Differentiation-Associated Expression of the Epstein-Barr Virus

Transcription

1 JOURNAL OF VIROLOGY, June 1991, p X/91/ $02.00/0 Copyright 1991, Amerian Soiety for Mirobiology Vol. 65, No. 6 Differentiation-Assoiated Expression of the Epstein-Barr Virus BZLF1 Transativator Protein in Oral Hairy Leukoplakia L. S. YOUNG,'* R. LAU,2 M. ROWE,' G. NIEDOBITEK,l G. PACKHAM,2 F. SHANAHAN,2 D. T. ROWE,2 D. GREENSPAN,3 J. S. GREENSPAN,3 A. B. RICKINSON,l AND P. J. FARRELL2 Department of Caner Studies, Caner Researh Campaign Laboratories, The Medial Shool, University of Birmingham, P.O. Box 363, Birmingham B15 2TT,1 and Ludwig Institute for Caner Researh, St. Mary's Hospital Medial Shool, London W2 IPG,2 United Kingdom, and Oral AIDS Center and Department of Stomatology, Shool of Dentistry, University of California, San Franiso, California Reeived 6 Deember 1990/Aepted 7 February 1991 The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein whih has been shown to disrupt virus lateny in EBV-infeted B ells. We have generated a monolonal antibody, BZ1, to BZLF1 whih reats in immunohistology, immunoblotting, and immunopreipitation and whih reognizes both the ative, dimeri form and the inative, monomeri form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-assoiated lesion haraterized by high-level EBV repliation, were examined by immunohistohemistry using the BZ1 monolonal antibody. A differentiation-assoiated pattern of BZLF1 expression was observed, BZ1 reating with nulei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A omparison of in situ hybridization to EBV DNA and viral apsid antigen staining with BZ1 reativity suggested that BZLF1 expression preedes rampant virus repliation. The inability to detet EBV in the lower epithelial layers of oral hairy leukoplakia raises questions onerning the nature of EBV lateny and persistene in stratified squamous epithelium. Oral hairy leukoplakia (OHL) is a reently desribed epithelial lesion ourring in assoiation with AIDS (7). OHL presents as raised white areas usually on the lateral border of the tongue. The histologial appearane is haraterized by an intraellular edema of spinous ells, alterations of the nulei suh as basophili nulear inlusions, and superfiial parakeratosis (11). Although this histology resembles that observed in papillomavirus-assoiated infetions, there has been no reliable demonstration of suh an infetion in OHL. Initial examination of OHL under the eletron mirosope showed the presene of a herpes-type virus partile whih was later onfirmed as Epstein-Barr virus (EBV) both by DNA hybridization studies and by immunohistohemial analysis with antibodies direted against EBV antigens of the virus lyti yle, e.g., viral apsid antigen (VCA) and early antigen (EA-D and EA-R) (8). On treatment with aylovir, an inhibitor of the herpesvirus DNA polymerase, OHL was found to regress (4), impliating EBV as an etiologial agent in the development of this nonmalignant epithelial proliferation. In situ hybridization to EBV DNA has revealed repliating EBV distributed foally in the more differentiated layers of OHL (2, 11). This is in agreement with the pattern of immunostaining observed by using either polylonal human sera to late viral antigens or monolonal antibodies (MAbs) to VCA, early antigens (EA-D and EA-R), and membrane antigen. The assoiation of EBV repliation with the terminally differentiating tongue epithelial ells of OHL is onsistent with previous studies of epithelial EBV infetion (23) and suggests that OHL may represent an exaggerated form of the natural epithelial ell infetion with EBV. In this study we have generated a MAb to the key immediate-early protein BZLF1, whih has been shown to disrupt virus lateny in B ells (12). This MAb has been fully * Corresponding author. haraterized and used to analyze the