Zurich Open Repository and Archive. {beta}1 Integrin Expression Increases Susceptibility of Memory B-Cells to EBV Infection

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1 University of Zurich Zurich Open Repository and Archive Winterthurerstr. 0 CH-0 Zurich Year: 00 {beta} Integrin Expression Increases Susceptibility of Memory B-Cells to EBV Infection Dorner, M; Zucol, F; Alessi, D; Haerle, S K; Bossart, W; Weber, M; Byland, R; Bernasconi, M; Berger, C; Tugizov, S; Speck, R F; Nadal, D Dorner, M; Zucol, F; Alessi, D; Haerle, S K; Bossart, W; Weber, M; Byland, R; Bernasconi, M; Berger, C; Tugizov, S; Speck, R F; Nadal, D (00). {beta} Integrin Expression Increases Susceptibility of Memory B-Cells to EBV Infection. Journal of Virology, ():-. Postprint available at: Posted at the Zurich Open Repository and Archive, University of Zurich. Originally published at: Journal of Virology 00, ():-.

2 JVI Accepts, published online ahead of print on April 00 J. Virol. doi:0./jvi.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. JVI0-0 Version 0 0 β Integrin Expression Increases Susceptibility of Memory B- Cells to EBV Infection Marcus Dorner,, Franziska Zucol, Davide Alessi, Stephan K. Haerle, Walter Bossart, Markus Weber, Rahel Byland,, Michele Bernasconi, Christoph Berger, Sharof Tugizov, Roberto F. Speck, and David Nadal * Experimental Infectious Diseases and Cancer Research, University Children s Hospital of Zurich, University of Zurich, Zurich, Switzerland, Department of Otorhinolaryngology, Head and Neck Surgery, University Children s Hospital of Zurich, Zurich, Switzerland, Institute for Medical Virology, University of Zurich, Zurich, Switzerland, Department of Surgery, University Hospital Zurich, Zurich, Switzerland Department of Medicine, University of California San Francisco, San Francisco, USA, and Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland Present address: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York * Corresponding author: David Nadal, M.D., Division of Infectious Diseases and Hospital Epidemiology, University Children's Hospital of Zurich, Steinwiesstrasse, CH-0 Zürich, Switzerland Phone +, FAX + 0; david.nadal@kispi.uzh.ch Word count abstract Word count text,0 Running Title: β integrin, memory B-cells and EBV infection Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

3 JVI0-0 Version 0 ABSTRACT The Epstein-Barr virus (EBV) uses nasopharyngeal-associated lymphoid tissue (NALT) as portal of entry to establish life-long persistence in memory B-cells. We previously showed that naïve and memory B-cells from NALT are equally susceptible to EBV infection. Here, we show that memory B-cells from NALT are significantly more susceptible to EBV infection than those from remote lymphatic organs. We identify β integrin, which is expressed highest by naïve B-cells of distinct lymphoid origin and by memory B-cells from NALT, as mediator of increased susceptibility to infection by EBV. Furthermore, we show that BMRF--β integrin interaction and the downstream signal transduction pathway is critical for post-binding events. Increase of β integrin expression in peripheral blood memory B-cells provoked by CD0 stimulation plus B- cell receptor cross-linking increased the susceptibility of non-nalt memory B-cells to EBV infection. Thus, EBV seems to utilize the increased activation status of memory B-cells residing in the NALT to establish and ensure persistence. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

4 JVI0-0 Version 0 0 INTRODUCTION The Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is transmitted via saliva and infects more than 0% of the world s population (). Much of EBV s medical importance relates to its association with B-cell malignancies including Burkitt s lymphoma, Hodgkin s lymphoma, and post-transplant lymphoproliferative disease (). The oncogenic potential of EBV is clearly illustrated by its unique capability to growth-transform B-cells in vitro (). In the current paradigm, EBV infects naïve B-cells in vivo in tonsils (). EBV is mainly present as a latent virus; upon infection EBV expresses distinct patterns of its latency genes depending upon distinct B-cell differentiation stages, varying from expression of all 0 known EBV latent genes in naïve B-cells to complete absence of EBV mrna expression in resting memory B-cells. This has led to the model that EBV, by virtue of expression of its latency genes, provides cell survival signals in naïve B-cells (). Recent data suggested that EBV, in particular, expedites the antigen-driven somatic hypermutation and selection process of B-cells taking place in germinal centers (GC) (). Chaganti et al. challenge the current paradigm showing in patients with primary EBV infection that EBV avoids GC transit and infects directly memory B-cells (). This report is consistent with in vitro experiments showing that EBV is able to infect memory B- cells (, 0) besides the well accepted susceptibility of naïve and GC B-cells to EBV. Irrespective of what B-cell subset is the primary target of EBV, it s propagation within the host is linked to proliferation of infected B-cells which delivers latent EBV to daughter cells or, more rarely, to switching of EBV to lytic infection (). This latter process can eventually be triggered by the differentiation of infected memory B-cells to plasma cells, and results in the release of virions that may subsequently infect new B-cells (). Importantly, transmission of EBV to naïve hosts is thought to occur via droplets loaded with virions (). Thus, lytic replication of EBV best takes place in nasopharyngeal-associated lymphoid tissue (NALT) which Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

5 JVI0-0 Version 0 0 will release EBV into the saliva, generating infectious droplets. Therefore, the NALT is the neuralgic point of EBV transmission, i.e., as portal of entry of EBV as well as shedding organ for further transmission (). The attachment of EBV to B-cells is mediated by the direct interaction of EBV glycoprotein gp0/0 with cellular CD, initiating receptor-mediated endocytosis. After binding to CD, EBV gp can interact with host HLA class II, leading to a conformational change in the viral glycoproteins and triggering fusion with the host cell membrane (, ). Nevertheless, experimental data suggests that CD and HLA class II are dispensable for the infection of B-cells (). Notably, in polarized oropharyngeal epithelial cells which lack CD, interactions between the EBV glycoprotein BMRF- via its Arg-Gly-Asp (RGD) motif with β integrin is critical for infection (,, ). The role of β integrin in mediating EBV infection of memory B-cells from NALT or non-nalt is unknown. We have recently demonstrated that tonsillar memory B-cells are much more susceptible to EBV infection than those from the peripheral blood, originating from various lymphoid tissues (). Thus, tonsillar memory B-cells seem to express properties which render them more susceptible to EBV infection compared to their counterparts from other lymphatic origin. Here, we hypothesized that memory B-cells from the NALT exhibit specific properties rendering them highly susceptible for EBV infection. Indeed, in this work, we found that memory B-cells from the NALT are distinguished from memory B-cells of other lymphoid tissue by their β integrin expression levels and thus their activation status and that this higher expression level is a critical factor for their higher susceptibility to EBV infection. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

