Proc. Natl. Acad. Sci. USAVol. 89, pp. 10292-10296, November 1992Cell Biology

Localization of Epstein-Barr virus-encoded RNAs EBER-1 andEBER-2 in interphase and mitotic Burkitt lymphoma cells

(double-stranded RNA-dependent protein kinse/in situ hybridization/laser scnning miroscopy/rough endoplasmic retlculum/m is)

MARTIN SCHWEMMLEt, MICHAEL J. CLEMENSU§, KURT HILSEt, KARIN PFEIFER¶, HELMUT TR6STERI,WERNER E. G. MULLER0, AND MICHAEL BACHMANN¶tInstitut fMr Biologie III, Universitit Freiburg, Schinzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany; *Division of Biochemistry, St. George'sHospital Medical School, Cranmer Terrace, London SW17 ORE, United Kingdom; and lAbteilung Mr Angewandte Molekularbiologie, Institut fUrPhysiologische Chemie, Universitfit Mainz, Duesbergweg 6, D-6500 Mainz, Federal Republic of Germany

Communicated by George Klein, July 2, 1992 (received for review March 3, 1992)

ABSTRACT The subcellular distribution of the small Ep-stein-Barr virus-encoded RNAs EBER-1 and EBER-2 has beeninvestigated by using a high-resolution in situ hybridizationtechnique. The distribution prns in Raji cells of fluorescentoligodeoxynucleotides complementary to each RNA were de-tected by confocal laser nning microscopy. Both RNAs werefound in the cytoplasm as well as in the nuclei of interphasecells. In contrast, use of the same technique indicated anexclusively nuclear location for cellular U2 RNA. In thecytoplasm distribution of the EBERs was similar to that of thedouble-stranded RNA-dependent protein kinase, to whichthese RNAs can bind, and was coincident with the roughendoplasmic reticulum. In cells undergoing mitosis the EBERsbecame localized around the chromosomes, whereas the pro-tein kinase remained uniformly distributed in the cytoplasm. Acytoplasmic location for EBER-1 and EBER-2 in interphaseceils is consistent with the evidence for a role for these smallRNAs in translational control.

Epstein-Barr virus (EBV) is a human herpesvirus with arestricted host-cell range, infecting mainly B lymphocytesand epithelial cell types (1). EBV is strongly mitogenic forresting B cells and is also associated with a number ofmalignancies, such as Burkitt lymphoma and nasopharyngealcarcinoma (2). In most infected B cells the virus does notreplicate but enters a latent state in which a very limitednumber of viral genes are expressed. In addition to a smallnumber of protein-coding mRNAs, two noncoding RNAspecies, EBER-1 and EBER-2, are expressed and accumu-late to high levels (3, 4). Although these small RNAs (167 and172 nucleotides, respectively) are ubiquitously present inBurkitt lymphoma and lymphoblastoid cell lines, they do notappear required for either cell immortalization or viral rep-lication per se (5). It is, however, possible that they areneeded in vivo during the establishment of latent infections,when host defense mechanisms, such as the interferon re-sponse, must be overcome (6, 7).We have previously shown that, in vitro, EBER-1 can bind

to the interferon-induced protein kinase that is a double-stranded RNA (dsRNA)-activated inhibitor of protein syn-thesis (also known as p68; DAI) (8). This enzyme is activatedby dsRNA and inhibits protein synthesis as a result of thephosphorylation of the polypeptide chain initiation factoreIF-2 (9). When EBER-1 is present in vitro, it preventsactivation of DAI by dsRNA and thus protects proteinsynthesis from inhibition by dsRNA in the reticulocyte lysatesystem (10, 11). Similar effects are also seen when EBER-2is used in these assays in place of EBER-1 (T. Sharp andM.J.C., unpublished observations).