pattern of BZLF1 expression in OHL. We demonstrate, for the first time, the differentiation-assoiated expression of BZLF1 in a biologially relevant ellular environment, i.e., human epithelial ells. MATERIALS AND METHODS Colletion of OHL biopsies. The subjets hosen for tongue biopsies were individuals with symptomati human immunodefiieny virus disease (CDC IV) in whom OHL had persisted for at least 3 months. Subjets were evaluated for the presene of OHL by two physiians before being submitted to tongue biopsy. Under loal anestheti, an elliptial inisional biopsy (2 mm wide by 3 mm long) was taken from the side of the tongue with OHL. The tissue sample was snap-frozen in liquid nitrogen and subsequently stored at -70 C. Four of the biopsies analyzed were from British patients, and two biopsies were from Amerian patients. Generation of MAbs. A fusion protein ontaining part of Staphyloous aureus protein A at its N terminus joined to the full-length BZLF1 sequene (prex-bzlf1) was used for immunization (3). Hybridomas were generated as previously desribed (19), and ulture supernatants were sreened for relevant antibodies by using an enzyme-linked immunosorbent assay (ELISA) with a different BZLF1 fusion protein (pex-bzlf1), ontaining part of the CII protein of phage X fused to BZLF1, as the positive target antigen and with phage lambda ontrol protein (NpeX) as an irrelevant target antigen (17). The hybridoma ultures that produed apparently BZLF1 speifi antibodies in the ELI- SAs were subsequently tested for reativity on virus-produtive and nonprodutive ell lines in an indiret immunofluoresene test. At this stage, hybridomas of interest were reloned by limiting dilution and haraterized further by immunoblotting experiments. In this way an immunoglobu- 2868

2 VOL. 65, 1991 BZLF1 EXPRESSION IN ORAL HAIRY LEUKOPLAKIA 2869 lin (Ig) G1 MAb, designated BZ1, speifi for BZLF1 was isolated. Immunofluoresene and immunohistohemial staining of ells and tissues. For immunofluoresene, a three-step double-onjugate indiret method was used. Methanol-fixed ell spreads or ryostat setions were rehydrated with phosphate-buffered saline (PBS) ontaining 20% normal rabbit serum and 10% EBV-seronegative human serum and inubated with various dilutions of hybridoma ulture supernatant for 2 h at 37 C. Bound MAb was deteted by inubation with fluoresein isothioyanate (FITC)-onjugated goat antimouse IgG (Sigma) for 1.5 h at 37 C, washing in PBS, and then inubation for 1 h at 37 C with FITC-onjugated rabbit anti-goat IgG (Sigma). The slides were finally washed in PBS, mounted in DABCO (1,4-diazabiylo[2,2,2]otane)- based antifading mountant, and visualized by fluoresene mirosopy. For double staining, an indiret immunofluoresene tehnique was used. This proedure relied on the different sublass speifiities of the primary MAbs and used a mixture of FITC-onjugated goat anti-mouse IgGl and rhodamine-onjugated goat anti-mouse IgG2a (Southern Biotehnology Assoiates) as the seond step. Immunohistology was performed by the alkaline phosphatase-antialkaline phosphatase (APAAP) tehnique as previously desribed (1). In situ hybridization. In situ hybridization was performed as desribed previously (13), using the "S-labeled plasmid pba-w (3.1-kb BamHI-W internal repetitive fragment of EBV strain M-ABA, subloned in pbr322) kindly provided by G. W. Bomkamm, Munih, Germany (16). In brief, frozen setions were fixed in 4% paraformaldehyde for 20 min at room temperature (RT), inubated in 0.02 N HCl for 10 min at RT, and then subjeted to a proteolyti digestion in mg of pronase (Boehringer, Mannheim, Germany) per ml for 10 min at RT. After extensive washing in PBS, 25 RI of hybridization mixture ontaining 1 ng of the labeled probe was applied to a setion. Cell and probe DNA were denatured together for 2 min at 95 C, and