INTRODUCTION. Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, UK

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1 Journal of General Virology (24), 85, DOI 1.199/vir Capacity of Epstein Barr virus to infect monocytes and inhibit their development into dendritic cells is affected by the cell type supporting virus replication Andre Ortlieb Guerreiro-Cacais, 1 3 LiQi Li, 1 3 Daria Donati, 2 Maria Teresa Bejarano, 1,2 Andrew Morgan, 3 Maria G. Masucci, 1 Lindsey Hutt-Fletcher 4 and Victor Levitsky 1 Correspondence Victor Levitsky Victor.Levitsky@mtc.ki.se 1,2 Microbiology and Tumor Biology Center 1 and Center for Infectious Medicine, Huddinnge Hospital 2, Karolinska Institutet, Nobels väg 16, S Stockholm, Sweden 3 Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, UK 4 Department of Microbiology and Immunology, Louisiana State University, Health Science Center, Shreveport, LA, USA Received 28 March 24 Accepted 24 June 24 Epstein Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular components of the innate immune system are poorly characterized in terms of their function in the development of EBV-specific immunity or interaction with the virus. This study demonstrates that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different ratios of the gp42 and gh glycoproteins in the envelope of virions that were derived from major histocompatibility complex class II-positive or -negative cells accounted primarily for the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation. The purified gh/gl heterodimer binds efficiently to monocytes and DCs, but not to B cells, suggesting that high expression levels of a putative binding partner for gh contribute to virus entry. This entry takes place despite very low or undetectable expression of CD21, the canonical EBV receptor. These results indicate that the site of virus replication, either in B cells or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the virus s immunomodulating capacity and may have important implications for the regulation of virus host interactions during primary and chronic EBV infection. INTRODUCTION Epstein Barr virus (EBV), a ubiquitous human herpesvirus, causes infectious mononucleosis and is associated with a number of malignancies of different cellular origins (Rickinson & Kieff, 1996). EBV establishes latent infection predominantly, if not exclusively, in B lymphocytes (Thorley-Lawson et al., 1996). B lymphocytes also represent the only documented site of EBV replication in healthy virus carriers (Tao et al., 1995). Whether EBV replicates in oropharyngeal epithelial cells during primary infection remains a matter of controversy (Sixbey et al., 1984; 3These authors contributed equally to this work. Niedobitek et al., 1997, 2). It is clear, however, that EBV can replicate in polarized epithelial cells in vitro and in immunocompromised individuals with hairy leukoplakia, a benign, hyperplastic, epithelial lesion that is composed of EBV-infected cells producing large amounts of the virus (Greenspan et al., 1985; Conant, 1987). Specific T lymphocytes inhibit growth of EBV-transformed B cells in vitro. In vivo, T cells suppress virus reactivation and EBV-induced tumorigenesis, as demonstrated by the effects that they mediate upon adoptive transfer to immunodeficient patients (Rooney et al., 1995; Heslop et al., 1996; Heslop & Rooney, 1997; Gustafsson et al., 2). Little is known, however, about the mechanisms 8-14 G 24 SGM Printed in Great Britain 2767 On: Tue, 13 Nov 218 :42:19

2 A. O. Guerreiro-Cacais and others that are responsible for the induction of EBV-specific immunity and the capacity of the virus to interfere with this process. Antigen presentation by dendritic cells (DCs) is believed to be required for primary T-cell stimulation. Consistent with the critical function of DCs in anti-virus immunity, many viruses are known to infect different subsets of DCs and affect their differentiation, survival, migration and/or T cellstimulatory capacity (reviewed by Becker, 23; Kobelt et al., 23; Schneider-Schaulies et al., 23; Sevilla et al., 23; Steinman et al., 23). DCs were recently shown to crosspresent virus antigens that are derived from EBV-infected cells and to be necessary for inhibition of EBV-induced B- cell