The subcellular location of EBER-1 and EBER-2 is amatter of some controversy. Howe and Steitz (12) publishedan in situ hybridization analysis of Raji Burkitt lymphomacells that concluded that these small RNAs are predomi-nantly in the cell nucleus. Other studies, however, haveindicated the presence of EBERs in cytoplasmic RNA prep-arations, where they may be associated with polysomal RNA(13, 14). The subcellular location of the EBERs is clearlycritical to their proposed role in translational control becauseinitiation of protein synthesis is exclusively a cytoplasmicevent and DAI is, at least partially, a ribosome-associatedenzyme (15, 16). We have, therefore, reinvestigated thedistribution of EBER-1 and EBER-2 within the cell by usinga technique involving in situ hybridization with fluorescentoligodeoxynucleotides and confocal laser scanning fluores-cence microscopy (17). This method is capable of higherresolution than previous in situ hybridization approaches; italso allows comparison with the subcellular localization ofproteins such as DAI, which can be detected by immunoflu-orescence with equally high resolution. This paper reports onthe location of the EBERs and DAI during interphase and atdifferent stages of mitosis.

MATERIALS AND METHODSMaterials. Species-specific anti-mouse or anti-human IgG

conjugated with fluorescein isothiocyanate (FITC) and anti-rabbit IgG conjugated with rhodamine B isothiocyanate(RITC) were purchased from Medac (Hamburg). The mono-clonal antibody against DAI (18) was from A. G. Hovanes-sian (Institut Pasteur, Paris). Monospecific anti-La antibod-ies from a patient (Ma), used in earlier studies (19), wereprepared by adsorption to recombinant La protein (from W.van Venrooij, University ofNijmegen, The Netherlands). Tostain the endoplasmic reticulum (ER) we used rabbit anti-bodies against the Ca2+ binding protein CaBP-3 (from H.Soling, Gottingen, F.R.G.); these antibodies have recentlybeen shown to react preferentially with the rough ER (20).Oligodeoxynucleotides were made with an Applied Biosys-tems model 394 DNA synthesizer. The sequences synthe-sized were 5'-GGGTGGCTACAGCCACACACGTCTCCT-CCCTAGCAAAACCTCTAGGGCAGC-3' (antisense to nu-cleotides 10-60 of EBER-1) and 5'-GTCGGTAGCAC-CGCACTGAGCGTTGGCGGTGTGTCCGAAACCACTA-GGGCAACGGCT-3' (antisense to nucleotides 6-62 ofEBER-2). Fluorescein labeling was done by using the one-

Abbreviations: DAI, double-stranded RNA-activated inhibitor ofprotein synthesis; dsRNA, double-stranded RNA; EBV, Epstein-Barr virus; EBER, EBV-encoded RNA; ER, endoplasmic reticulum;FITC, fluorescein isothiocyanate; RITC, rhodamine B isothiocya-nate; CaBP-3, Ca2+-binding protein 3.§To whom reprint requests should be addressed.

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step labeling technique (Fluor Prime, Pharmacia) during theautomated synthesis as described by Schubert et al. (21).Subsequent purification of the oligodeoxynucleotides wasachieved by reverse-HPLC.

Cell Culture. The EBV-positive Burkitt lymphoma-derivedRaji cell line (22), the EBV-negative BL41 cell line (23), andother Burkitt lymphoma and lymphoblastoid cell lines weregrown in RPMI 1640 medium/10%o fetal calf serum in ahumidified 5% CO2 atmosphere to a density of 106 cells perml. Then 200-Al aliquots of cell suspension were centrifugedonto coverslips with a Heraeus cell centrifuge (250 x gfor2-5min at 370C). The cells were fixed with ice-cold methanol/0.02% (wt/vol) EGTA for 1 hr.In Situ Hybridization. This hybridization was as described

by Carmo-Fonseca et al. (17) with minor modifications.Briefly, the fixed cells were rinsed in 6x standard salinephosphate/EDTA (1x SSPE is 0.18 NaCl/10mM phosphate,pH 7.4/1 mM EDTA) (SSPE) and incubated with E. colitRNA (1 mg/ml in 6x SSPE/5x Denhardt's solution). Thefluorescent oligodeoxynucleotides were diluted in 6xSSPE/5 x Denhardt's solution to a final concentration of 1-5pmol/p.l. Incubation was performed for 1 hr in a humidifiedchamber at room temperature. The cells were washed ex-tensively by gentle shaking in 6x SSPE at room temperature.Finally the cells were washed with 20 mM Hepes, pH7.9/0.15 M KCI/0.05% Tween 20 and were mounted.