hybridization was arried out overnight at 37 C. Slides were then washed in 50% formamide-o.lx SSC (SSC is 0.15 M NaCl plus sodium itrate)-10 mm dithiothreitol for 4 h, rinsed in 2x SSC, dehydrated through graded ethanols, dipped in Ilford G5 emulsion, exposed at 4 C, developed, and ounterstained. Double-labeling immunohistology and in situ hybridization. For the simultaneous detetion of EBV DNA and the BZLF1 protein in the same tissue setion, frozen setions were fixed sequentially in aetone and hloroform for 30 min eah at RT. Immunohistology was arried out as desribed above. After APAAP staining, slides were subjeted to in situ hybridization without any additional treatment. After hybridization overnight and washing as desribed above, slides were air dried briefly, dipped into Ilford G5 emulsion, and exposed for 3 days. The slides were then developed, ounterstained with hematoxylin, and embedded with glyerol gelatin (Dakopatts, Glostrup, Denmark). SDS-PAGE and immunoblotting. Protein extrats of appropriate ell lines were analyzed by sodium dodeyl sulfate (SDS)-polyarylamide gel eletrophoresis (PAGE) on 10% polyaylamide Laemmli gels, blotted onto nitroellulose filters, probed with speifi antibodies, and deteted with 125I-protein A, all as desribed previously (20). Epitope mapping of the BZLFl-speifi MAb. Various deletion and point mutants of BZLF1 were transribed and translated in vitro. The radioative protein (labeled with [35S]ysteine) was then immunopreipitated with the BZLF1-speifi MAb BZ1, and 30 RI of in vitro translation reation mixture was mixed with 25 RI of BZ1 ulture supernatant and 220 p1 of ELB (250 mm NaCI, 50 mm N-2- hydroxyethylpiperazine-n'-2-ethanesulfoni aid [HEPES; ph 7.0], 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 0.1% Nonidet P-40) at 4 C for 30 min. Then 12.5 pi of a 1:1 slurry of protein A-Sepharose beads that had been preoated with a rabbit anti-mouse Ig antibody (Z259; Dakopatts) was added, the solution was mixed for 30 min at 4 C, and the beads were olleted by entrifugation and washed three times in ELB. The bound proteins were eluted in SDS-gel sample buffer and analyzed on a 17.5% polyarylamide gel. The protein A beads were preoated by mixing 200 RI of a 1:1 slurry of beads in ELB with 12 p1 Z259. After 30 min at 4 C, the beads were washed three times in ELB. Supershift experiment to demonstrate that the BZLFlspeifi MAb reognizes the ative dimer of BZLF1. Fusion proteins of SmaI-C BZLF1 loned at the BamHI-EoRI sites of pgex and a two-repeat lone of the EBNA-LP (as a ontrol) were indued and purified by hromatography on glutathione agarose. The onentration of eah fusion protein was determined by estimating the intensity of the Coomassie-stained gel band on a polyarylamide gel in omparison with known amounts of bovine serum albumin. Standard gel retardation reations were performed as desribed previously (3). Eah reation mixture ontained 6 ng (=0.5 pmol) of 32P-labeled M 19-mer (double-stranded GAAGCACTGACTCATGAAG), 100,ug of poly(di-dc) per ml, and, where indiated, 0.1 prg (=1 pmol) of fusion protein, 1 pi of BZ1 asites fluid, or 1 RI of PE2 (a MAb to EBNA2; 22) asites fluid. The fusion protein was allowed to bind to the radioative DNA for 20 min at RT prior to addition of the appropriate antibody and inubation for a further 20 min. Controls were inubated for the same length of time. Complexes were resolved by gel eletrophoresis on a 4% polyarylamide gel in 0.5 x Tris-borate-EDTA and were deteted by autoradiography. RESULTS Charaterization of the BZLFl-speifi MAb (BZ1). An IgGl hybridoma (designated BZ1) was isolated whih stably produed antibody speifially reative against produtively infeted EBV ell lines, as assessed in immunofluoresene staining. This MAb reated strongly in immunoblotting with the B95.8 and P3HR1 produer ell lines