6 JVI0-0 Version 0 0 MATERIALS AND METHODS Cell culture and viruses. Cells were maintained in RPMI0 supplemented with 0% FBS, % L-glutamine and % penicillin/streptomycin (all from Gibco, Basel, Switzerland). The EBV strains used were B., B.EBfaV-GFP carrying a CMV-EGFP cassette (, 0) (kindly provided by Richard Longnecker, Northwestern University Feinberg School of Medicine, Chicago, Illinois), and B expressing low BMRF- levels (). B.EBfaV-GFP and B were constantly cultivated in the presence of G- (Sigma, Buchs, Switzerland) to maximize the yield of recombinant virus. Flow cytometry analysis after staining for EBER () showed that among the recombinant EBV producer cells (B.EBfaV-GFP and B) less than % were positive for EBER but did not express grenn fluorescence protein (GFP). Virus production was performed as described previously (). The same virus stocks were used for all experiments in order to exclude bias from batch-dependent viral titers, except where indicated. Inactivation of EBV was performed either by heat-inactivation at C or C for 0 min. DNAse treatment. To remove non-encapsidated EBV DNA before EBV DNA quantification, viral stocks were treated with DNAse according to the instructions of the manufacturer (Ambion, Austin, TX). Quantitative PCR (qpcr) assay. Quantification of EBV DNA copies in viral stocks was carried out after DNAse treatment using the TaqMan (Applied Biosystems, Rotkreuz, Switzerland) real-time PCR technique with a PCR primer-probe-system targeting the conserved EBV BamHI W region as reported (). Serial dilutions of EBV DNA calibrated to an EBVspecific plasmid were included in every PCR run (). Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

7 JVI0-0 Version EBER in situ hybridization. The presence of EBER in single cells was checked for by in situ hybridization and flow cytometry as reported (), with the modification that the EBER hybridization probe was linked to Atto0 (Microsynth, Balgach, Switzerland). EBV-negative Ramos Burkitt lymphoma cells and lymphoblastoid cell line (LCL) cells were used as negative and positive controls, respectively. 0 0 Antibodies and reagents. Antibodies to CD, CD0, CD, CD, or CDL were from BD Biosciences (Allschwil, Switzerland). Blocking antibodies against α β integrin and EBV gp0/0 were from Abcam (Cambridge, United Kingdom), the EBV gp (clone F.) blocking antibody was kindly provided by Dr. L. Hutt-Fletcher (Louisiana State University, Shreveport, Louisiana), recombinant β integrin was from Thermo Scientific (Surrey, United Kingdom) and blocking antibodies against CD or HLA-DR,DP,DQ from Biolegend (Luzern, Switzerland). The inhibitors PP, Wortmannin, Cytochalasin D and AG- were from Calbiochem (Dietikon, Switzerland) and Ly-00 from Sigma-Aldrich. Antibodies detecting py FAK or FAK were from BD Biosciences. Antibodies detecting py c-src, c-src, py pik pα, PIK pα, ps cofilin, cofilin and all HRP-conjugated secondary antibodies were from Cell Signaling (Allschwil, Switzerland). Anti-human IgM and CD0L were from R&D Systems (Abingdon, United Kingdom). Cells were stimulated with concentrations recommended by the manufacturer. Isolation of cells. Human mononuclear cells were isolated from tonsils of donors undergoing routine tonsillectomy, from peripheral blood, and from mesenteric lymph nodes of donors undergoing abdominal surgery. Donors were EBV-seronegative. Tonsillar and mesenteric lymph node mononuclear cells were prepared as previously described (). B-cell subsets separation was Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

8 JVI0-0 Version 0 0 performed by using the B-cell isolation kit II and CD microbeads according to the manufacturer s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the isolated subsets was determined by flow cytometry and was above 0% in all preparations. This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University Children s Hospital of Zurich (IRB study # StV /0) and University Hospital of Zurich (IRB study # EK 0). All subjects provided written informed consent for the collection of samples and subsequent analysis. Infection of cells. EBV infection was performed as described earlier () with minor modifications. Briefly, supernatants of B., B.EBfaV-GFP, or B BMRF- low cells were centrifuged at 0.000xg for h at C. The pellet was resuspended in PBS and 0% of high-titer EBV (0.-. x 0 EBV DNA copies/ml after DNAse treatment) was added to the memory B- cells. The cells were then subjected to spinoculation at C at 00g for 0 min as conventional inoculation with EBV-containing supernatants normally does not yield sufficient numbers of infected cells to permit detailed susceptibility studies (). All infection frequencies shown using B.EBfaV-GFP were quantified using flow cytometry h post inoculation of cells. EBV binding assay. Isolated B-cells or monocytes from peripheral blood were incubated with either wild-type B. or B BMRF- low EBV at either multiplicity of infection (moi) or per cell for h at C. Following extensive washing of the cells with ice-cold PBS, RNA was extracted and the number of bound EBV DNA copies was determined by qpcr as previously described (). Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

9 JVI0-0 Version 0 0 Inhibition of EBV infection. Inhibition of EBV receptors was performed by incubating memory B-cells with either anti-human CD (:00), anti-human HLA-DR,DP,DQ (:0), dilutions of anti-human CD0, or dilutions of anti-β integrin for h at C followed by spinoculation of the cells. Inhibition of EBV glycoproteins was performed by incubation of the EBV-containing supernatants with either anti-gp0/0 (:00), or dilutions of anti-gp or soluble recombinant β integrin for h at C prior to spinoculation of memory B-cells. Inhibition of the integrin signaling was performed by incubating the cells with either PP (0µM), wortmannin (0nM), Ly-00 (0µM), AG- (0µM) or cytochalasin D (0µM) for 0 min prior to spinoculation with EBV. Western blotting. Cells were resuspended in 00µl modified RIPA buffer. Total amounts of protein in each sample were quantified and normalized using Nanodrop technology (Willington, DE) and samples were separated on a -% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, California). Fluorescence microscopy. Cells were washed with ice-cold PBS and were cytospun on microscopy slides. Fixation was performed in % paraformaldehyde for 0 min prior to permeabilization for min using 0.% TritonX-00. Actin was stained using FITC-Phalloidin (Sigma-Aldrich) for h at room temperature. Flow cytometry and fluorescence-activated cell sorting. Flow cytometry was performed using a Cytomics FC 00 (Beckman Coulter, Nyon, Switzerland) and fluorescence-activated cell sorting was done using a MoFlo (Dako Cytomation, Glostrup, Denmark) Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

