Protection from Interferon-Induced Apoptosis by Epstein-Barr Virus Small RNAs Is Not Mediated by Inhibition of PKR

The Epstein-Barr virus (EBV) EBER transcripts are small, highlystructured RNAs able to bind to and inhibit activation of thedouble-stranded RNA-dependent protein kinase PKR in cell-freesystems, and within latently infected B-cell lines they inhibitalpha interferon-induced apoptosis that is believed to be mediatedthrough PKR. Here, we address the consequences of EBER expressionfor PKR activation in vivo in response to alpha interferon.In agreement with published findings, either EBV infection orthe EBERs alone protected Burkitt lymphoma cells from alpha-interferon-inducedapoptosis. However, utilizing multiple phosphorylation state-specificantibodies to monitor PKR activation within cells in responseto interferon, we demonstrate that the EBERs are unable to inhibitphosphorylation of either cytoplasmic or nuclear PKR. Concordantly,a direct substrate of PKR, the ${alpha}$ subunit of eukaryotic initiationfactor 2 (eIF-2 ${alpha}$ ), was equally phosphorylated in EBV-positiveand EBV-negative cells following interferon treatment. Therefore,EBER inhibition of alpha-interferon-induced apoptosis, and potentiallyother PKR-mediated events, is unlikely to be mediated throughdirect inhibition of PKR, as previously thought.

INTRODUCTION

Top

Introduction

The Epstein-Barr virus (EBV) small RNAs EBER-1 and EBER-2 (167and 172 nucleotides, respectively) are highly structured noncodingRNA polymerase III transcripts expressed at very high levels( $~$ 10⁷ copies per cell) within the nuclei of cells latently infectedby EBV (1, 6, 8, 9, 20, 31). Though not essential for EBV immortalizationof B lymphocytes in vitro (39), a hallmark property of thisoncogenic herpesvirus, the EBER RNAs can nonetheless enhanceimmortalization (45) and contribute to the tumorigenic potentialof Burkitt lymphoma (BL) and other B-lymphoma cell lines, aswell as the growth of cells derived from gastric and nasopharyngealcarcinomas (10, 11, 14, 32, 46). The precise contribution(s)of the EBER RNAs to the tumorigenic potential of B cells isunclear (32), while enhanced growth of epithelial tumor cellshas been attributed to up-regulation of insulin-like growthfactor 1, though the mechanism through which this occurs isalso unknown (10, 11, 32).

The EBER RNAs also enhance interleukin-10 expression (13) andinhibit apoptosis induced by alpha interferon (IFN- ${alpha}$ ) (24), functionslikely to promote long-term persistence of the virus. Whereasthe mechanism through which the EBERs promote interleukin-10expression is unknown, inhibition of IFN- ${alpha}$ -induced apoptosishas been reported to be mediated through inhibition of the double-strandedRNA (dsRNA)-dependent protein kinase PKR (24). PKR has alsobeen implicated in various signal transduction pathways andresponse to stress signals (44), and dominant-negative formsof PKR have transforming potential (16, 23). Thus, an inhibitionof PKR by the EBER RNAs has implications for their oncogenicrole in EBV infection, as well.

PKR is a latent, IFN-inducible Ser/Thr kinase activated by dsRNAvia two dsRNA-binding motifs (dsRBM I and II) present in eachPKR monomer. This promotes protein dimerization and conformationalchanges that result in autophosphorylation-mediated activationof PKR and the subsequent phosphorylation by PKR of the ${alpha}$ subunitof eukaryotic initiation factor 2 (eIF-2 ${alpha}$ ) on Ser⁵¹, resultingin cessation of protein synthesis (reviewed in reference 44).Because viral infections can result in the production of dsRNAscapable of activating PKR, PKR is considered a significant componentof the innate antiviral response, though numerous viruses havedeveloped mechanisms to circumvent PKR's antiviral functions(5). Specifically, some viruses encode dsRNAs capable of beingbound by PKR but which have an inhibitory effect on its activation,circumventing the antiviral role of PKR. Notable examples arethe adenovirus VAI small RNA and (presumably) the EBV EBER RNAs(3, 17, 21, 34).