transformation in lymphocyte cultures that were established from EBV-seronegative, but not -seropositive, individuals (Subklewe et al., 21; Bickham et al., 23). These findings support a critical role for DCs in the induction of primary T-cell anti-virus responses. Whilst other human herpesviruses, such as cytomegalovirus (CMV) and herpes simplex virus, have been shown to inhibit the T cell-stimulatory function of DCs by a number of mechanisms, the capacity of EBV to interact with, and affect the function of, DCs or their precursors is poorly characterized. We have recently shown that EBV infection inhibits DC development from monocyte precursors in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL4), in a manner that is apparently independent of de novo expression of viral genes (Li et al., 22). It remains unclear, however, whether the virus is capable of entering monocytes or developing DCs and how the tropism of the virus for these cells is regulated at the molecular level. Cellular tropism of herpesviruses is determined by virusencoded proteins in the virion envelope that interact with various cellular receptors (reviewed by Spear & Longnecker, 23). Recent studies have shown that the canonical EBV receptor, CD21, which interacts with the major glycoprotein of the EBV envelope, gp35, is neither sufficient nor essential for B-cell infection and that fusion of the EBV envelope with the cell membrane requires components of the trimolecular complex, which includes glycoproteins gp85, gp42, and gp25 (Li et al., 1997; Wang & Hutt-Fletcher, 1998; Haan et al., 2). Abundance of another EBV envelope glycoprotein, gb (also referred to as gp11), has recently been shown to influence EBV tropism for different cell types (Neuhierl et al., 22). Whilst major histocompatibiliy complex (MHC) class II molecules have been identified as the cellular ligand of gp42, the interaction partners of gp85 and gp25 are currently unknown. These EBV proteins are homologous, respectively, to the gh and gl components of the herpes simplex virus and CMV membrane fusion complexes (Yaswen et al., 1993), are noncovalently associated in the virus envelope and are often referred to as the gh/gl heterodimer. The importance of the trimolecular assembly in EBV infection is demonstrated by the inability of recombinant viruses that are devoid of gp42 or gh to infect B cells (Wang & Hutt-Fletcher, 1998; Oda et al., 2). However, the presence of gp42 in the virus envelope impedes infection of epithelial cells that are infected efficiently by gp42-deficient virions (Wang et al., 1998). These data suggest that different modes of receptor ligand interactions are operational during EBV infection of different cell types (Wang et al., 1998). Thus, the bimolecular gh/gl complex mediates epithelial cell infection, whereas the trimolecular gp42/gh/gl complex is required for infection of B cells. The content of the biversus trimolecular complexes in the EBV envelope and the tropism of EBV virions can be affected by the type of cell that is supporting virus replication. Expression of human leukocyte antigen class II results in reduced levels of gp42 in the virion, due to association of these two proteins inside the cell and degradation of gp42 in the endosomal/lysosomal compartment. This may play an important role in the virus life cycle. Thus, EBV replication in epithelial cells generates virions that infect B cells preferentially, thereby establishing the EBV-carrier state in the infected individual, whereas EBV that is produced in B cells may be more efficient at mediating virus spread, due to more efficient infection of epithelial cells (Borza & Hutt-Fletcher, 22). The effect of EBV envelope composition on its interaction with monocytes or DCs has not been analysed. In this study, we demonstrate that, compared to B lymphocyte-produced virions, EBV from epithelial cells exhibits a significantly increased capacity to infect monocytes and inhibit their development into DCs. These findings may have important implications for our understanding of the immunoregulation and pathogenesis of EBV infection. METHODS Recombinant virus strains and virion isolation. All EBV virions used in the study were derived from the EBV strain originating from Akata, a Burkitt s lymphoma cell