but not with ell lines in whih EBV is latent (i.e., X50-7 and Raji) (Fig. 1). Furthermore, no reativity was observed in EBV-negative ell lines. The major protein reognized by BZ1 in B95.8 migrated with an Mr of about 39,000, whereas that observed in the P3HR1 ell line migrated with an Mr of approximately 42,000. This result is in agreement with previous studies using either polylonal human sera or a rabbit monospeifi antiserum (17, 18). The speifiity of BZ1 was further demonstrated by the reativity of the MAb with 293 ells transfeted with BZLF1 DNA and not with ontrol transfetants (Fig. 1). To map the epitope reognized by BZ1, we used a range of BZLF1 deletion mutants (Fig. 2a) loned into the transription vetor SP64. When linearized and transribed with SP6 polymerase, RNA was produed whih yielded BZLF1 proteins of the expeted size when translated in the retiuloyte lysate (Fig. 2b). MAb BZ1 was then used in immunopreipitations of these proteins in vitro translated in the presene of [35S]ysteine. Whereas the N-Pstl deletion

3 2870 YOUNG ET AL. 116_ N Z2t a) a o "' C C + I C 0 CL Ci Co 0) Lo CO) 1 -j 0) 0 _ b, FIG. 1. Immunoblot showing the reativity and speifiity of MAb BZ1 with BZLF1 protein in produtively infeted ell lines (B95.8 and P3HR1) and BZLF1 transfetants (293 + BZ). No reativity was observed with EBV-positive latently infeted EBVpositive lines (X50-7 and Raji), an EBV-negative line (BL40), or ontrol transfetants (293 + puc). a BZL F 1 N H-1l IHndll delta N BEIr-.l Pstid C B3s.nT PJst delta 46 * 30 * DNA eeneng domain Dimer satson domat' Point mulants a <- z LL U, 02 0D 0D a. z ) 11i K RL M _r b d mutant of BZLF1 failed to be reognized by BZ1, all of the other deletion mutants were readily deteted (Fig. 2 and d). This finding suggested that the epitope reognized by BZ1 resided in the dimerization domain of BZLF1. Point mutations within the dimerization region did not affet BZ1 reognition. The M point mutant was immunopreipitated effiiently in two suh experiments (one illustrated Fig. 2), even though its preipitation seems to be redued in Fig. 2d. The KL mutant is unable to dimerize under these onditions (9), so the reativity of BZ1 with the dimerization domain of BZLF1 and the effiient reognition of these mutants raised the question of whether this MAb would reognize the dimerized, funtional form of the protein (10) or might be reating only with the inative, monomeri form. To examine this point, gel retardation assays were performed using the AP-1 site upstream of BMLF1 as the target and glutathione S-transferase (GST) fusion proteins of either BZLF1 (GST-SmaI-C) or, as a ontrol, EBNA-LP (GST- LP). These assays demonstrated speifi binding of GST- SmaI-C to the AP-1 site, as shown previously (Fig. 3, traks 7 to 9). Addition of MAb BZ1 to the gel retardation assay shifted the BZLF1-DNA omplex (trak 8), indiating that the MAb was able to reognize the funtional ative dimerized form of the BZLF1 protein. No effet was observed with use of a MAb against the EBNA2 protein (trak 9), and no shift was seen with the GST-LP fusion protein (traks 4 to 6). Reativity of BZ1 with OHL biopsies. The OHL biopsy material was initially sreened for EBV infetion by in situ hybridization to viral DNA and by immunostaining for VCA. Six biopsies were seleted for subsequent analysis. Serial setions were then analyzed by in situ hybridization and stained with MAb BZ1 and the anti-vca MAb V3 (Fig. 4). In situ hybridization with an EBV-speifi probe resulted in m E _ 0D u - - o C -J r E E N, U) 0) j oi z m z m Y Y i _ 0,J r mi z mo z a Ye Ye.j Nl "'W.. _ r J. VIROL S4_ FIG. 2. Epitope mapping of MAb BZ1. (a) Diagrammati representation of the various deletion and point mutations used; (b) in vitro translations without immunopreipitation; ( and d) two experiments with immunopreipitation.