Several lines of experimental evidence support an inhibitoryeffect of the EBER RNAs on PKR through direct interaction withPKR. Within reticulocyte lysates, the EBERs, like the structurallycomparable adenovirus VAI small RNA, bind PKR and inhibit itsrepression of translation in a dose-dependent manner (3, 34).Concordantly, stem-loop IV of EBER-1 is bound in a specificmanner by PKR in vitro through its dsRBM I, but not dsRBM II(42), a feature apparently distinct from activating dsRNAs thatinteract with both sites, presumably relieving an autoinhibitoryinfluence of dsRBM II on the PKR catalytic domain (36). TheEBER RNAs, furthermore, are able to rescue replication of adenovirusesthat lack the VAI RNA gene (2), suggesting that the EBERs sharewith the VAI RNA the ability to inhibit PKR in vivo. Finally,protein kinase assays performed with B-cell lysates have indicatedthat PKR activation is inhibited in the presence of endogenousEBERs in latently infected cells or by EBERs stably expressedin an EBV-negative B-cell background (24, 46).

While the EBER RNAs are able to bind to and inhibit PKR in cell-freesystems and compensate for VAI RNA loss in the context of acytolytic adenovirus infection, the different principal locationsof PKR (cytoplasmic) and the EBERs (nuclear) within cells latentlyinfected by EBV prompted us to ask whether the EBER RNAs aresignificant inhibitors of PKR in vivo. Here, we show, throughthe use of recently available phosphospecific antibodies toPKR, as well as phosphospecific antibody to its substrate eIF-2 ${alpha}$ ,that the EBER RNAs are incapable of inhibiting IFN- ${alpha}$ -inducedexpression and activation of PKR within BL cells. Thus, theability of the EBERs to protect against IFN- ${alpha}$ -induced apoptosis,as shown here and previously (24), must occur independentlyof a direct inhibition of PKR.

MATERIALS AND METHODS

Top

Materials and Methods

Cell culture. Cells were maintained in RPMI 1640 medium containing 2 mM L-glutamine(Mediatech) and 10% defined fetal bovine serum (HyClone). AkataA.15 and A.2 cell lines are EBV-positive and -negative derivatives,respectively, of the parental Akata BL cell line (33). AkataAk^– (EBV-negative) and Ak⁺ (EBV-positive) cells were kindlyprovided by K. Takada (Hokkaido University, Sapporo, Japan).Daudi, KemI, and MutuI are EBV-positive BL cell lines, the lasttwo of which maintain a latency I program of EBV gene expressioncomparable to that of Akata BL cells. A.2.EBER and A.2.Vectorare derivatives of A.2 Akata cells engineered to express EBER-1and EBER-2 or that contain an empty EBER expression vector,respectively, as previously described (32). These two linesalso express the EBV genome maintenance protein EBNA-1, whichcontributes to high-level EBER expression and maintenance ofthe extrachromosomal EBER expression plasmid that contains atruncated EBV origin of latent DNA replication (oriP).

Cell death assays. Cells were seeded at 2.5 x 10⁵ per ml in standard growth medium.At 24 h postseeding, human IFN- ${alpha}$ (R&D Systems) was addedto a final concentration of 100 or 500 U/ml. Cell viabilitywas monitored daily by trypan blue dye exclusion, while aliquotsof cells were simultaneously harvested for analysis of poly(ADP-ribose)polymerase (PARP) cleavage by immunoblotting (Roche).

RNA analysis. Total cellular RNA was isolated from 10⁷ cells (treated for48 h with IFN- ${alpha}$ [100 U/ml]) with RNA-Bee as recommended by themanufacturer (TelTest), followed by extraction with an equalvolume of phenol-chloroform and then chloroform prior to ethanolprecipitation. For RNA (Northern) blot analysis, duplicate samplesof 10 µg of RNA were fractionated by electrophoresis ina 1.2% agarose-2.2 M formaldehyde gel and blotted onto GeneScreenPlus membrane (DuPont). ³²P-labeled DNA probes specific foreither EBER-1 or EBER-2 were generated by random-priming labeling.Following hybridization, the blots were washed and processedby autoradiography.