10 JVI0-0 Version DNA microarrays. RNA from isolated tonsillar or peripheral blood B-cells was extracted using the TRIzol method (Invitrogen). Quality was verified on a 00 Bioanalyzer and Nanodrop. RNA was transcribed to crna using the NuGEN System. The synthesized crna was hybridized to UPlus.0 GeneChips (Affymetrix) and scanned using an Affymetrix GeneChip Scanner 000 per Affymetrix protocols. Results were analyzed using Genespring software (Agilent). Statistics. Statistical analyses were performed using Graphpad Prism Software (La Jolla, CA). Dual comparisons were analyzed using a two-tailed Mann-Whitney t-test. P values below 0.0 were considered as being statistically significant. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

11 JVI0-0 Version 0 0 RESULTS Susceptibility of memory B-cells from various lymphoid tissues to EBV infection..even though peripheral blood resting memory B-cells are the main reservoir for EBV persistence in vivo (), memory B-cells from peripheral blood appear to be less susceptible to EBV infection in vitro than their tonsillar counterparts (, ). This may indicate that EBV preferentially infects memory B-cells from the NALT rather than from other lymphoid tissues. To investigate tissue-specific EBV infection susceptibility in detail, we performed infection assays of lymphoid cells from tonsils, peripheral blood, and mesenteric lymph nodes by spinoculation of the EBV strain B.EBfaV-GFP to quantify the EBV-infected memory B-cells by flow cytometry as previously described (). Notably, following spinoculation EBV enters B- cells by the well known EBV receptors, i.e., CD and HLA-DR (). Memory B-cells were identified by expression of CD and CD (). Memory B-cells from tonsils, which are part of the Waldeyer s ring forming the NALT, showed the highest susceptibility to EBV infection with. ±. % of infected cells hours post inoculation (Fig. A). In contrast, memory B-cells from peripheral blood which represent a mixture of memory B-cells from all lymphatic compartments exhibited lower infection frequencies of 0. ±. % (p = 0.00) and memory B-cells from mesenteric lymph nodes exhibited even lower infection frequencies of..0 ±. % (p = 0.0). This suggested that EBV preferentially infects memory B-cells originating from NALT (Fig. A). As the expression of L-selectin (CDL) on memory B-cells is strongly associated with NALT origin () we used this cell surface marker to further characterize the origin of EBVinfected memory B-cells from the various lymphoid tissues. Tonsils, where NALT-originating memory B-cells are generated, contained. ±. % CD + CD + memory B-cells expressing CDL indicating that less than 0 % of the memory B-cells have homed to the tonsil from Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00 0

12 JVI0-0 Version 0 0 remote lymphatic organs (Fig. B). In contrast only. ±. % (p = 0.00) and.0 ±. % (p = 0.0) of the CD + CD + peripheral blood and mesenteric lymph node memory B-cells, respectively, expressed CDL (Fig. B), documenting that only a minority of the memory B- cells in these lymphoid tissues expresses the NALT-associated cell surface marker. CD + CD + CDL + memory B-cells isolated from tonsils, peripheral blood, or lymph nodes showed higher EBV infection susceptibility frequencies (. ±.0 %,. ±. %, and.0 ±. %, respectively) than their CD + CD + CDL - counterparts (0.0 ±.0 %,.0 ±. %, and. ±. %, respectively; Fig. C). Importantly, neither exposure to nor infection with EBV in vitro did alter the levels of CDL expression on memory B-cells (data not shown). Thus, the presence of CDL on the cell surface was associated with a significantly higher susceptibility to EBV infection compared with the absence of CDL. To investigate whether CDL might be a cellular receptor for EBV in addition to the identified EBV receptors on B-cells, i.e. CD and HLA class II, we performed EBV infection experiments using tonsillar memory B-cells and CDL blocking antibodies. We could not observe any inhibition of EBV infection by CDL-blocking antibodies, even at very high antibody concentrations (Fig. D). Thus, CDL defines a subset of NALT-originating memory B-cells that is phenotypically characterized by increased susceptibility to EBV infection but appears not to be involved in the cell infection process. Expression of α β integrin on memory B-cells from various lymphoid tissues. Next, we were interested in identifying among cell surface structures potential candidates contributing to preferential infection of memory B-cells from NALT compared to those of other secondary lymphoid tissues. EBV is able to infect epithelial cells despite their lack of CD, the main receptor for EBV on B-cells. Thus, we hypothesized that epithelial cell surface structures Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

13 JVI0-0 Version 0 0 facilitating or mediating EBV entry are present and active on memory B-cells. Recently, it has been described that the integrin receptor family, including the α β integrin, plays an important role in EBV attachment to epithelial cells (, ). This process is mediated by the EBV glycoprotein BMRF- (). To identify whether the differential susceptibility of memory B-cells from NALT and non-nalt might be due to dissimilar α β integrin expression levels, we measured the expression profile of integrins in B-cells from tonsils and peripheral blood, respectively. Here, especially the integrin subunits α and β and the integrin-related genes TIF and PTK were found to be significantly up-regulated in tonsillar B-cells (Fig. A), indicating that signaling via α β integrin is particularly important in B-cells originating from the tonsils. To confirm these data at the protein level, we isolated memory B-cells from tonsils, peripheral blood and mesenteric lymph nodes and subjected them to immunofluorescent staining for α β integrin and flow cytometry. Immunofluorescence showed a weaker expression of α β integrin on memory B-cells from peripheral blood compared to tonsils even though all cells expressed at least low levels of α β integrin (Fig. B and C). This observation was further validated by quantification of the α β integrin expression levels using flow cytometry showing that memory B-cells from tonsils, peripheral blood, and mesenteric lymph nodes were α β -positive in. ±. %,. ±. %, and.0 ±. %, respectively. Notably, two distinct populations could be distinguished based on their α β expression levels (Fig. D, G and J). Cells with high expression levels of α β integrin were predominant in tonsils with only a minority of cells expressing low amounts of α β (Fig. D and E). In contrast, in peripheral blood the majority of α β -positive memory B-cells expressed low integrin levels (Fig. G and H) and in mesenteric lymph nodes virtually all α β -positive memory B-cells showed low expression levels (Fig. J, K). Comparing the Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