Immunolabeling. Double immunostaining was done byusing a murine monoclonal antibody to DAI or a monospe-cific human autoantibody to the nuclear antigen La, togetherwith a rabbit antibody directed against Ca2+-binding protein3 (CaBP-3). The bound antibodies were detected with spe-cies-specific secondary antibodies that were conjugated witheither FITC or RITC. First, the methanol-fixed cells wererehydrated in phosphate-buffered saline (PBS) for 5 min.Then the cells were incubated with either the anti-DAIantibody for 30 min or the monospecific anti-La antibodiesfor 15 min. After washing with PBS (twice at 5 min each time),the cells were incubated with anti-mouse or anti-human IgGantibodies conjugated with FITC for 15 min. After washingwith PBS as above, cells were stained with the anti-CaBP-3antibody for 15 min. Unbound antibodies were removed bywashing with PBS, and the bound antibodies were detectedwith anti-rabbit IgG antibodies conjugated with RITC. Afterwashing with PBS, the stained cells were mounted andanalyzed. Control experiments established that the second-ary anti-IgG antibodies did not cross-react with the primaryantibodies from the other species (data not shown).

Confocal Laser Sanning Microscopy. After in situ hybrid-ization or immunolabeling, the cells were analyzed by con-focal laser scanning microscopy with an LSM 10 microscope(Zeiss) that had the following characteristics: objectivelenses, Plan-Neofluar 40x /1.3 oil; laser lines, Ar (488 nm) orHe/Ne (514 nm); FITC filter systems, bandpath (BP) 450-490; Towpath (LP) 520; beamsplitter (FT) 510; RITC filtersystems, BP 546; LP 590; FT 580. Optical section images (512pixels) were automatically generated, and evaluation ofstored stacks of optical sections was done with the LSM 10image-processing unit. Images were photographed from thescreen with Agfa-Pan 25 or Kodak 100 color slide film.

RESULTSLocalization of EBERs in Interphase Cells. In situ hybrid-

ization of fluorescent oligodeoxynucleotides complementaryto either EBER-1 or EBER-2 in Raji cells gave a strongpattern of staining, as illustrated in Fig. 1. Both probesrevealed an essentially identical subcellular distribution forthe two RNAs, with staining of both nucleus and cytoplasm.The nuclei showed a typical speckled-type staining pattern.The cytoplasmic compartment also exhibited a nonhomoge-

FIG. 1. In situ hybridization of EBER-1 and EBER-2 in inter-phase Raji cells. In situ hybridization was done as described withfluorescent oligodeoxynucleotides complementary to EBER-1 (a-c)or EBER-2 (d-f). (a and d) FITC fluorescence. (band e) Differentialinterference contrast images of cells in a and d, respectively. (c)Color overlay of a and b. (f) Color overlay of d and e. (Bar in a =10 ,um.)

neous pattern with the perinuclear region being preferentiallystained. In this cell type, this region corresponds to that partofthe cytoplasm where the rough ER and Golgi apparatus arelocated. Similar cytoplasmic staining for the EBERs was alsoseen in other EBV-positive Burkitt lymphoma cell lines (e.g.,Daudi) and in the EBV-immortalized simian and humanlymphoblastoid cell lines M-ABA and M-ABA/CBL, respec-tively (data not shown).Because such strong cytoplasmic staining contradicts ear-

lier reports localizing the EBERs exclusively to the nucleus(12, 24), it was important to rule out the possibility of eithernonspecific staining or leakage of RNA from the nucleus inthese experiments. EBV-negative BL41 cells, lackingEBERs, were examined in parallel with the Raji cells. Fig. 2shows that the fluorescent oligonucleotides complementaryto the EBERs did not stain the BL41 cells, indicating that theability of the oligonucleotides to hybridize was specific forEBER-1 and EBER-2 under the conditions used. Further-more, preincubation of these probes with La antigen-associated RNAs prepared from Raji cells (which include theEBERs) strongly reduced the in situ hybridization signal(data not shown); this result is further evidence of specificity.We also performed in situ hybridization with a fluorescentoligonucleotide complementary to the U2 small nuclearRNA. In this case, staining of only the nuclei was observedin both Raji and BL41 cells (Fig. 2). This result suggests that