line. Generation of viruses carrying a cassette that encodes both neomycin resistance and green fluorescent protein (GFP), inserted into the open reading frame of the non-essential tyrosine kinase gene, has been described previously (Molesworth et al., 2). EBV-negative Akata and gastric carcinoma AGS cells infected with recombinant viruses were grown in medium that was supplemented with 5 mg geneticin sulphate ml 21 (G418) (Sigma Aldrich). AGS cells expressing the MHC class II transactivator (AGS CIITA cells) were grown in the presence of?4 mg puromycin ml 21 (Borza & Hutt-Fletcher, 22). Recombinant Akata virus (B-EBV) was produced by inducing the EBV lytic cycle in Akata cells with anti-human IgG, as described previously (Wang & Hutt-Fletcher, 1998). Virus lytic cycle in AGS (E-EBV) and AGS CIITA (E-II-EBV) cells was induced by incubating the cells for 24 h with 3 ng 12-O-tetradecanoylphorbol-13-acetate ml 21 and?5 mm sodium butyrate (Calbiochem). These chemicals were removed by exchange of culture medium and virus-containing supernatants were harvested 5 days later. Viruses were concentrated from filtered supernatants (?8 mm) by centrifugation at 16 g for 9 min at 4 uc and the pellets were resuspended in RPMI medium with 1 mg bacitracin ml 21 (Sigma). Mock preparations were produced from EBV-negative AGS cells, following the same procedures Journal of General Virology 85 On: Tue, 13 Nov 218 :42:19

3 Altering tropism of EBV for monocytes and DCs Quantification of EBV virions. DNA slot-blot analysis and measurement of cell-associated virus by fluorescence-activated cell sorting (FACS) analysis or real-time PCR were used to equalize virus stocks for virion content. Details of the virus-binding assay and FACS analysis for both virus quantification and comparison of envelope glycoproteins are described below. Slot-blot analysis was performed as described previously (Wang & Hutt-Fletcher, 1998). The quantity of EBV DNA in virion particles was measured by hybridization with a 32 P-labelled BamHI W fragment of EBV DNA by means of a random-primed DNA labelling kit (Boehringer Mannheim). Samples (2 ml of a 16 concentrated virus stock) were digested for 1 min at room temperature with DNase I.?5 M EDTA (1 ml) was added to stop digestion and virus particles were sedimented for 1 h at 16 g. Sedimented virus was digested overnight at 56 uc with proteinase K (2?5 mgml 21 in?2 M EDTA) and serial dilutions were made in PBS. Sample DNA was denatured by the addition of?1 vol. 5 N NaOH, neutralized with 2 M ammonium acetate, applied to a nylon membrane and cross-linked by UV irradiation. Hybridizations were carried out as described previously (Mackett et al., 199) and quantified by scanning with a Storm PhosphorImager (Molecular Dynamics). EBV-negative Akata cells were incubated with different dilutions of virus stocks for 1 h at 4 uc to determine the number of virions that were capable of binding to CD21-positive cells. Cells were washed three times in cold PBS and DNA was extracted with a QIAamp DNA blood mini kit (Qiagen), eluted in sterile water and stored at 22 uc. The TaqMan PCR reagent system and ABI PRISM 77 sequence detection system (Applied Biosystems) were used to measure EBV DNA (Heid et al., 1996). The EBV target gene chosen was latent membrane protein (LMP)1 and the amplification primer sequences were: forward primer, 59-AAGGTCAAAGAACAAGGCCAAG-39; reverse primer, 59- GCATCGGAGTCGGTGGG-39; probe, 59-AGGAGCGTGTCCCCGT- GGAGG-39. The probe was labelled with 6-carboxyfluorescein (FAM; reporter) and 6-carboxytetramethylrhodamine (TAMRA; quencher). A standard curve was created to calculate the number of EBV genomes (3) by using serial dilutions of DNA extracted from the EBV-positive human cell line Namalwa, which carries two integrated EBV copies per cellular genome (Klein et al., 1972). The standard and the DNA samples were run in triplicate. Amplification mixtures (25 ml) contained 16 TaqMan Universal PCR master mix (Applied Biosystems) and 9 nm of each EBV primer, 2 nm EBV probe, water and 5 ml sample DNA. Cycling parameters were as follows: 5 uc for 2 min, 95 uc for 1 min and 45 cycles of 95 uc for 15 s and 6 uc for 1 min. Preparation of monocytes and DCs and EBV infection. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by centrifugation on Ficoll density gradients. Monocytes were purified from PBMCs by centrifugation on Percoll density gradients, as described by Kouwenhoven et al. (21). Alternatively, CD14 + monocytes were purified by using a monocyte-negative isolation kit (Dynal Biotech) or MACS CD14 MicroBeads (Miltenyi Biotec) according to the manufacturers instructions. The negatively selected population contained >98 % CD14 + cells, as determined by staining and FACS analysis. Monocytes were resuspended in culture medium (RPMI 164 medium supplemented with 1 % heat-inactivated fetal calf serum, 2 mm L-glutamine, 1 U penicillin ml 21 and 1 mg streptomycin ml 21 )at a density of cells ml 21 and cultured for the indicated time in medium supplemented with 1 U recombinant GM-CSF ml 21 and 1 U recombinant IL4 ml 21 (a kind gift from the Schering- Plough Research Institute, New Jersey, USA). Changes in cell morphology were monitored daily by visual inspection using an inverted microscope. EBV infection was performed by resuspending monocytes in culture medium containing 1 arbitrary unit (AU) (see below) of the indicated EBV preparation. Cells were incubated for 2 h at 37 uc, washed and cultured in medium that contained IL4 and GM-CSF to induce DC development. EBV-specific antibodies and binding assays. The mabs F-2-1 (Strnad et al., 1982), which is specific for the EBV envelope glycoprotein gp42 (Li et al., 1995), E1D1 (Oba & Hutt-Fletcher, 1988) [reacting with the EBV gh/gl complex (Li et al., 1995)], 72A1 (Hoffman et al., 198), specific for the EBV gp35/22 glycoprotein, and CL59 (Molesworth et al., 2), specific for gh, were purified from culture supernatants of the corresponding hybridoma cell lines. The mab 5B2, which is specific for gb, was purchased from Virusys. FACS analysis of virus bound to the surface of Burkitt s lymphoma Raji cells, which express high levels of the CD21 molecule, was used to measure virus in different preparations and analyse the content of different glycoproteins in the EBV envelope. To compare virus stocks, Raji cells were fixed in?1 % paraformaldehyde, resuspended in 1 ml RPMI medium that contained the indicated EBV preparation and incubated for 1 h. Cells were washed in PBS and incubated in 5 ml 2L1 gp35-specific mab or isotype control antibody diluted in PBS. Antibody binding was revealed by allophycocyanin (APC)-conjugated goat anti-mouse antibody (BD Biosciences) or R-phycoerythrin (R-PE)-conjugated rabbit anti-mouse antibody (Dako). All procedures were carried out on ice. Cells that had not been exposed to EBV were used as a control. The amount of virus that generated a mean fluorescence intensity (MFI) value with gp35-specific antibody in the binding assay that was comparable to that obtained with 1 ml B95.8 supernatant was expressed as 1 AU of EBV virions. B95.8 virus (?5 AU) was used in binding assays that were performed for equalization of virus stocks. To analyse the protein composition of the envelope, EBV was reduced to?25 AU, virus glycoprotein-specific antibodies were used at 1 mg per sample (2?5 mg per sample for mab 5B2) and the biotinylated anti-mouse antibody and PE- or APC-conjugated streptavidin were used at a 1 : 1 dilution (all from BD Biosciences). Detection of apoptosis and GFP expression. Efficiency of Annexin V binding to the surface of EBV-infected and control cells was measured by using an Annexin V PE apoptosis detection kit I (Pharmingen). GFP expression in cells infected by the recombinant EBV strains was monitored either by measuring the intensity of green fluorescence in infected cells using FACS analysis or by immunoblotting of total cell lysates using a mixture of two GFP-specific mabs, 7.1 and 13.1 (Roche). gh/gl binding assay. gh was co-expressed with gl in SF9 insect cells and precipitated as a gh/gl heterodimer. SF9 cells were infected at an m.o.i. of 3 with baculovirus expressing gh/gl (Pulford et al., 1995). Five days later, the culture medium was clarified by low-speed centrifugation to remove cells and then centrifuged at 16 g for 9 min to remove virus. PEG 35 was added to a final concentration of 2 % (w/v), the solution was stirred for 1 h at 4 uc and then centrifuged at 14 g for 2 min. The pellet was resuspended in?1 vol. PBS and the precipitated