Immunoblot analysis of PKR and eIF-2 ${alpha}$ phosphorylation. For each sample, 5 x 10⁶ cells were washed once in phosphate-bufferedsaline and frozen at –86°C. The thawed cell pelletswere lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA,1% Triton X-100, protease inhibitor (Roche Complete Mini), andphosphatase inhibitor (Calbiochem; phosphatase inhibitor cocktailset II). Following 10 min on ice, extracts were clarified bymicrocentrifugation at 13,200 rpm for 10 min. The protein concentrationwas determined by Bradford assay (Bio-Rad), and 20 µgof protein was fractionated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis, transferred to an Immobilon P membrane(Millipore), and immunoblotted using an enhanced-chemiluminescencedetection system (Amersham). The blots were initially probedusing phosphorylation site-specific antibodies; subsequentlystripped of antibody in 62.5 mM Tris-HCl (pH 6.8), 100 mM 2-mercaptoethanol,2% sodium dodecyl sulfate (50°C for 30 min); and reprobedwith phosphorylation state-independent antibodies. The blotswere stripped a second time and reprobed with antibody to ß-actinas a control for protein loading. The primary antibodies utilizedwere phosphorylation site-specific antibodies for PKR (Thr⁴⁴⁶and Thr⁴⁵¹; Cell Signaling Technology) and eIF-2 ${alpha}$ (Ser⁵¹; CellSignaling), total PKR (Cell Signaling Technology), total eIF-2 ${alpha}$ (provided by R. Kaufman, University of Michigan), and ß-actin(Oncogene Research Products). Secondary antibodies conjugatedto horseradish peroxidase were obtained from Chemicon. To assessthe phosphorylation status of PKR in response to IFN- ${alpha}$ , cellswere seeded at 2.5 x 10⁵ per ml, and IFN- ${alpha}$ was added as describedabove. For analyses of eIF-2 ${alpha}$ phosphorylation, an initial celldensity of 1.5 x 10⁵ was employed to reduce the background levelof phospho-eIF-2 ${alpha}$ (19). For analysis of cytoplasmic and nuclearextracts, cells were untreated or treated with IFN- ${alpha}$ at 500 U/ml;10⁷ cells were then fractionated using the NE-PER kit (Pierce)according to the manufacturer's instructions. The integrityof the nuclear and cytoplasmic fractions was assessed by immunoblottingwith an antibody to ß-tubulin (provided by N. Morrissette,University of California, Irvine).

RESULTS AND DISCUSSION

Top

Results and Discussion

EBV and EBER inhibition of IFN- ${alpha}$ -induced apoptosis. To determine whether the EBERs inhibit PKR in the context ofa cellular response to IFN- ${alpha}$ , we first assessed their abilityto protect Akata BL cells from IFN- ${alpha}$ -induced apoptosis. For unknownreasons, Akata cells can spontaneously lose the EBV genome inculture, resulting in the loss of tumorigenic potential andresistance to various proapoptotic stimuli that can be restoredby reinfection with EBV (15, 33, 35). Further, EBV protectionof BL cells (including Akata) from IFN- ${alpha}$ -induced apoptosis hasbeen attributed to the inhibition of PKR by the EBERs (24).Thus, the EBV-positive/negative Akata cell system offers anideal model with which to assess EBER functions.