14 JVI0-0 Version 0 0 frequencies of α β -posititve memory B-cells in the two complementary lymphoid tissues, tonsils and mesenteric lymph nodes, with the percentage of CDL + NALT-originating memory B-cells it is obvious that the α β -positive phenotype is restricted to memory B-cells selected in the NALT. Analysis of the mean fluorescence intensity (MFI) of α β integrin on these cells revealed that α β high memory B-cells from tonsils and peripheral blood expressed - to 0-fold higher levels of α β integrin than α β low memory B-cells (Fig. F, I), whereas no α β high cells were found in mesenteric lymph nodes (Fig. L). By contrast, virtually all naïve B-cells from tonsils, peripheral blood, and mesenteric lymph nodes invariably expressed amounts of α β as high as the α β high tonsillar memory B-cells (Fig. G, H, I, M, N, O, S, T, U). Thus, tonsillar memory B- cells differ from their counterparts from the other lymphatic tissues investigated by the virtue of their α β high expression, a characteristic common to the naïve B-cells from these tissues. Binding of EBV to α β -expressing memory B-cells. Next, we addressed the question, whether EBV uses α β integrin to attach to NALT-originating memory B-cells as it does with epithelial cells. Two cellular receptors are important for EBV binding and entry into B-cells: CD and HLA class II (, ). Notably, both receptors are similarly expressed by memory B-cells from NALT and non-nalt (). CD (CR, CR) which is a member of the complement receptor family acts as the main attachment factor for EBV on B-cells. Attachment of EBV to CD via the viral glycoprotein gp0/0 has been shown to induce receptor-mediated endocytosis. Within the endosomal compartment, a trimer of EBV glycoproteins consisting of gh, gl and gp binds to HLA class II. This interaction, in turn, triggers a ph-dependent membrane fusion of EBV with the endosomal membrane and allows nuclear transport of the capsid (, ). Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

15 JVI0-0 Version First, we aimed at identifying whether the α β high memory B-cell subpopulation in tonsils is indeed more susceptible to EBV infection. Infection of tonsillar memory cells, unsorted or sorted by flow cytometry, showed a 0% - 0% higher infection frequency of α β high as compared to α β low memory B-cells (p = 0.0; Fig. A). FACS sorting was performed by gating on CD + CD + cells followed by selection of α β high and low expressing populations. 0 0 Thereby, we could guarantee that the Ig isotype composition of the sorted cell populations high reflected that of unsorted memory B-cells. This preferential infection susceptibility of α β high memory B-cells was also evident for memory B-cells from peripheral blood, where α β low memory B-cells exhibited a 0% higher infection frequency compared to their α β high counterparts (p = 0.0; Fig. B). Although the observed infection frequencies in the α β low memory B-cell compartment were only increased by 0% to 0% compared to the α β memory B-cell compartment these differences might be sufficient in vivo and allow preferential infection of α β high memory B-cells. Next, to test whether EBV uses α β integrin on NALT memory B-cells for attachment, we performed experiments using antibodies blocking EBV binding to α β integrin or soluble recombinant β integrin to compete with EBV binding to cellular β integrin. As expected, blocking of CD or blocking of HLA class II molecules showed a significant reduction of EBV infection frequency legitimizing the use of spinoculation as a means to test EBV entry, as already shown in previous work () (Fig. C). Using antibodies to CD0 which is expressed by B-cells but is irrelevant for EBV infection, we found no changes in EBV infection frequencies compared to mock treatment. When using either anti-α β blocking antibodies alone or in combination with anti-cd blocking antibodies, however, a dose-dependent reduction of the EBV infection frequency could be observed in tonsillar memory B-cells (Fig. C). As the combination of CD Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

16 JVI0-0 Version 0 0 and α β blocking antibodies showed a nearly complete inhibition of EBV entry it is unlikely that receptors other than CD, HLA-DR and α β integrin play a pivotal role in EBV entry into memory B-cells. Finally, recombinant soluble β integrin showed a similar dose-dependent inhibition of EBV infection of memory B-cells from tonsils in the presence of additional inhibition of the EBV glycoprotein gp0/0 (Fig. D). As positive controls, we also included blocking of gp0/0 alone or in combination with dilutions of anti-gp to block EBV penetration of the endosomal membrane. Thus, we could show by either blocking cellular α β integrin or by competing with recombinant β integrin that EBV indeed uses β integrin as factor to attach to memory B-cells. Lack of BMRF- impairs infection but not binding to B-cells. As α β integrin has recently been shown to be critical for the infection of epithelial cells by EBV (, -) we chose to employ the described BMRF- low EBV strain B to test whether the enhanced infection susceptibility of α β integrin-expressing memory B-cells is due to BMRF--α β integrin interactions. The BMRF- low B and the B.EBfaV-GFP strains did not contain significant amounts of wild-type EBV (<%; see materials and methods), therefore we can exclude any significant interference of wild-type EBV. EBV produced from both, B.EBfaV-GFP and B as well as wild-type B. cells was used to infect B-cells isolated from tonsils. EBV infection was determined by flow cytometry for GFP fluorescence h following inoculations of the cells. As expected, inoculation with wild type EBV showed no GFP fluorescence (Fig. A) and inoculation with B.EBfaV-GFP showed an increase of GFP fluorescence in around 0 % of the inoculated cells (Fig. B). In contrast, less than % GFP fluorescence was observed hours after inoculation of the cells with BMRF- low B EBV, Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

17 JVI0-0 Version 0 0 despite using the same amount of EBV DNA copies as measured by qpcr after DNAse treatment of the viral stocks preparation (Fig. C). Next, we compared the ability of B BMRF- low and wilt-type EBV to bind to B-cells by performing binding experiments followed by quantification of bound EBV DNA copies by quantitative PCR to determine the number of EBV particles bound per cell. Both wild-type and BMRF- low EBV were able to bind to B-cells with comparable, dose-dependent efficiency (Fig. D). These results indicate that binding of EBV to B-cells is not influenced by BMRF--α β integrin interaction, and are in line with CD being the main receptor for EBV binding to B- cells. To verify that, we performed binding experiments on monocytes which lack the expression of CD. Binding to monocytes was impaired when using BMRF- low EBV as compared to wildtype EBV (Fig. E). Surprisingly, wild-type EBV binding to B-cells and monocytes was similar. This might indicate that additional binding sites for EBV may exist that partially compensate for the lack of CD. Taken together, these data indicate that BMRF- is non-essential for the formation of enveloped EBV particles which are still able to bind to B-cells via CD and/or HLA-DR but exhibit reduced binding capacity to monocytes lacking expression of CD. Role of integrin signaling for EBV entry into NALT memory B-cells. Even though the requirement of α β integrin for EBV binding and infection of epithelial cells has been shown previously (, ), the participation of signaling events downstream of α β integrin initiated through EBV binding and their contribution to EBV entry has not been elucidated. First evidence that α β integrin may be involved in the EBV infection process of B-cells originating from the tonsil arises from our microarray analysis of tonsillar B-cells showing significant up-regulation of TIF and PTK (Fig. A), two genes downstream of α β integrin. We therefore dissected the Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