gh/gl was dialysed against PBS in dialysis tubing with a molecular mass cut-off of 25 Da, as described previously (Pulford et al., 1995). The indicated cells were resuspended at cells in 2 ml PBS containing 1 % serum from an EBV-seronegative individual and were incubated on ice for 1 h in the presence of 5 mg purified gh/gl. Where indicated, 1 mg E1D1 mab or isotype control antibody were added to the reaction. Cells were then washed in PBS and gh/gl binding was revealed by CL59 mab staining, followed by incubation with anti-mouse IgG fluorescein isothiocyanate (FITC)- labelled secondary antibody. Cells were washed and analysed by FACS On: Tue, 13 Nov 218 :42:19

4 A. O. Guerreiro-Cacais and others RESULTS EBV virions produced in different cell types differ in their capacity to induce apoptosis in developing DCs The same recombinant EBV strain, which was generated by using virus that was originally derived from the EBVpositive Burkitt s lymphoma cell line Akata, has been produced in either Akata cells, gastric carcinoma AGS cells or AGS CIITA cells that express MHC class II molecules constitutively. These virus preparations, referred hereafter to as B-EBV (for B cells), E-EBV (for epithelial cells) or E-II-EBV, respectively, were normalized according to the intensity of gp35-specific staining in a virus-binding assay using CD21 + Raji cells. The number of virions in each virus stock was expressed in AU, as described in Methods. The amount of gp35 has previously been shown to correlate with viral DNA content (Neuhierl et al., 22). In confirmation, we detected comparable amounts of viral DNA in the normalized virion preparations by using DNA slotblot analysis (data not shown). Purified monocytes were infected for 2 h (1 AU in monocytes) with E-EBV, E-II-EBV or B-EBV and cultured in the presence of IL4 and GM-CSF to stimulate their differentiation into DCs. To assess the pro-apoptotic activity of different EBV preparations, cells were stained with Annexin V and analysed by flow cytometry. After infection with E-EBV, the proportion of apoptotic cells in developing DC cultures reached 5 % on day 3 and >7 % on day 5 post-infection. In contrast, only 9?9 and 6?4% of cells were apoptotic in control cultures at the same time points. Infection with B-EBV only slightly increased the proportion of Annexin V-positive cells, whilst E-II-EBV exhibited an intermediate activity, as compared with B- EBV and E-EBV (Fig. 1). Similar relative pro-apoptotic activities of different EBV virions were revealed by monitoring the recovery of viable cells in DC cultures at day 6 or 7 post-infection (Table 1). Composition of virion envelope in different EBV preparations We set out to analyse the expression of virus-encoded proteins in the envelope of EBV virions, in order to uncover why there were differences in the pro-apoptotic activity of EBV preparations. We could not exclude, however, that the presence of gp35-containing membrane-like structures that were devoid of viral genomes, non-enveloped capsids in concentrated virus preparations or variability in the number of repeats present in the BamHI W genome fragment affected our evaluation of virion content in EBV stocks. To circumvent these potential problems, we used an EBV-specific real-time PCR to compare the numbers of EBV genomes that were associated with EBV-negative Akata cells after preincubation with different virion preparations that had been equalized for gp35 content. This analysis revealed that the number of virions that were able to bind to CD21 was similar in E-EBV and E-II-EBV preparations, whereas B-EBV stocks contained approximately 2?5-fold more virus genomes, suggesting that these virions have a relatively decreased amount of gp Fig. 1. EBV virions produced in different cell lines differ in their capacity to induce apoptosis in developing DCs. Viruses from supernatants of AGS (E-EBV), AGS-CIITA (E-II-EBV) and Akata (B-EBV) cells were concentrated by ultracentrifugation and numbers of virions in the resulting preparations were equalized by using a binding assay to CD21 + cells and slot-blot analysis. Purified monocytes were infected with 1 AU of the indicated virions, cultured in the presence of GM-CSF and IL4 and tested for their capacity to bind PE-conjugated Annexin V on day 3 or 5 post-infection. Control cells were mock-infected with preparations obtained from EBV-negative AGS cells as described in Methods. The results shown are one representative of five experiments performed with cells isolated from different donors. 277 Journal of General Virology 85 On: Tue, 13 Nov 218 :42:19