As shown in Fig. 1A (left), survival of EBV-positive (A.15)Akata cells in the presence of IFN- ${alpha}$ (100 U/ml) was significantlyhigher than for EBV-negative (A.2) Akata cells. We next askedwhether the EBERs alone were capable of protection. As illustratedin Fig. 1A (right), expression of EBERs 1 and 2 within EBV-negativeAkata cells, at levels comparable to those of EBV-positive BLcell lines (Fig. 2), likewise promoted cell survival. Interestingly,we consistently observed an intermediate protective effect ofthe EBERs relative to the protection afforded by EBV infection,suggesting that another gene(s) expressed within Akata cellsmay also contribute to survival. Nonetheless, significant protectionfrom IFN- ${alpha}$ -induced cell death could be attributed to the EBERs.Virtually identical results were obtained when an IFN- ${alpha}$ concentrationof 500 U/ml was used in parallel (data not shown). To confirmthat protection afforded by EBV infection and EBERs alone wasdue to inhibition of apoptosis, we monitored cleavage of PARP,a caspase-mediated event characteristic of apoptotic cell death(25). As shown in Fig. 1B, induction of PARP cleavage by IFN- ${alpha}$ was nearly completely inhibited in the EBV-positive cells (A.15)and notably reduced in the presence of the EBERs alone (A.2EBER). This was consistent with the relative levels of protectionfrom IFN- ${alpha}$ -mediated cell death observed in Fig. 1A and an earlierreport demonstrating an antiapoptotic effect of the EBERs withinAkata and other BL lines (24). Comparable results were obtainedwith a higher concentration (500 U/ml) of IFN- ${alpha}$ , and little orno cleavage of PARP was observed in the absence of IFN- ${alpha}$ overthe 72-h course of the experiment (data not shown).

View larger version (35K):
[in this window]
[in a new window]
FIG. 1. EBV infection and EBERs protect BL cells against IFN- ${alpha}$ -induced apoptosis. (A) Protection of Akata BL cells from IFN- ${alpha}$ (100 U/ml)-induced cell death by EBV infection (left) or by stable expression of the EBERs in the absence of EBV infection (right). A.2 and A.15 are EBV-negative and -positive Akata cell lines, respectively. Cell viability was measured by trypan blue dye exclusion. Un, uninduced; Ind, induced with IFN- ${alpha}$ ; vec, vector-only control. The error bars indicate standard errors of the mean. (B) Inhibition of IFN- ${alpha}$ -induced cleavage of PARP by EBV infection (left two blots) and stable EBER expression in EBV-negative Akata cells (right two blots). Cells (from panel A) were exposed to IFN- ${alpha}$ for the times indicated prior to detection of PARP by immunoblotting. Full-length (113-kDa) and the 89- and 24-kDa cleavage products of PARP are indicated. Virtually identical results were obtained with 500 U/ml IFN- ${alpha}$ for both assays (data not shown).

View larger version (76K):
[in this window]
[in a new window]
FIG. 2. EBER-1 and EBER-2 expression in BL cells. Expression levels of the two EBERs were assessed by RNA (Northern) blot analysis of total cell RNA from the EBV-negative Akata cell lines A.2 and Ak^–, EBV-positive Akata lines A.15 and Ak⁺, A.2 Akata cells that contain the empty EBER expression vector (A.2 vec) or that stably express the EBER RNAs (A.2 EBER), and the non-Akata BL cell lines KemI, MutuI, and Daudi. The level of rRNA in each lane that was detected by ethidium bromide staining of the gel prior to blotting is shown as an indication of RNA loading; note that the A.2 EBER lane was underloaded.