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FIG. 2. Absence of EBER hybridization in EBV-negative cellsand pattern of U2 nuclear RNA hybridization in EBV-positive and-negative cells. In situ hybridization was performed on EBV-negativeBL41 Burkitt lymphoma cells (ad) and EBV-positive Raji cells (eand f) as in Fig. 1, by using either a mixture of the fluorescentoligodeoxynucleotides complementary to EBER-1 and EBER-2 (b)or a fluorescent oligodeoxynucleotide complementary to U2 nuclearRNA (d andf). (a, c, and e) Differential interference contrast imagesof the cells in b, d, and f, respectively. (b) BL41 cells: FITCfluorescence pattern with EBER probes. (d) BL41 cells: FITCfluorescence pattern with U2 RNA probe. (f) Raji cells: FITCfluorescence pattern with U2 RNA probe. (Bars = 10 A&m.)nuclear RNA did not leak appreciably during fixation andsubsequent handling of cells.The EBERs belong to a class ofRNAs known to associate

with the La antigen (3), and previous reports have describedthe rapid leakage of the La protein from nuclei duringsubcellular fractionation (25, 26). To test for the possibility ofleakage of La-containing ribonucleoprotein particles from thenucleus as a cause of the strong cytoplasmic EBER signals,Raji cells were stained with monospecific anti-La antibodiespurified by adsorption to recombinant La protein. Theseantibodies stained only the nucleus and did not stain any partofthe cytoplasm (Fig. 3c). Thus, within the limits ofdetectionof the La antigen, our results suggest that no leakage of thisprotein from the nucleus occurred and that the cytoplasmicEBERs may not be associated with this protein.To test whether the EBER-specific oligonucleotides hy-

bridized to a region of the cytoplasm containing the rough ERand/or Golgi apparatus, the anti-La stained cells were dou-ble-stained with antibodies to a well-known ER protein,CaBP-3 (20). It is evident that this antibody stained the ERbut did not stain the nucleus (Fig. 3a). The pattern anddistribution of staining with anti-CaBP-3 is similar to thepattern of in situ hybridization shown by the EBER-specificoligonucleotides (Fig. 1).Because EBER-1 can bind to the interferon-inducible

protein kinase DAI (8) and this enzyme associates withribosomes (15, 16), it was of interest to examine the subcel-lular location of DAI by immunofluorescent staining. Fig. 3d andf shows that costaining of cytoplasmic structures wasobtained with antibodies against DAI and CaBP-3. Thus, DAIis apparently largely localized to the rough ER region whereextensive hybridization to the EBERs is also seen.

FIG. 3. Double immunofluorescent staining of the La antigen orDAI with the rough ER in Raji cells. Interphase cells (a-f) andmitotic cells (g-i) were double-stained for the La antigen (c and i) orDAI (d and g) together with the rough ER protein CaBP-3 (a andf)with FITC- and RITC-conjugated antibodies as described. (b, e, andh) Corresponding differential interference contrast images for cellsshown in a and c, d and f, and g and i, respectively. Note absenceof perichromosomal staining for La or DAI in the mitotic cells.

Localuation of EBERs in Mitotic Cells. During fluorescentstaining of the Raji cell population, a number of cells inmitosis were identified by their appearance under phasecontrast. The pattern of in situ hybridization of the EBERprobes to these cells was quite different (Fig. 4). Intensivestaining of the perichromosomal regions of cells in latemetaphase and anaphase was seen, particularly for EBER-1(Fig. 4 b and c). Perichromosomal staining of EBER-2 wasless intense (Fig. 4 g-i). In contrast, immunostaining exper-iments indicated that neither the La protein nor DAI becamelocalized to the perichromosomal regions in mitotic cells (Fig.3, g and i). Thus during mitosis the EBERs in the perichro-mosomal region appear not to be associated with either theLa protein or DAI.