5 Altering tropism of EBV for monocytes and DCs Table 1. Recovery of DCs in cultures of monocytes infected with EBV virions produced in different cell lines Purified monocytes were infected with the indicated preparations of EBV (the cell lines used to produce the relevant virus are indicated in parentheses) and cultured in the presence of GM-CSF and IL4. Recovery of viable cells was monitored at day 6 or 7 of culture. NT, Not tested. EBV virion type DC recovery (%) in experiment no.*: Mean±SD B-EBV (Akata) NT ?3 5?3 88?5 81±19 E-EBV (AGS) 14?3 34?8 35?5 1?7 14?4 26?2 22?6±11 E-II-EBV (AGSCIITA) 22?5 46?4 63?2 38? ?8 43?2±15?3 *Results of six independent experiments are shown as a percentage of viable cells relative to the number of cells in cultures of mock-infected monocytes. (Fig. 2a). In fact, B-EBV, E-EBV and E-II-EBV exhibited similar gb contents after normalization according to the number of genomes, as measured by real-time PCR. Some of the stocks of B-EBV contained up to twofold higher levels of gh, as compared to E-EBV or E-II-EBV (Fig. 2b), but this difference was not observed with every virus preparation (Fig. 2d and data not shown). In contrast, the level of gp35-specific signal was decreased consistently in B-EBV virions by two- to threefold. Normalization of virus stocks by using gp35 signal intensity resulted in a corresponding increase of fluorescence intensity obtained with gh or gb specific antibody in a binding assay with B-EBV (Fig. 2b). Analysis of independent experiments demonstrated that the ratio between the intensities of ghand gp35-specific signals was, on average, 2?5-fold higher in B-EBV than in E-EBV or E-II-EBV (Fig. 2d). In addition, E-EBV and E-II-EBV contained comparable amounts of gp35 and gh, whereas the level of gp42 was significantly decreased in E-II-EBV, resulting in an approximately twofold increase in the gh : gp42 ratio. B-EBV expressed a barely detectable level of gp42 that was reflected in a fivefold increase in the gh : gp42 ratio (Fig. 2c, e). Collectively, these data demonstrated that the three types of EBV virions contained comparable amounts of gh and gb in the virion envelope, the content of gp35 was approximately 2?5-fold lower in B cell-derived virions than in E-EBV- and E-II-EBV-derived virions and the content of gp42 was decreased to a different extent in B-EBV and E-II-EBV, as compared to E-EBV. Decreased gp42 content and decreased pro-apoptotic activity of EBV virions correlates with their impaired capacity to enter monocytes To test to what extent the lower inhibitory activity of B-EBV and E-II-EBV can be compensated for by increasing the m.o.i., monocytes were infected with E-EBV (1 AU) or with the indicated increasing amounts of the other two virus preparations. Induction of apoptosis in developing DCs was monitored by Annexin V staining on day 4 postinfection. In this and all subsequent experiments, numbers of virions were normalized by using the gp35 staining intensity in the virus-binding assay, which underestimated the number of B-EBV genomes by approximately 2?5-fold. We have chosen to use gp35-based normalization as purified gp35 was shown to mediate a number of biological and biochemical changes in human monocytes (D Addario et al., 1999). Infection of monocytes with 1 AU B-EBV did not induce any significant increase in the proportion of Annexin V-positive cells in DC cultures. The proportion of apoptotic cells increased gradually with increasing amounts of B-EBV used for infection, but even cultures that were infected with 9 AU B-EBV (approx fold more EBV genomes than in 1 AU E-EBV) contained relatively fewer apoptotic cells than cultures that were infected with 1 AU E- EBV (Fig. 3a). Infection of monocytes with 1 AU E-II-EBV resulted in only a slight increase in the percentage of apoptotic cells (1?4-fold), whereas the numbers increased by 1?7- and 2?2-fold after infection with 2 or 3 AU E-II-EBV, respectively. The latter