Activation of PKR by IFN- ${alpha}$ in the presence EBV. To determine whether inhibition of IFN- ${alpha}$ -induced apoptosis wasactually due to inhibition of PKR by the EBERs, the phosphorylation(and therefore activation) status of PKR within Akata BL cellsupon exposure to IFN- ${alpha}$ was monitored by immunoblotting with antibodiesspecific for PKR phosphorylated on either Thr⁴⁴⁶ or Thr⁴⁵¹.Mutational analyses have indicated that Thr⁴⁴⁶ is critical forPKR function, while Thr⁴⁵¹ is essential (30, 47). We first assessedPKR activation in two independently derived sets of EBV-negativeand -positive Akata lines—one set was derived within ourlaboratory (A.2 and A.15), and the other (designated Ak^–and Ak⁺) was a gift from K. Takada (Hokkaido University, Sapporo,Japan). As demonstrated in Fig. 3A, relative to total PKR, boththe kinetics and levels of PKR phosphorylation at either Thr⁴⁴⁶or Thr⁴⁵¹ were comparable in EBV-negative and -positive cellsin response to IFN- ${alpha}$ . Likewise, expression of the EBERs alonewithin EBV-negative Akata cells could not prevent phosphorylationof PKR (Fig. 3B). As expected, total PKR protein levels werehigher following IFN- ${alpha}$ treatment, as the PKR gene is an IFN-stimulatedgene (22). Identical results were obtained with a third phosphospecificPKR antibody (phospho-PKR Thr^446/451; Cell Signaling) (datanot shown). Comparable activation of PKR was observed in threeother EBV-positive BL lines analyzed (Daudi, KemI, and MutuI),as shown in Fig. 4, each of which expresses EBER-1 and EBER-2at levels comparable to those of EBV-positive Akata cells (Fig.2). KemI and MutuI cells, like their Akata cell counterparts,maintain a strict type I latency program with respect to EBVlatency gene expression. Therefore, we concluded from theseresults that, in vivo, the EBV EBERs are unlikely to preventsignificant activation of PKR in response to IFN- ${alpha}$ . The mechanismthrough which PKR is activated in response to IFN- ${alpha}$ is unknown,though IFN- ${alpha}$ / ${gamma}$ -dependent signaling to the IRF-1 promoter requiresPKR, implying activation, and phosphorylation of PKR occurredwhen the effect of IFN- ${gamma}$ on PKR was assessed (18). dsRNA-independentactivation of PKR by the PKR-interacting protein PACT (proteinactivator of PKR) occurs under various stress conditions (26,44); however, PACT itself is not activated by IFNs (26, 27).

View larger version (89K):
[in this window]
[in a new window]
FIG. 3. IFN- ${alpha}$ -induced phosphorylation of PKR is unaffected by EBV infection or the EBERs. (A) Phosphorylation of PKR at Thr⁴⁴⁶ and Thr⁴⁵¹ (pThr⁴⁴⁶ and pThr⁴⁵¹) in response to IFN- ${alpha}$ (100 U/ml) was monitored within two independently derived lines of EBV-negative (A.2 and Ak^–) and EBV-positive (A.15 and Ak⁺) Akata cells by immunoblotting with phosphospecific PKR antibodies. The blots were subsequently stripped and reprobed with antibodies to total PKR and ß-actin (loading control). (B) Phosphorylation of PKR monitored in EBV-negative Akata BL cells either containing the empty EBER expression vector (A.2 vec) or stably expressing EBER-1 and EBER-2 (A.2 EBER) at levels comparable to EBV-infected Akata cells.

View larger version (42K):
[in this window]
[in a new window]
FIG. 4. IFN- ${alpha}$ induces phosphorylation of PKR in EBV-positive BL cell lines. Phosphorylation of PKR was monitored as described in the legend to Fig. 3.

Our finding is in apparent disagreement with that of Nanbo andcolleagues, who found that phosphorylation of PKR is inhibitedin the presence of the EBER RNAs (24). There are two plausiblereasons for this. First, instead of assessing phosphorylationof endogenous PKR in response to IFN- ${alpha}$ (as in Fig. 3), PKR phosphorylationin the presence and absence of the EBERs was assessed by invitro kinase assay following immunoprecipitation of PKR transientlyoverexpressed in BL cells. Thus, the observed phosphorylationof PKR from EBV-negative BL cells, which was inhibited in thepresence of the EBERs, may not have been representative of themechanism through which PKR is activated in response to IFN- ${alpha}$ .Notably, a number of stress signals, including overexpression,result in activation of PKR. Second, under the conditions ofimmunoprecipitation and the in vitro kinase assay, corruptionof EBER subcellular location and/or disruption of the interactionbetween the EBER RNAs and ribosomal protein L22 (formerly EBER-associatedprotein [EAP]) (40, 41), which has been reported to excludeinteraction of EBER-1 with PKR in vitro (4), may have permittedan EBER interaction with PKR sufficient to inhibit activation.This possibility also raises concern over the physiologic relevanceof an earlier report of EBER inhibition of poly(I) ·poly(C)-induced phosphorylation of PKR in vitro (46).