4 VOL. 65, 1991 BZLF1 EXPRESSION IN ORAL HAIRY LEUKOPLAKIA ww FIG. 3. Supershift experiment demonstrating that MAb BZ1 reognizes the ative dimer of BZLF1. The gel retardation reations were performed as previously desribed (2), using the AP-1 site upstream of BMLF1 as the target. Traks: 1 to 3, reation mixtures ontaining no fusion protein; 4 to 6, reation mixtures ontaining EBNA-LP (2R) fusion protein; 7 to 9, reation mixtures ontaining BZLF1 SmaI-C fusion protein; 2, 5, and 8, inubated with the MAb BZ1; 3, 6, and 9, inubated with the EBNA2-speifi MAb, PE2. an aumulation of grains over nulei of the upper layers of the epithelium (Fig. 4a). This signal was usually very intense, leading to saturation of the emulsion even after short exposure times (2 to 3 days). Several lusters of positive ells separated by apparently noninfeted ells were identified in eah speimen, resulting in a foal distribution of EBV repliation. Oasional ells adjaent to foi of viral repliation displayed a weaker signal. No signal was observed over the basal ell layers, and no signal was obtained with unrelated probes (not shown). The reativity of both MAb BZ1 and V3 was onsistent with this pattern of viral repliation. Immunohistology with BZ1 MAb produed an intense staining of nulei in the upper layers of OHL epithelium (Fig. 4b). Interestingly, the stratum orneum demonstrated a markedly redued level of EBV repliation as assessed by all three riteria, i.e., in situ hybridization, BZ1 staining, and V3 staining. To assess the relative assoiation of BZLF1 and VCA expression, double immunofluoresene staining was performed. In these experiments, oloalization of BZLF1 and VCA proteins was observed in ertain ell layers (Fig. 5). However, a population of ells that were BZLF1 positive but VCA negative were also observed. These ells were present in the upper spinous layer and generally appeared as a single ell layer immediately preeding VCA expression. This observation was onfirmed in experiments in whih setions of OHL were stained with MAb BZ1 and then hybridized for EBV DNA (Fig. 4 and d). This approah permits loalization of antigen and viral DNA on a single setion. A layer of BZLF1-positive ells displaying a weak EBV-speifi signal was observed below ell layers in whih the strong signal generated by high levels of virus DNA obsured BZ1 staining. No BZ1 staining was observed lower in the epithelium. DISCUSSION We have generated a speifi MAb, BZ1, against the BZLF1 protein whih reats in immunohistology, immunoblotting, and immunopreipitation assays. Apart from the usefulness of BZ1 in the analysis of BZLF1 expression in ells and tissues, the ability of this reagent to reognize the ative, dimeri form of the BZLF1 protein suggests that it will also be important in funtional studies. Epitope mapping experiments loalized the region of BZLF1 reognized by BZ1 to the dimerization domain. Supershift assays learly demonstrated that BZ1 reognized the funtional dimerized form of BZLF1. However, point mutations whih prevent the homodimerization of BZLF1 did not prevent reognition of the protein by BZ1, suggesting that the MAb reats with both the ative, dimeri form and the inative, monomeri form of the protein. Thus, BZ1 must reognize either (i) an epitope within the dimerization domain whih does not interfere with the funtional dimerization of BZLF1 or (ii) an epitope outside of the ore dimerization domain. These possibilities are urrently being investigated by using other mutants of BZLF1. The reativity of BZ1 with nulei of the upper spinous layer of OHL onfirms previous studies demonstrating a differentiation-assoiated pattern of EBV repliation in this lesion (11, 23). The influene of squamous differentiation on BZLF1 expression may be ontrolled at the BZLF1 promoter level by differentiation-assoiated regulatory fators. BZLF1 appears to be expressed prior to rampant virus repliation, as assessed either by double staining for BZLF1 and VCA or by in situ hybridization and BZLF1 staining. Interestingly, the superfiial parakeratoti layer appears