was comparable with the proapoptotic activity of 1 AU E-EBV (Fig. 3b). A similar pattern of relative pro-apoptotic activities in the three virus preparations was observed by monitoring the recovery of viable cells in infected DC cultures (data not shown). The virus used in this study contains a cassette that confers both neomycin resistance and GFP expression; the latter can be used for monitoring virus entry. Total cell lysates of monocytes that were infected with 1 AU of the indicated viruses were tested by immunoblotting with GFP-specific antibodies to compare the efficiency of virus entry 48 h after infection. At this time point, the highest intensity of GFP fluorescence was detected in E-EBV-infected monocytes and Raji cells by using time kinetics experiments (data not shown). GFP-specific band intensity was significantly stronger in E-EBV-infected than in E-II-EBVinfected monocytes, whereas the level of GFP expression was below the detection limit in B-EBV-infected cells (Fig. 3c). Expression of GFP in monocytes that were infected with B-EBV increased with the increaing virus dose that was used for infection. However, the level of GFP expression in monocytes that were infected with 9 AU On: Tue, 13 Nov 218 :42:19

6 A. O. Guerreiro-Cacais and others FL2-H FL2-H FL2-H B-EBV was still significantly lower than that observed in cells infected with 1 AU E-EBV (Fig. 3d). Next, we used FACS analysis to determine the proportion of monocytes that were susceptible to EBV entry. In different preparations of monocytes, a large proportion (>6 %) of cells that were infected with 1 AU E-EBV became positive for green fluorescence, as assessed by FACS analysis. A lower proportion of positive cells with a lower MFI was detected in E-II-EBV-infected cells, whereas monocytes that were infected with B-EBV at the same m.o.i. were negative or only marginally positive for GFP expression (Fig. 3e,f). Immature DCs remain permissive for EBV entry In our previous study, we showed that immature DCs become resistant to the pro-apoptotic effect of the virus at day 5 6 of their development in culture in vitro (Li et al., 22). To analyse whether the capacity of EBV virions 2772 Journal of General Virology 85 On: Tue, 13 Nov 218 :42:19

7 Altering tropism of EBV for monocytes and DCs Fig. 2. Protein composition of the envelope differs in EBV virions isolated from different cell lines. Raji cells were preincubated with each of the EBV virions indicated. The gp35, gh and gp42 contents in the virus envelope were estimated by using the relevant specific mabs. Binding of antibodies to cell surface-bound EBV was revealed by sequential incubation with biotinylated anti-mouse antibody and APC-conjugated streptavidin. (a) The left panel shows histogram plots obtained by FACS analysis of Raji cells pre-incubated with the indicated preparations of virions and detected by gp35-specific antibody. MFIs are indicated and the numbers in parentheses are the same values expressed as percentages relative to the MFI obtained with E-EBV. The same virus stocks were diluted, as indicated, in the graph on the right panel and pre-incubated with EBV-negative Akata cells for 1 h at 4 6C. DNA was extracted from cells after washing away unbound virus and the number of cell-associated virus genomes was evaluated by EBV-specific real-time PCR. (b) gp35, gh and gb contents were analysed for E-EBV (X), E-II-EBV (&) and B-EBV (m), using the relevant specific antibodies in the EBV-binding assay. B-EBV was used at?5 and?2 AU, respectively corresponding to the virion normalization based on either gp35 content or genome copy numbers. (c) The histogram plots show comparative analysis of the gp35 (dotted line), gh (thin line) and gp42 (thick line) content in the indicated virion preparations. (d) Ratios between MFIs obtained in the virus-binding assay with the gh- or gp35-specific antibody and calculated for each of the indicated virion preparations are shown as the mean±sd of four independent experiments. (e) Ratio of MFIs obtained with the gh- or gp42-specific antibodies was calculated and presented as described for (d). to inhibit DC development correlates with the capacity of EBV virions to enter cells, we infected monocytes or developing DCs with B-EBV, E-EBV, or E-II-EBV