Phosphorylation of nuclear PKR is unaffected by EBV. Approximately 20% of the total cellular pool of PKR is presentwithin the nucleus (12). While the role of nuclear PKR is unclear,one function may be to phosphorylate protein substrates otherthan eIF-2 ${alpha}$ , such as DRBP76, itself a dsRNA-binding protein (28).Because the EBER RNAs reside almost exclusively within the cellnucleus (9), possibly as a consequence of stable associationvia their 3' termini with the La nuclear protein (6, 20), theEBER RNAs may primarily target nuclear PKR. Because the nuclearpool of PKR is minor relative to that present in the cytoplasm,a negative effect of EBV on PKR phosphorylation within the nucleusmight be masked in experiments that assess phosphorylation oftotal PKR (as in Fig. 3 and 4), the bulk of which would originatefrom the cytoplasm. We therefore fractionated EBV-positive and-negative Akata cells into nuclear and cytoplasmic fractionsfollowing IFN- ${alpha}$ treatment (500 U/ml for 24 h) and assessed thephosphorylation of PKR on Thr⁴⁵¹. Phosphorylation of nuclearPKR, however, mirrored that of cytoplasmic PKR, with no decreasein phosphorylation associated with EBV infection (Fig. 5). Althougha small amount of the cytoplasmic ß-tubulin was detectedin the nuclear fractions, this could not account for a significantdegree of cytoplasmic PKR contamination.

View larger version (78K):
[in this window]
[in a new window]
FIG. 5. Phosphorylation of nuclear PKR is unaffected by EBV infection. Phosphorylation of PKR (pThr⁴⁵¹) within the nuclear and cytoplasmic fractions of EBV-negative (A.2) and EBV-positive (A.15) Akata cells was monitored in the absence or presence of IFN- ${alpha}$ treatment. The blots were reprobed with an antibody to ß-tubulin, which is predominately cytoplasmic. The amount of protein loaded per lane was based on cell equivalents times 10⁵ instead of actual protein, since protein levels within nuclear fractions were substantially lower than in corresponding cytoplasmic fractions. Un, uninduced; Ind, induced with IFN- ${alpha}$ .

IFN- ${alpha}$ -induced phosphorylation of eIF-2 ${alpha}$ . We further examined the likelihood that the EBER RNAs do notinhibit PKR in vivo by assessing the influence of EBV on phosphorylationof eIF-2 ${alpha}$ , a principal substrate of PKR. If the EBER RNAs areindeed capable of inhibiting PKR function, a decrease in phosphorylationof eIF-2 ${alpha}$ in response to IFN- ${alpha}$ should be observed within EBV-positiveAkata cells relative to their EBV-negative counterparts. However,we detected no influence of EBV infection on the IFN- ${alpha}$ -inducedphosphorylation of eIF-2 ${alpha}$ on Ser⁵¹ (Fig. 6). The same resultwas obtained with a different phosphospecific eIF-2 ${alpha}$ antibody(data not shown) and is consistent with our observation thatneither EBV infection nor the EBERs alone are capable of preventingphosphorylation of PKR on Thr⁴⁴⁶ or Thr⁴⁵¹ (Fig. 3).

View larger version (42K):
[in this window]
[in a new window]
FIG. 6. IFN- ${alpha}$ -induced phosphorylation of eIF-2 ${alpha}$ is unaffected by EBV infection. EBV-negative (A.2) and EBV-positive (A.15) Akata cells were untreated or exposed to IFN- ${alpha}$ (100 U/ml) for up to 24 h. Phosphorylation of eIF-2 ${alpha}$ was monitored at the indicated times by immunoblotting to detect phosphorylated (pSer⁵¹) relative to total eIF-2 ${alpha}$ . The blot was reprobed with an antibody to ß-actin to monitor protein loading.