18 JVI0-0 Version 0 0 integrin signaling pathway (, ) following EBV binding to memory B-cells. One of the initial steps in integrin signaling involves activation of focal adhesion kinase by phosphorylation of Tyr followed by downstream signaling through PIK pα, c-src kinases and cofilin to finally activate the depolymerization of the actin cytoskeleton (Fig. A) (,,,, 0). To investigate whether EBV itself activates integrin signaling, thereby enhancing infection efficiency, we (i) followed the phosphorylation status of FAK, PIK and cofilin over time upon binding of EBV to tonsillar memory B-cells treated or not with inhibitors of FAK, c-src or PIK pα activation prior to infection with EBV and (ii) investigated the specificity of the BMRF- /integrin interaction by employing a recombinant EBV strain containing only trace amounts of BMRF- protein (, ). Binding of EBV tonsillar memory B-cells led to up-regulation of pfak within 0 minutes, followed by phosphorylation of c-src, PIK pα, and cofilin (Fig. B). The kinetic of c-src phosphorylation, which is much faster compared to that of PIK pα, indicates that c-src actually phosphorylates PIK pα in the process of EBV entry. Furthermore, protrusions in the actin cytoskeleton similar to those observed upon activation of integrin signaling via fibronectin were readily detectable in memory B-cells when EBV binding took place (Fig. D). This was abolished when using heat-denatured EBV or heat-inactivated EBV, indicating that a conformationally-active BMRF- might be required for interaction with α β integrin (Fig. D). We also conducted the experiments using a recombinant EBV containing only trace amount of BMRF- (). Following challenge of memory B-cells with B-BMRF- low EBV, activation of FAK and all downstream effectors was abolished (Fig. C), as well as rearrangements of the actin cytoskeleton did not occur (Fig. D). Given that the B-BMRF- low EBV strain still contains residual BMRF- efficient activation of integrin signaling obviously requires a certain threshold amount of incorporated BMRF-, that is present in wild-type but not Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

19 JVI0-0 Version 0 0 BMRF- low EBV. Memory B-cells could not be infected with B-BMRF- low as determined by flow cytometry after staining for EBER () hours after spinoculation, even if B-BMRF- low was used in up to -fold higher DNA concentration than wild-type EBV B. (data not shown). These results indicate that interaction between EBV BMRF- and integrins on memory B-cells is crucial for the activation of the integrin signaling pathway. To further investigate the dependence of EBV entry into memory B-cells on integrin signaling, we used small-molecule phosphorylation inhibitors of FAK (AG-), PIK (Wortmannin and Ly-00) or c-src (PP) prior to infection of memory B-cells by EBV-EGFP (Fig. A, B). These experiments showed that, indeed, the whole integrin signaling pathway is crucial for EBV entry as all used inhibitors significantly reduced the susceptibility of the cells to infection with EBV. Dependence on the actin cytoskeleton rearrangement was tested by pretreating the memory B-cells with Cytochalasin D which disrupts the actin microfilaments () showing increased EBV infection of memory B-cells (Fig. A, B). Taken together these results indicate that EBV binds to α β integrin on NALT memory B-cells via its glycoprotein BMRF- and initiates activation of the integrin signal transduction pathway. Starting with the phosphorylation of FAK, c-src, PIK pα and cofilin, this leads to depolymerization of the actin cytoskeleton and thus to a more efficient nuclear translocation of the EBV capsid. Induction of β integrin expression in memory B-cells isolated from non-nalt increases their susceptibility to EBV infection and transformation. As high expression of β integrin is linked to increased susceptibility to EBV infection of memory B-cells from NALT, we aimed at inducing β integrin expression in memory B-cells from non-nalt to investigate whether this would increase the susceptibility to EBV infection. Given that expression and activity of integrins Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

20 JVI0-0 Version 0 in B-cells is regulated mainly via B-cell-activating signals received by CD0 () or the B-cell receptor (), we triggered memory B-cells, either from tonsils or peripheral blood, with CD0L plus anti-igm antibody prior to EBV infection. Even though IgM-positive memory B-cells only make up a minority of tonsillar memory B-cells, stimulation with anti-igm and CD0L has been shown to be sufficient for activation (). The expression of β integrin was monitored by flow cytometry hours post stimulation of cells. Whereas the percentage of memory B-cells expressing high levels of β integrin could not be increased by triggering tonsillar memory B- cells (Fig. A), a three-fold increase was observed upon triggering of peripheral blood memory B-cells with CD0L plus anti-igm (Fig. B, C). The susceptibility of memory B-cells from tonsils to EBV infection was not increased following in vitro triggering of these cells as demonstrated by spinoculation of B.EBfaV-GFP (Fig. D), but EBV infection of memory B- cells from peripheral blood triggered with CD0L plus anti-igm could be increased to percentages (0 %) matching those of their tonsillar counterparts (Fig. E). Thus, the proportion of memory B-cells from peripheral blood expressing high levels of β integrin as well as the susceptibility to EBV infection can substantially be increased in vitro. In contrast, tonsillar memory B-cells show already maximal percentages of cells expressing high levels of β integrin and susceptibility to EBV infection prior to in vitro triggering. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

21 JVI0-0 Version 0 0 DISCUSSION In this work, we studied the mechanisms underlying the increased susceptibility to EBV infection of memory B-cells from tonsils as opposed to memory B-cells from other lymphoid sites. Our experimental data indicate that i) high expression of α β integrin is unique to memory B-cells from the NALT, and EBV indeed uses β integrin as co-factor to attach to memory B-cells; ii) triggering the α β integrin signaling pathway is a key event for EBV entry and thus for tonsillar B-cells susceptibility to in vitro infection; and iii) inducing expression of β integrin by activating memory B-cells with CD0L plus anti-igm antibody increases the susceptibility of memory B-cells to EBV infection. We conclude that memory B-cells from tonsils exhibit increased susceptibility to EBV infection by virtue of their high basal activation status reflecting their high β integrin expression which mediates increased attachment and entry of the virus compared to memory B-cells from other lymphatic tissues where B-cell-activating factors or antigen are less abundant. The starting point of our hypothesis was the observation that tonsillar memory B-cells are particularly susceptible to ex vivo EBV infection () and that Janz et al. recently reported the possible existence of additional receptors for EBV on B-cells besides CD and HLA class II (). Thus, we hypothesized that memory B-cells from the NALT exhibit specific properties rendering them highly susceptible for EBV infection, and we assumed that memory B-cells primed in the tonsils would keep their susceptibility to EBV infection when circulating in the blood. To address this hypothesis we employed the unique CD + CD + CDL + phenotype of NALT-originating memory B-cells (, ) to compare the relative infection susceptibilities of NALT-originating memory B-cells at distinct lymphoid compartments to that of memory B-cells selected at other lymphoid sites. Although NALT-originating (CDL + ) memory B-cells exhibit a higher susceptibility to EBV infection irrespective of whether they are isolated directly from 0 Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

