autophosphorylation by SDS gel elec- trophoresis followed by autoradiography. The labelled PKR band is indicated. (C) L22 par- tially relieves the inhibitory effect of EBER-1 on the activation of PKR by dsRNA. PKR was incubated with [32P]ATP[cP] in the pres- ence of the indicated concentrations of poly(I). poly(C), EBER-1 and L22, and the samples were analysed as in (A) and (B). The upper panel shows the autophosphorylated PKR band and the lower panel shows the phos- phorylated L22 band.

Fig. 5. Lack of effect of L22 on protein kinase R (PKR) activation in the absence of EBER-1. (A) Wild-type PKR was incubated with [32P]ATP[cP] for 20 min at 30 (cid:2)C in the presence of the indicated concentrations of poly(I).poly(C) and L22. The samples were analysed by SDS gel electro- phoresis followed by autoradiography. The upper panel shows the autophosphorylated PKR band and the lower panel shows the phosphorylated L22 band. (B) Phosphorylation of L22 by PKR. The protein kinase was incubated with [32P]ATP[cP] in the presence of the indicated concentrations of poly(I).poly(C) and L22. The samples were analysed by SDS gel electrophoresis followed by semidry transfer to a poly(vinylidene difluoride) (PVDF) membrane. The blot was incubated with a polyclonal antibody against L22, followed by an anti-rabbit secondary antibody linked to alkaline phosphatase. Development of the blot with Nitro Blue tetrazolium reagent was performed as previously described [26]. The immunoblot was then subjected to autoradiography and the two images were aligned.

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Fig. 6. Modulation of reporter gene expression by EBER-1, L22 and protein kinase R (PKR). Wild-type murine embryonic fibroblasts and equivalent cells from PKR–/– animals were transfected with an expression vector for b-galactosidase, together with plasmids encoding EBER-1, L22 and PKR in various combinations, as described in the Materials and methods. After 48 h, extracts were prepared and b-galactosidase activity was determined. The results are calculated as relative light units per unit of protein. (A) b-galactosidase activity in wild-type cells; (B) b-galactosidase activity in PKR–/– cells; and (C) effect of EBER-1 on b-galactosidase activity in wild-type and PKR–/– cells in the presence or absence of transfected PKR and L22 (expressed as percentage increase relative to the values in equivalent cells not transfected with EBER-1).

Discussion

protein [7]. More recently, Vuyisich et al. [30] have sugges- ted that stem–loop IV of EBER-1 is the probable binding site for PKR (Fig. 7B). On the other hand, L22 does not contain any dsRBM-like motif within its sequence [35–37] and binds poorly to dsRNA (Fig. 2). The ribosomal protein is clearly an RNA-binding protein, however, as it has been shown to associate with ligands as diverse as 28S rRNA [17], human telomerase RNA [18] and the 3¢ X region of the hepatitis C virus genomic RNA [19], as well as EBER-1.

The small EBV-encoded RNA, EBER-1, can bind to at least three cellular protein ligands in infected cells, viz. the protein kinase PKR, ribosomal protein L22 and the La antigen. There are distinct binding sites for these three proteins on the RNA (Fig. 7B), but at least one of them (stem–loop IV) may constitute a site for either PKR or L22 [17,29,30]. Previous work suggests that there is also a binding site for L22 comprising part of stem–loop III [17,29]. Thus, it is possible that EBER-1 may bind two L22 molecules, or one L22 and one PKR molecule simulta- neously. We have shown, by UV cross-linking studies, that L22 strongly reduces the binding of PKR to the RNA. Conversely, PKR reduces the amount of L22 bound to EBER-1, but only by (cid:1) 50% (Fig. 1B). These results are consistent with a model in which PKR competes with L22 for only one of two separate sites on EBER-1 that bind the ribosomal protein.

An important consequence of the dual ability of EBER-1 to bind both PKR and L22 is that the activation of the protein kinase by dsRNA can potentially be regulated by a protein which cannot directly bind to PKR-activating dsRNA ligands. Several other cellular and viral proteins, including TAR-BP, E3L, NF-90 and PACT/Rax, have previously been shown to regulate PKR, but these proteins are, themselves, dsRNA-binding proteins that either seques- ter dsRNA or interact with PKR via binding to the dsRBMs of the kinase [38–44]. Although L22 can interact directly with PKR, at least when the latter is activated by dsRNA (Fig. 3B), on its own it has no apparent ability either to activate or inhibit the kinase (Fig. 5A). This confirms the indirect nature of the regulatory effect of the ribosomal protein. Neither the molecular basis nor the significance of the direct interaction between L22 and PKR is known, but because L22 has no dsRNA-binding ability, a bridging effect of dsRNA between the two proteins seems unlikely.