DISCUSSIONThe evidence for a cytoplasmic location of EBER-1 andEBER-2 in interphase cells, provided by the in situ hybrid-ization studies reported in this paper, is consistent with ourprevious observations that these small RNAs may play a rolein translational regulation. EBER-1 can bind to the cytoplas-mic dsRNA-activated protein kinase DAI (8) and is able torescue protein synthesis from inhibition by dsRNA in vitro(10, 11). Recent data indicate that EBER-2 has similarproperties (M.S. and T. Sharp, unpublished observations).We have also shown that the ability ofDAI to phosphorylatepolypeptide chain initiation factor eIF-2 is inhibited in thepresence of either of these small RNAs (I. Jeffrey, M.S., andM.J.C., unpublished observations). This result suggests thatthe mode of action of the EBERs is highly analogous to thatof the adenovirus-encoded RNA, VA1 (7, 27). A cytoplasmicsite of action of the EBERs is also consistent with earlierreports that these RNAs are associated with polysomal RNA(13, 14) and that DAI is itself a ribosome-associated protein

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FIG. 4. In situ hybridization of EBER-1 and EBER-2 in mitoticRaji cells. In situ hybridization was done as described in Fig. 1 byusing fluorescent oligodeoxynucleotides complementary to EBER-1(a-f) or EBER-2 (g-4). Hybridization patterns are shown for cells inmetaphase (a, d, g, andj), late metaphase/anaphase (b, e, h, and k)and late anaphase/telophase (c, f, i, and 1). (a-c, g-i) FITC fluores-cence. (d-f) Differential interference contrast images of the cells ina-c, respectively. (j-i) Differential interference contrast images ofthe cells in g-i, respectively. Note the particularly strong staining ofperichromosomal regions with the EBER-1 probe in cells in latemetaphase/anaphase. (Bars = 10 Am.)

(15, 16). The latter finding is supported by the results of ourdouble-immunolabeling experiments that use anti-DAI andanti-CaBP-3 antibodies.

Previous work by Howe and Steitz (12), using in situhybridization and autoradiography, concluded that theEBERs were predominantly localized in the cell nucleus inRaji cells. However, although this study produced clearevidence for the presence of the EBERs in the nucleus, thedata do not indicate an exclusively nuclear localization forthese RNAs. We have used the same cell type in the presentstudies but have used a method of in situ hybridization thatis both more sensitive and of higher resolution than was

previously possible. The use of fluorescent oligodeoxynucle-otide probes combined with confocal laser scanning micros-copy reveals a high degree of detail in the analysis of thedistribution of these abundant RNAs. This is particularly thecase when the fixed cells are well spread because the ratio ofnuclear-to-cytoplasmic volume is high in lymphoid cells. Ourdata suggest that very little leakage of nuclear materialoccurred during cell fixation, staining, or mounting becauseboth U2 RNA and La antigen remained entirely in the nucleiof interphase cells after these procedures. Although we havefound a strong cytoplasmic signal for the EBERs, we alwaysdetect some in the nucleus also, suggesting that some fractionof both EBER-1 and EBER-2 does, indeed, have a nuclearlocation. However, no nuclear function for the EBERs hasyet been identified. The utility of EBER-1 as an abundanttarget for the diagnostic identification of EBV-infected cellsin human tumors by in situ hybridization (24) is, of course,not dependent on a particular subcellular distribution.

Distribution of the EBERs in the cytoplasm of Raji cellsduring interphase is very similar to that of DAI. The patternof cytoplasmic staining for the EBERs coincides with that ofthe rough ER, and in double-antibody-labeling experimentsDAI clearly colocalizes with the rough ER. A substantialfraction of the DAI is probably associated with ribosomes inthis region. Previous low-resolution studies of DAI distribu-tion in interferon-treated HeLa cells have shown a uniformcytoplasmic staining pattern, changing to a picture of peri-nuclear concentration after heat shock or infection withencephalomyocarditis virus (16, 28).