22 JVI0-0 Version 0 0 tonsils, peripheral blood, or mesenteric lymph nodes we excluded that CDL itself functions as co-receptor for EBV by using neutralizing antibodies against CD. We conclude that CDL phenotypically marks a population of memory B-cells that have been selected in the NALT and have retained or obtained receptors required for an efficient infection with EBV. Recently, it has been found that the interaction of an EBV envelope protein, BMRF-, with integrins α β plays a role in the infection of polarized epithelial cells (() and Speck R, unpublished data) which do not express the known B-cellular receptors CD and HLA class II, both crucial for EBV entry. Here, we found that memory B-cells from the NALT express α β integrin at least at 0-fold higher levels as compared to their counterparts from peripheral blood high or mesenteric lymph nodes. Notably, we identified two distinct populations, defined as α β and α β low, and found most tonsillar memory B-cells expressing high amounts of α β integrin and most peripheral blood or mesenteric lymph node memory B-cells expressing low amounts of α β integrin. Given that blocking of and saturating α β integrin by antibodies and the recombinant extracellular domain of β integrin, respectively, resulted in a clear decrease of EBV entry into memory B-cells, β integrin has a key role as co-factor in the viral entry process. Notably, memory B-cells from distinct lymphatic tissue express equal amounts of the cell surface molecules CD and HLA class II () which constitutes the EBV receptor complex: that consequently does not explain the distinct susceptibility to EBV infection. In contrast, the level of α β integrin expression discriminates between degrees of susceptibility to EBV infection of memory B-cells. As we show, the susceptibility to EBV infection correlating to α β integrin expression levels is mediated by binding of EBV s structural protein BMRF- to α β integrin.. Further, we show that each step of β integrin-mediated signaling leading to the activation of the downstream Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

23 JVI0-0 Version 0 0 targets (0) is crucial for EBV entry into B cells. Very importantly, activation of memory B-cells from peripheral blood with CD0L plus anti-igm resulted in vigorous upregulation of β integrin expression. Thereby, the susceptibility of peripheral blood memory B-cells to infection by EBV, i.e. of memory B-cells which were not from the NALT was increased. Stimulation of the B-cell receptor results in increasing the expression levels of integrin, whereas CD0L, which binds to CD0 on B-cells and in addition to α β integrin () may activate the down-stream integrin signaling pathway and in turn may lead to an increased uptake of EBV via actin cytoskeleton reorganizations. Thus, these data demonstrate clearly that the susceptibility of memory B-cells to EBV infection is greatly dependent on their activation status and the resulting high expression levels of β integrin. In fact, in a primary immune reaction, the CD0L expressed on activated T cells has a key role in subsequent activation of B-cells by binding to CD0 (). Thus, the events described above may be operative during primary or secondary immune responses in the NALT explaining its marked susceptibility to EBV infection and its central role in EBV pathogenesis. Whether the interaction of EBV s essential envelope protein gh with integrins α V β or α v β, which very recently have been shown to trigger epithelial cell fusion by EBV (), plays a role for B-cell infection remains to be investigated. We propose a model in which EBV takes advantage of targeting activated CDL + high α β memory B-cells which preferentially reside in or home to NALT and, following circulation, are likely to home back to NALT. We have previously shown that naïve B-cells from distinct tissues are equally highly susceptible to EBV infection ex vivo (). Here, we report that naïve B-cells from distinct tissues essentially express α β high high, contrasting memory B-cells expressing α β preferentially when originating from NALT. Our data speak in favor of a direct infection of tonsillar memory B-cells with EBV which, in addition to primary infection of naïve () and germinal center B-cells (, ), contributes to a more efficient establishment of EBV persistence. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

24 JVI0-0 Version 0 In NALT, the memory B-cells may encounter recall antigen and subsequently either propagate EBV as latent infection to progeny cells or undergo differentiation to plasma cells resulting in lytic replication of EBV which then can be transmitted via saliva to new susceptible hosts. Triggering of CD0 and the B-cell receptor mediated by exposure to microorganisms may be involved in this process, as such triggering increased the proportion of cells expressing high levels of β integrin. NALT is readily accessible to a wide variety of antigen explaining why memory B-cells generated within and homing back to the NALT are much more readily found in an activated, i.e. β integrin high expressing state compared to remote lymphoid organs which are not accessible by antigen or pathogens directly but only following initial immune recognition by sentinel immune cells such as dendritic cells. The idea that EBV establishes persistence by direct infection of memory B-cells has also been proposed by Kurth et al. who examined EBV-infected B-cells in infectious mononucleosis (, ). Our findings give a detailed insight of EBV B-cell infection and biology. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

25 JVI0-0 Version ACKNOWLEDGEMENTS We thank Dr. Lindsey M. Hutt-Fletcher and Dr. Richard Longnecker for reagents and R. Herrera for technical assistance. This work was funded by the Swiss National Foundation (#000-), the Forschungskredit of the University of Zurich, and the Velux Foundation. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

26 JVI0-0 Version 0 0 REFERENCES. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson.. EBV persistence in memory B cells in vivo. Immunity :-0.. Bechtel, D., J. Kurth, C. Unkel, and R. Kuppers. 00. Transformation of BCRdeficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas. Blood 0:-0.. Berger, C., P. Day, G. Meier, W. Zingg, W. Bossart, and D. Nadal. 00. Dynamics of Epstein-Barr virus DNA levels in serum during EBV-associated disease. J Med Virol :0-.. Brandtzaeg, P., I. N. Farstad, and G. Haraldsen.. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol Today 0:-.. Brandtzaeg, P., and F. E. Johansen. 00. Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties. Immunol Rev 0:-.. Chaganti, S., E. M. Heath, W. Bergler, M. Kuo, M. Buettner, G. Niedobitek, A. B. Rickinson, and A. I. Bell. 00. Epstein-Barr virus colonisation of tonsillar and peripheral blood B cell subsets in primary infection and persistence. Blood.. Chesnokova, L. S., S. L. Nishimura, and L. M. Hutt-Fletcher. 00. Fusion of epithelial cells by Epstein-Barr virus proteins is triggered by binding of viral glycoproteins ghgl to integrins alphavbeta or alphavbeta. Proc Natl Acad Sci U S A 0:0-.. Crouch, J., D. Leitenberg, B. R. Smith, and J. G. Howe.. Epstein-Barr virus suspension cell assay using in situ hybridization and flow cytometry. Cytometry :0-. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