It is probable that different structural features of the EBER-1 molecule are recognized by PKR and L22. The protein kinase is a classical dsRNA-binding protein [31], with two double-stranded RNA-binding motifs (dsRBMs) in its N-terminal half [32–34] (Fig. 7A). Our previous data have shown that EBER-1 competes with dsRNA for association with PKR, suggesting that one or more stem– loop structures within the viral RNA are recognized by the

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PKR-interacting partners is another ribosomal protein, L18, which can function as a PKR inhibitor [25]. However, there is no evidence that L18 is phosphorylated by PKR. It is not known whether L22 can be phosphorylated by PKR in intact ribosomes. We and others have previously shown that PKR tightly associates with ribosomes, both in vivo [48] and in vitro [49,50]. However the molecular basis of this association has not been established and previous evidence has suggested that ribosome-bound PKR is inactive [49,51]. Our earlier data showed that PKR dissociates readily from ribosomes upon its activation by dsRNA. It would now be of interest to determine whether L22 becomes phosphoryl- ated during this process and whether such phosphorylation might play a role in the release of PKR from ribosomes. L22 has also been shown to be a substrate for the protein kinase CKII, in vitro [52]. However, the physiological significance of phosphorylation by this enzyme also remains unclear at the present time.

L22 can also serve as a substrate for PKR in vitro (Figs 4C and 5). A number of other PKR substrates have been identified in addition to the classical physiological target (the a subunit of polypeptide chain initiation factor eIF2), and some of these proteins can also act as regulators of the enzyme [43,45–47]. Amongst the list of direct

The ability of L22 to reverse the inhibition by EBER-1 of the dsRNA-mediated activation of PKR, has several implications. Recent reports have shown that high-level expression of EBER-1 can lead to the resistance of cells to physiological stresses and pro-apoptotic stimuli, can confer aspects of the transformed phenotype, and may result in outright tumorigenicity in some cases [9–12]. There is evidence to suggest that these effects are at least partially a consequence of the ability of the RNA to block the activation of PKR (which is strongly pro-apoptotic) [53–58]. When EBV infects cells, a substantial proportion of the EBER-1 that is synthesized binds to L22 [14,29,35], and this binding may therefore attenuate the antiapoptotic and potentially tumorigenic effects of EBER-1 by seques- tering much of the RNA away from PKR (Fig. 7C). The extent to which this occurs in vivo will clearly depend on the relative levels of EBER-1, L22 and PKR, as well as on the affinities of the two proteins for the viral RNA and on the accessibility of L22 and PKR to EBER-1 in different subcellular compartments. L22 is obviously highly abundant as a component of ribosomes, but whereas it can interact with EBER-1 in the cell nucleus

Fig. 7. Model for the proposed mechanism of regulation of protein kinase R (PKR) by double-stranded RNA (dsRNA), EBER-1 and L22. (A) PKR is present in cells in an inactive form in which one or both N-terminal double-stranded RNA-binding motifs (dsRBMs) (shaded rectangles) interact with the kinase domain (filled rectangle) and block the activity of the latter [62]. The kinase is activated when dsRNA binds to both dsRBMs, producing a conformational change in the protein that leads to homodimerization and relief of the auto-inhibi- tion. Autophosphorylation of PKR and phosphorylation of eIF2a and various other substrates can then take place [27,63,64]. Data in this study suggest that L22 can also be a substrate for phosphorylation by PKR. (B) The structure of EBER-1. The proposed binding sites for PKR, L22 and the La antigen are boxed. (C) Small inhibitory RNAs, such as EBER-1, bind to a single dsRBM of PKR in competition with dsRNA, thus preventing the conformational change required for activation of the enzyme [34]. However, in the presence of L22, EBER- 1 binds to the ribosomal protein and is sequestered away from PKR. Under these conditions the inhibitory effect of the small RNA on PKR is reduced and the ability of the protein kinase to be activated by dsRNA is restored.

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4. Clemens, M.J. & Elia, A. (1997) The double-stranded RNA- dependent protein kinase PKR: structure and function. J. Inter- feron Cytokine Res. 17, 503–524.

[14], it does not appear to be able to bind the viral RNA when it is incorporated into mature ribosomes (M. J. Clemens, unpublished observations). PKR occurs in both the cytoplasm and nucleoli [48,59] and its levels rise in the former, but not in the latter, compartment following interferon treatment [48]. EBER-1 has been found in both nuclear and cytoplasmic locations [60,61], and L22 relocates from the nucleolus to nucleoplasm upon infec- tion with EBV [14].

5. Clarke, P.A., Sharp, N.A. & Clemens, M.J. (1990) 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. 193, 635–641.

6. Clarke, P.A., Schwemmle, M., Schickinger, J., Hilse, K. & Clemens, M.J. (1991) Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI. Nucleic Acids Res. 19, 243–248.