Nanbo and colleagues, however, did observe a decrease in eIF-2 ${alpha}$ Ser⁵¹ phosphorylation within EBER-expressing BL cells when phosphorylationwas assessed by immunoblotting at a single time point of 12h posttreatment with IFN- ${alpha}$ (24). We did observe some variationin eIF-2 ${alpha}$ phosphorylation between EBV-positive and -negativecells at certain time points, e.g., an apparently smaller amountof phosphorylation in EBV-positive cells at 2 h but a greateramount of phosphorylation in the same cells at 24 h (Fig. 6).Overall, however, we found no evidence of significant inhibitionin eIF-2 ${alpha}$ phosphorylation over the course of these experiments,consistent with the lack of an effect of either EBV infectionor the EBER RNAs alone on PKR phosphorylation (Fig. 3). Anotherexplanation for this apparent discrepancy may be the differentcell densities at which cells were seeded in the current andprevious studies (1.5 x 10⁵ and 5 x 10⁴ per ml, respectively).In our hands, even in the presence of 10% serum, BL cells seededat densities lower that 10⁵ per ml typically exhibit an extendedlag period before they are able to begin proliferating. Lowcell density alone, or possibly in combination with IFN- ${alpha}$ asused in the previous study, therefore, may have contributedto stress-induced phosphorylation of eIF-2 ${alpha}$ by another eIF-2 ${alpha}$ kinase that may be negatively regulated by the EBER RNAs. Currently,it is not known if either of the EBER RNAs directly or indirectlytargets another member of this family of stress-responsive kinases.

Conclusions. Previous studies have concluded, based largely on cell-freesystem-based assays, that the EBER RNAs can function by directinhibition of PKR. Here, by directly monitoring phosphorylationof endogenous PKR and its substrate, eIF-2 ${alpha}$ , within BL cellstreated with IFN- ${alpha}$ , which induces PKR-dependent apoptosis (24),we found no evidence that the antiapoptotic effect of the EBERs(or EBV infection in general) in this system is mediated bydirect inhibition of PKR. Our findings, furthermore, are consistentwith three previous observations that suggest PKR is not a directtarget of the EBERs in vivo. First, replication of vesicularstomatitis virus, a virus sensitive to the antiviral functionof PKR (37), is equivalent in B-cell lines immortalized witheither a wild-type EBV or a mutant virus lacking the EBER genesand is equivalently repressed in both lines by IFN- ${alpha}$ treatment(38). Second, the EBERs are normally in a complex with rpL22,an interaction that in vitro can exclude binding by PKR (4),and third, activation of PKR is not prevented upon infectionof EBV/EBER-positive cells with mutant adenovirus that doesnot express the PKR-inhibiting VAI small RNA (43). We conclude,therefore, that EBER inhibition of IFN- ${alpha}$ -induced apoptosis inlatently infected cells must occur downstream of PKR activation.One possible target in this respect may be the nuclear dsRNA-bindingprotein DRBP76, a putative substrate of PKR (28).

Finally, while our findings and those discussed above argueagainst direct inhibition of PKR during latent EBV infection,as yet we have not ruled out EBER inhibition of PKR upon activationof lytic infection. Specifically, disruption of nuclear membraneintegrity and/or latency-associated EBER-protein interactionsupon EBV reactivation may enable the EBERs to bind to, and thusinhibit subsequent activation of, PKR, e.g., by abundant structuredviral transcripts produced during the lytic cycle. AlthoughEBER gene transcription is reduced upon reactivation, EBER levelsremain relatively high through at least 72 h postinduction oflytic infection (7). Potentially, this could ensure inhibitionof PKR prior to adequate expression of the EBV early proteinSM, itself an inhibitor of PKR (29).

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grantsCA056639 and CA073544 (to J.T.S.) and Cancer Center SupportGrants CA21765 (to St. Jude Children's Research Hospital) andCA62203 (to the University of California, Irvine) from the NationalCancer Institute and by the American Lebanese Syrian AssociatedCharities (ALSAC). K.A.L. was a University of California UROPFellow.

We thank K. Takada, R. Kaufman, and N. Morrissette for giftsof reagents and D. Henson for excellent technical assistance.

FOOTNOTES

* Corresponding author. Present address for I. K. Ruf: Department of Molecular Biology and Biochemistry, 3236 McGaugh Hall, University of California, Irvine, CA 92697. Phone: (949) 824-4485. Fax: (949) 824-8551. E-mail: iruf{at}uci.edu. Mailing address for J. T. Sample: Department of Biochemistry, MS340, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105. Phone: (901) 495-3467. Fax: (901) 525-8025. E-mail: jeff.sample{at}stjude.org.

REFERENCES

Top