27 JVI0-0 Version 0 0. Dorner, M., F. Zucol, C. Berger, R. Byland, G. T. Melroe, M. Bernasconi, R. F. Speck, and D. Nadal. 00. Distinct ex vivo susceptibility of B-cell subsets to epsteinbarr virus infection according to differentiation status and tissue origin. J Virol : Ehlin-Henriksson, B., J. Gordon, and G. Klein. 00. B-lymphocyte subpopulations are equally susceptible to Epstein-Barr virus infection, irrespective of immunoglobulin isotype expression. Immunology 0:-0.. Hehlgans, S., M. Haase, and N. Cordes. 00. Signalling via integrins: implications for cell survival and anticancer strategies. Biochim Biophys Acta :-0.. Hutt-Fletcher, L. M. 00. Epstein-Barr virus entry. J Virol :-.. Huveneers, S., H. Truong, and H. J. Danen. 00. Integrins: signaling, disease, and therapy. Int J Radiat Biol :-.. Janz, A., M. Oezel, C. Kurzeder, J. Mautner, D. Pich, M. Kost, W. Hammerschmidt, and H. J. Delecluse Infectious Epstein-Barr virus lacking major glycoprotein BLLF (gp0/0) demonstrates the existence of additional viral ligands. J Virol :0-.. Kurth, J., M. L. Hansmann, K. Rajewsky, and R. Kuppers. 00. Epstein-Barr virusinfected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction. Proc Natl Acad Sci U S A 00:0-.. Kurth, J., T. Spieker, J. Wustrow, G. J. Strickler, L. M. Hansmann, K. Rajewsky, and R. Kuppers EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency. Immunity :-. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

28 JVI0-0 Version 0 0. Laichalk, L. L., and D. A. Thorley-Lawson. 00. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J Virol :-0.. Leveille, C., M. Bouillon, W. Guo, J. Bolduc, E. Sharif-Askari, Y. El-Fakhry, C. Reyes-Moreno, R. Lapointe, Y. Merhi, J. A. Wilkins, and W. Mourad. 00. CD0 ligand binds to alphabeta integrin and triggers cell signaling. J Biol Chem :-.. Mancao, C., M. Altmann, B. Jungnickel, and W. Hammerschmidt. 00. Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus. Blood 0:-. 0. Oh, M. A., E. S. Kang, S. A. Lee, E. O. Lee, Y. B. Kim, S. H. Kim, and J. W. Lee. 00. PKCdelta and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin alpha but not its tailless mutant. J Cell Sci 0:- 0.. Rickinson, A., and E. Kieff. 00. Epstein-Barr virus. In: Knipe D. Howley P., eds. Fields Virology. th ed Lippincott Williams & Wilkins:-.. Rose, D. M., R. Alon, and M. H. Ginsberg. 00. Integrin modulation and signaling in leukocyte adhesion and migration. Immunol Rev :-.. Rubtsova, S. N., R. V. Kondratov, P. B. Kopnin, P. M. Chumakov, B. P. Kopnin, and J. M. Vasiliev.. Disruption of actin microfilaments by cytochalasin D leads to activation of p. FEBS Lett 0:-.. Shi, C., and D. I. Simon. 00. Integrin signals, transcription factors, and monocyte differentiation. Trends Cardiovasc Med :-.. Sixt, M., M. Bauer, T. Lammermann, and R. Fassler. 00. Beta integrins: zip codes and signaling relay for blood cells. Curr Opin Cell Biol :-0. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

29 JVI0-0 Version 0 0. Souza, T. A., B. D. Stollar, J. L. Sullivan, K. Luzuriaga, and D. A. Thorley-Lawson. 00. Influence of EBV on the peripheral blood memory B cell compartment. J Immunol :-0.. Spaargaren, M., E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, S. Middendorp, R. W. Hendriks, and S. T. Pals. 00. The B cell antigen receptor controls integrin activity through Btk and PLCgamma. J Exp Med :-0.. Speck, P., K. M. Haan, and R. Longnecker Epstein-Barr virus entry into cells. Virology :-.. Speck, P., K. A. Kline, P. Cheresh, and R. Longnecker.. Epstein-Barr virus lacking latent membrane protein immortalizes B cells with efficiency indistinguishable from that of wild-type virus. J Gen Virol 0 ( Pt ): Speck, P., and R. Longnecker.. Epstein-Barr virus (EBV) infection visualized by EGFP expression demonstrates dependence on known mediators of EBV entry. Arch Virol :-.. Steiniger, B., E. M. Timphus, R. Jacob, and P. J. Barth. 00. CD+ B cells in human lymphatic organs: re-evaluating the splenic marginal zone. Immunology :-.. Thorley-Lawson, D. A. 00. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol :-.. Thorley-Lawson, D. A., and A. Gross. 00. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med 0:-.. Tugizov, S. M., J. W. Berline, and J. M. Palefsky. 00. Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells. Nat Med :0-.. van Kooten, C., and J. Banchereau CD0-CD0 ligand. J Leukoc Biol :-. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

30 JVI0-0 Version 0. Wortis, H. H., M. Teutsch, M. Higer, J. Zheng, and D. C. Parker.. B-cell activation by crosslinking of surface IgM or ligation of CD0 involves alternative signal pathways and results in different B-cell phenotypes. Proc Natl Acad Sci U S A :-.. Xiao, J., J. M. Palefsky, R. Herrera, J. Berline, and S. M. Tugizov. 00. EBV BMRF- facilitates cell-to-cell spread of virus within polarized oral epithelial cells. Virology :-.. Xiao, J., J. M. Palefsky, R. Herrera, J. Berline, and S. M. Tugizov. 00. The Epstein- Barr virus BMRF- protein facilitates virus attachment to oral epithelial cells. Virology 0:0-.. Xiao, J., J. M. Palefsky, R. Herrera, and S. M. Tugizov. 00. Characterization of the Epstein-Barr virus glycoprotein BMRF-. Virology :-. 0. Yee, K. L., V. M. Weaver, and D. A. Hammer. 00. Integrin-mediated signalling through the MAP-kinase pathway. IET Syst Biol :-. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