Despite the coincident distribution of DAI and the EBERSin interphase cells our data suggest that the EBERs are notdependent on the presence ofDAI for their localization. Thecells used here were not pretreated with interferon andcontained only basal levels of DAI; moreover, in many cellsno DAI could be detected by immunofluorescence (data notshown). In contrast most cells were EBER-positive andshowed a common pattern of distribution of hybridizingmaterial. Thus, it is probable that factors other than thepresence of DAI determine localization of the EBERs inthese cells.At least two other proteins have been shown to bind to

EBER-1; these are the La antigen (3) and the EBER-associated protein (29). Both are believed to be predomi-nantly nuclear proteins, although in some cases La antigencan also be found in the cytoplasm (30). We cannot excludethe possibility that the subcellular distribution of the EBERsmay be a result of their association with low levels ofcytoplasmic La. However, the La antigen detected in thepresent study was exclusively nuclear.

Significance of the localization of the EBERs to perichro-mosomal regions during mitosis is not yet clear. It is not fromthe presence of either DAI or the nuclear form of the Laantigen in this region during mitosis because these proteinsdid not show similar perichromosomal staining. There may bea role for the EBER-associated protein in promoting associ-ation of the EBERs with mitotic chromosomes, but thus farno immunofluorescence studies have been conducted onEBER-associated protein distribution. Alternatively, theEBERs may have yet other ligands (including RNA mole-cules as well as proteins) that result in the changes in theirbehavior observed at different times during the cell cycle.

In spite of the time that has elapsed since the EBERs werefirst discovered and characterized (3, 4, 13), knowledge oftheir precise role in the physiology of EBV has remainedelusive. The in vitro evidence for their ability to regulate DAIactivity and to protect protein synthesis from the inhibitoryeffects of dsRNA (8, 10), together with the genetic evidencethat multiple copies of the EBERs can substitute for VA1RNA during the growth of adenovirus (31), strongly suggestthat EBERs are important in the control of translation in theinfected cell. Nevertheless, the recent demonstration that amutant EBV that lacks the EBER genes can infect andimmortalize B lymphocytes in culture and can replicate in anapparently normal fashion (5) suggests that these normallyabundant small RNAs are not essential gene products. It ismore likely that they play a role in allowing the virus to infectcells and establish itself in a latent form in vivo in the face ofhost defense mechanisms, such as the production of inter-feron and activation of immune responses. Thus, the EBERsmay form part of a panoply of viral gene products, includingthe EBNA-2 protein (32) and the product of the BCRFJ gene(33), that may serve to damp down host responses to infec-tion. In this respect, it is of interest that there is an almostcomplete absence of EBER expression in EBV-infectedkeratinocytes in oral hairy leukoplakia lesions in AIDSpatients (34). However, the EBERs are still strongly ex-

pressed in EBV-associated lymphomas that develop in theseseverely immunosuppressed individuals. Identification of the

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subcellular distribution of the EBERs and of the cellularconstituents to which they bind at different phases in the lifecycle of the cell should contribute to an improved under-standing of their function during infection.

We are grateful to Dr. A. G. Hovanessian and Prof. H. Soling forgenerous gifts of antibodies to DAI and CaBP-3, respectively, and toDr. G. Igloi for synthesis of the fluorescent oligodeoxynucleotides.This investigation was supported by grants from the DeutscheForschungsgemeinschaft to M.B. (Ba 1145/1-1) and K.H. (Hi 188/5-1) and from the Cancer Research Campaign, the Wellcome Trust,and the Leukaemia Research Fund to M.J.C. Collaboration wasfacilitated by the British-German Academic Research Collaborationprogram. M.B. is a recipient of a Hermann and Lilly Schillingprofessorship granted by the Stifterverband fur die Deutsche Wis-senschaft.

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