to have a markedly redued level of both BZLF1 expression and viral DNA. This probably results from the redued availability of viral antigens and DNA in the nulei of this hyperparakeratoti layer, sine other antigens, suh as ytokeratins, are also less effiiently demonstrated at this site (15). Alternatively, EBV repliation may be down-regulated at the terminal stages of epithelial differentiation. This may have impliations for the amount of mature virus shed from OHL. The repliation of EBV in OHL, and by impliation probably in normal epithelium, is not typial of herpesviruses. Repliation of herpesviruses is usually aompanied by extensive damage to the affeted tissue, e.g., ulers in herpes simplex virus-indued stomatitis or intestinal ulers aused by ytomegalovirus. In ontrast, EBV repliation in the epithelial ells of OHL does not lead to uleration. Although some morphologial alterations indiative of ytopathi viral effets, e.g., koiloytosislike hanges and nulear inlusion bodies, are observed (11), virus repliation obviously does not lead to ell lysis. Moreover, the epithelial ells in whih EBV is repliating in OHL retain their ability to further differentiate, sine dysplasia is usually not observed in this lesion. Only the presene of parakeratosis indiates a minor impediment in the normal development of a fully mature epithelium. It seems, therefore, that EBV has evolved to exploit the normal proess of epithelial ell differentiation during its repliative yle, thereby avoiding signifiant ytopathi effets or assoiated inflammatory responses. It has been suggested that the behavior of EBV in OHL resembles that of human papillomavirus infetions in whih viral lateny is maintained in the basal and suprabasal epithelial layers (5, 23). However, in situ hybridization to EBV DNA in OHL failed to detet viral DNA in the lower layers of the lesion. This finding is in agreement with previous studies using biotinylated probes (2, 8). The highly sensitive in situ hybridization tehnique that we have employed in this study uses "S-labeled probes and is able to detet the single opy of the EBV genome present in the AW Ramos ell line (14). It may be, therefore, that EBV is not latent in the basal layers of OHL but that the foal areas of

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6 VOL. 65, 1991 BZLF1 EXPRESSION IN ORAL HAIRY LEUKOPLAKIA 2873 FIG. 4. (a) Demonstration of EBV DNA in an OHL by in situ hybridization using a 35S-labeled DNA probe (3 days of exposure; hematoxylin and eosin ounterstaining; x200). (b) Adjaent setion of the same sample stained with MAb BZ1 (APAAP tehnique; hematoxylin ounterstaining; x200). ( and d) Double-labeling immunohistology and in situ hybridization of an OHL biopsy showing a layer of BZLF1-positive ells underneath a fous of viral repliation (; 3 days of exposure; hematoxylin ounterstaining; x 100), whih display a weak EBV-speifi signal (d; x400). FIG. 5. Double immunofluoresene staining of an OHL setion for VCA (a; x200) and BZLF1 (b; x200), revealing a layer of BZLF1-positive but VCA-negative ells (arrows).

7 2874 YOUNG ET AL. EBV repliation represent regions of self-sustained reinfetion. If this is the ase, our ideas onerning the persistene of EBV in the epithelial ompartment may have to be revised. Indeed, other reent reports have questioned the role of epithelium as the permanent reservoir of EBV infetion (6, 13, 21). Further studies examining the pattern of EBV latent gene expression in OHL may help resolve this issue. ACKNOWLEDGMENTS This work was supported by the Caner Researh Campaign, London, United Kingdom, and by NIH grant P01 DE Support for G.N. was provided by the Deutshe Forshungsgemeinshaft. We are indebted to J. R. Harris and A. J. Pinhing for aess to their patients, to M. Birhall for olleting biopsies, and to H. Stein (Berlin, Germany) for kindly providing the APAAP omplex. REFERENCES 1. Cordell, J. L., B. Falini, W. N. Erber, A. K. Ghosh, Z. Abdulaziz, S. MaDonald, K. A. F. Pulford, H. Stein, and D. Y. 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