31 JVI0-0 Version 0 0 FIGURE LEGENDS FIG.. EBV preferentially infects memory B-cells expressing CDL. (A) EBV infection of memory B-cells from tonsils, peripheral blood and mesenteric lymph nodes, respectively. EBV infection was performed by spinoculation of B.EBfaV-GFP supernatants. Results shown are percentages of EGFP + cells gated for CD + CD + memory B-cells. (B) Percentage of memory B-cells (CD + CD + ) from tonsils, peripheral blood and mesenteric lymph nodes expressing CDL (C) EBV infection of CDL + and CDL - memory B-cells from tonsils, peripheral blood (PB) and mesenteric lymph nodes (LN), respectively. (D) EBV infection frequency susceptibility of tonsillar memory B-cells following blocking of CDL using blocking antibodies. Data are percentages of EGFP + cells gated for CD + CD + cells. Results shown are mean ± SD from - replicates. P values were calculated by using the Mann-Whitney t-test. * p = 0.0, ** p = 0.0, *** p = 0.0, **** p = 0.0, ***** p = FIG.. α β integrin is preferentially expressed on memory B-cells from NALT. (A) Gene expression profiling of integrin-related genes comparing tonsillar and peripheral blood B-cells. Scale indicates fold regulation. (B) Immunofluorescence of α β integrin on memory B-cells from tonsil and (C) peripheral blood (PB). Bar, 0 µm (D - U) Quantification of α β integrin expression on (D-F, J-L, P-R) memory B-cells and (G-I, M-O, S-U) naïve B-cells by flow cytometry. Data shown are percentages of α β -positive memory and naïve B-cells from (D-E, G-H) tonsils, (J-K, M-N) peripheral blood and (P-Q, S-T) lymph nodes and relative fluorescence intensity of β integrin on memory and naïve B-cells from tonsils (F, I), peripheral blood (L, O), and mesenteric lymph nodes (R, U), respectively. Data shown are mean ± SD from - biological replicates. *, not detected. P values were calculated by using the Mann-Whitney t-test. ** p = Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

32 JVI0-0 Version FIG.. Integrin α β is required for efficient attachment and entry of EBV on memory B-cells. (A) Infection frequency of α β low and α β high CD + CD + memory B-cells either by 0 0 inoculation of pooled tonsillar memory B-cells or fluorescence-activation cell-sorted memory B- low cell populations with B.EBfaV-GFP by spinoculation. (B) Infection frequency of α β and lhigh α β CD + CD + memory B by inoculation of pooled peripheral blood memory B-cells with B.EBfaV-GFP by spinoculation. (C) Inhibition of cellular receptors required for EBV infection (CD and HLA class II), α β integrin as well as non-relevant surface proteins (CD0) on memory B-cells from tonsils using different concentrations of blocking antibodies and combinations. (D) Inhibition of the viral glycoproteins gp0/0 and gp in combination with competition inhibition of recombinant soluble β integrin using tonsillar memory B-cells. Data shown in (A) and (B) are mean ± SD from biological replicates. P values were calculated using the Mann-Whitney t-test. Data shown in (C) and (D) are from one representative experiment out of three. * p = 0.0, ** p = 0.0. FIG.. Lack of BMRF- impairs infection but not binding to B-cells. (A-C) Flow cytometry to detect GFP fluorescence h after spinoculation of isolated tonsillar B-cells with either (A) wildtype B., (B) B.EBfaV-GFP or (C) B BMRF- low EBV. The viral stocks used for spinoculation were normalized to x 0 EBV DNA copies /ml after DNAse treatment. Shown is one of three representative experiments. (D-E) Binding efficiency of wild-type and B BMRF- low EBV to either (D) B-cells or (E) CD + monocytes as determined by quantitative PCR detecting EBV DNA copies bound per cell. Data shown are mean ± SD of three independent experiments. moi, multiplicity of infection. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

33 JVI0-0 Version 0 0 FIG.. EBV binding to integrin via BMRF- initiates signaling mandatory for virus entry into memory B-cells. (A) Proposed signal transduction cascade initiated by EBV BMRF- binding to α β integrin. Following binding of BMRF- to cellular α β integrin, a rapid phosphorylation of FAK initiates signal transduction via phosphorylation of the downstream mediators c-src, pα and cofilin and in between activation of the regulatory small GTPase RhoA, analogous to that initiated by binding of fibronectin to α β integrin (0). The resulting actin reorganization then facilitates EBV entry. (B, C) Phosphorylation (p-) of FAK, c-src, pα, and cofilin, following binding of (B) B. wild-type or (C) B-BMRF-low EBV to isolated tonsillar memory B- cells. (D) Initiation of actin filament reorganization (upper panel) triggered by binding of EBV, Fibronectin, heat-denatured EBV, heat-inactivated (h.i.) EBV, or B-BMRF-low EBV to isolated tonsillar memory B-cells. Actin was stained using FITC-Phalloidin. Cells were additionally stained with DAPI (, diamidino--phenylindole) to visualize nuclei (lower panel, merge). Bar, 0µm. FIG.. Activation of the integrin signal transduction pathway is crucial for EBV entry into memory B-cells. (A, B) Inhibition of signaling molecules c-src, PI kinase, FAK, activated downstream of integrin impact the susceptibility of tonsillar memory B-cells to EBV infection using B.EBfaV-GFP viral supernatants. Inhibitors used were AG- (FAK), PP (c-src), Wortmannin and Ly-00 (PIK). Cytochalasin D was included to show the dependence of infection susceptibility of memory B-cells on actin cytoskeleton depolymerization. Cells were counterstained for CD and CD to identify memory B-cells and determine their infection frequency by flow cytometry. Results shown are from one representative experiment out of two experiments. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

34 JVI0-0 Version FIG.. Augmenting β integrin expression on memory B-cells increases their susceptibility to EBV infection and transformation. (A-C) Expression of β integrin on memory B-cells isolated from (A) tonsils or (B, C) peripheral blood without or following h treatment with CD0L plus anti-igm. (D, E) Infection susceptibility to EBV of memory B-cells from (D) tonsils or (E) peripheral blood treated for h as described above as determined by flow cytometry h following inoculation of memory B-cells by B.EBfaV-GFP by spinoculation. Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

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37 anti-gp0 mock treatment anti-gp0 anti-gp (:000) anti-gp (:00) anti-gp (:0) rβ (0.0 µg/ml) rβ (0. µg/ml) rβ ( µg/ml) 0 EGFP + cells / CD + CD + cells (%) D 00 anti-cd mock treatment anti-cd anti-hla-dr,dp,dq anti-αβ (:000) anti-αβ (:00) anti-αβ (:0) anti-αβ (:000) anti-αβ (:00) anti-αβ (:0) anti-cd0 (:000) anti-cd0 (:00) anti-cd0 (:0) 0 EGFP + cells / CD + CD + cells (%) C 00 Tonsil Peripheral blood unsorted sorted unsorted high αβ low αβ high αβ low αβ high α β low αβ 0 0 EGFP + cells / CD + CD + cells (%) EGFP + cells / CD + CD + cells (%) 0 00 A B * 00 ** Figure JVI0-0 Version Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00

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39 JVI0-0 Version Figure Downloaded from jvi.asm.org at Universitaet Zuerich on May, 00