7. Sharp, T.V., Schwemmle, M., Jeffrey, I., Laing, K., Mellor, H., Proud, C.G., Hilse, K. & Clemens, M.J. (1993) Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VA1 RNA. Nucleic Acids Res. 21, 4483–4490.

8. Clemens, M.J., Laing, K., Jeffrey, I.W., Schofield, A., Sharp, T.V., Elia, A., Matys, V., James, M.C. & Tilleray, V.J. (1994) Regula- tion of the interferon-inducible eIF-2a protein kinase by small RNAs. Biochimie 76, 770–778.

It is clear from this and previous studies [9] that EBER-1 has one or more additional modes of action which can result in the enhancement of reporter gene expression, even in the complete absence of PKR, and this complicates the interpretation of data from in vivo transfection experiments. The molecular basis for this PKR-independent effect remains to be established. Interestingly, another small viral RNA (the VAI RNA of adenovirus, which inhibits PKR) is also able to stimulate gene expression when transfected into PKR–/– cells [65]. In this case, enhancement of the level of reporter gene mRNA was observed, possibly owing to increased RNA stability [66,67]. The possibility that EBER- 1 acts by a similar mechanism has not, to date, been investigated. Nevertheless, L22 clearly interferes with what- ever process is involved.

9. Laing, K.G., Elia, A., Jeffrey, I., Matys, V., Tilleray, V.J., Soub- erbielle, B. & Clemens, M.J. (2002) In vivo effects of the Epstein- Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation. Virology 297, 253–269.

In view of the regulatory effects of L22 in the presence of EBER-1, we suggest that the nuclear form of the ribosomal protein may be able to buffer cells against the effects of expression of the viral RNA. Thus, it is possible that L22, in addition to its function as a component of the ribosome, is able to protect cells against one mechanism of transformation by EBV. Although it is intrinsically unlikely that cells have evolved a mechanism specifically tailored to protection against this virus alone, the fact that L22 is relocated from the nucleolus to the nucleoplasm following infection [14] does suggest that this ribosomal protein is involved in a physiological response to the virus. EBV has several additional strategies by which it can [1–3]; nevertheless, the subvert cellular growth control possibility that enhancing the expression of L22 in cells may combat the transforming ability of EBER-1 or EBV should now be investigated.

10. Komano, J., Maruo, S., Kurozumi, K., Oda, T. & Takada, K. (1999) Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt’s lymphoma cell line Akata. J. Virol. 73, 9827–9831. 11. Yamamoto, N., Takizawa, T., Iwanaga, Y. & Shimizu, N. (2000) Malignant transformation of B lymphoma cell line BJAB by Ep- stein-Barr virus-encoded small RNAs. FEBS Lett. 484, 153–158. 12. Nanbo, A., Inoue, K., Adachi-Takasawa, K. & Takada, K. (2002) Epstein-Barr virus RNA confers resistance to interferon-a- induced apoptosis in Burkitt’s lymphoma. EMBO J. 21, 954–965. 13. Nucifora, G., Begy, C.R., Erickson, P., Drabkin, H.A. & Rowley, J.D. (1993) The 3;21 translocation in myelodysplasia results in a fusion transcript between the AML1 gene and the gene for EAP, a highly conserved protein associated with the Epstein-Barr virus small RNA EBER 1. Proc. Natl Acad. Sci. USA 90, 7784–7788. 14. Toczyski, D.P., Matera, A.G., Ward, D.C. & Steitz, J.A. (1994) The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes. Proc. Natl Acad. Sci. USA 91, 3463–3467.

Acknowledgements

15. Lerner, M.R., Andrews, M.C., Miller, G. & Steitz, J.A. (1981) Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc. Natl Acad. Sci. USA 78, 805–809.

16. Glickman, J.N., Howe, J.G. & Steitz, J.A. (1988) Structural ana- lyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J. Virol. 62, 902–911.

17. Dobbelstein, M. & Shenk, T. (1995) In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries. J. Virol. 69, 8027–8034.

We thank Dr Jonny Wood (Glasgow) for the plasmid for bacterial expression of L22, Dr Athanos Tzortzopoulos for help with the expression of PKR K296R in the baculovirus system, Dr Simon Morley (University of Sussex) for assistance in the purification of PKR, Dr Joan Steitz (Yale University) for antibody against L22 and Dr Eliane Meurs (Institut Pasteur) for antibody against PKR. This work was supported by grants from the Leukaemia Research Fund (9919) and the Cancer Prevention Research Trust and by a Medical Research Council Industrial Collaborative Studentship to J.V. 18. Le, S., Sternglanz, R. & Greider, C.W. (2000) Identification of two RNA-binding proteins associated with human telomerase RNA. Mol. Biol. Cell 11, 999–1010.

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