at days, 3 or 6 of culture in vitro in the presence of GM-CSF and IL4. Efficiency of EBV entry was assessed by monitoring green fluorescence intensity in monocytes 48 h after infection. Infection with 1 AU B-EBV resulted in only a slight increase in the MFI of infected cells, compared to controls (Fig. 4). In contrast, the majority of cells that were infected with either E-EBV or E-II-EBV became positive for green fluorescence. Infection with E-EBV was more efficient than with E-II-EBV, as judged by GFP signal intensity in infected cells (Figs 3 and 4), which was consistent with the differences in their capacity to inhibit DC development. This pattern was observed in cells that were infected at different time points Fig. 3. Comparative analysis of EBV virions for their pro-apoptotic activity in developing DCs and capacity to enter monocytes. Freshly isolated monocytes were infected with the indicated amounts of E-EBV, E-II- EBV or B-EBV and cultured in the presence of GM-CSF and IL4. Pro-apoptotic activity of EBV virions was assessed by Annexin V staining on day 5 of culture (a, b) and their capacity to enter monocytes was evaluated by monitoring GFP expression, either in total cell lysates by immunoblotting with GFPspecific antibodies (c, d) or by direct measurement of GFP-dependent fluorescence (e, f). In (c), B95.8 virus was used as an additional negative control. The histograms in (e) show green fluorescence of monocytes non-infected or infected with 1 AU of the indicated viruses. (f) Percentages of cells falling into the M1 region and the numbers above the graph represent MFIs of non-gated populations shown in (e). Each panel shows the results of one representative of two or three experiments On: Tue, 13 Nov 218 :42:19

8 A. O. Guerreiro-Cacais and others FL1-H Day (a) gh+e1d1 gh FL1-H FL1-H Day 3 Day 6 Fig. 4. Immature monocyte-derived DCs remain permissive for EBV entry. E-EBV (thick line), E-II-EBV (grey line) or B-EBV (thin line) were used to infect monocytes at the indicated days of culture in the presence of GM-CSF and IL4. GFP expression levels were monitored in mock-infected (control; grey shading) or EBV-infected cells by FACS analysis. Results are one representative of three experiments FL1-H (b) gh+e1d1 gh FL1-H (c) 5 4 gh+e1d1 3 gh FL1-H Developing DCs express a binding partner for soluble gh/gl Experiments with gp42- and gh-deficient EBV strains demonstrated that these two components of the virus fusion complex are required for EBV-induced apoptosis of monocyte-derived DCs (Li et al., 22). Whilst MHC class II molecules (which are expressed abundantly on monocytes) have been characterized as the binding partner of gp42, the identity and cellular distribution of structures that interact with gh are unknown. To test whether an interacting partner of gh is expressed on developing DCs, AGS cells, Akata cells and monocytes cultured overnight in GM-CSF and IL4 were incubated with soluble gh/gl. Protein binding to the cell surface was revealed by the gh-specific CL59 mab and the specificity of the binding was confirmed by the ability of E1D1 ghspecific blocking mab, but not control mab, to reduce the Fig. 5. Soluble gh/gl binds efficiently to AGS cells and developing DCs. EBV-negative Akata (a) and AGS (b) cells and purified monocytes cultured in the presence of GM-CSF and IL4 (developing DCs) (c) were incubated with purified soluble gh/gl for 3 min on ice (? cells in 5 ml, containing 5 mg gh/gl) in the absence (solid dark line) or the presence (solid grey line) of E1D1 antibody (5 mg). Binding of gh/gl was revealed by incubation with CL59 antibody followed by incubation with anti-mouse FITC-conjugated antibody. Filled and dotted histograms represent control samples cultured without gh/gl or with gh/gl-specific antibody alone, respectively. One representative of three experiments is shown. MFI of gh/gl-stained cells. As shown in Fig. 5, both AGS cells and developing DCs bound soluble gh/gl with comparable efficiency, whereas binding of the gh/gl protein to Akata cells was barely detectable Journal of General Virology 85 On: Tue, 13 Nov 218 :42:19

9 Altering tropism of EBV for monocytes and DCs DISCUSSION The results of this study demonstrate that the capacity of EBV to inhibit development of monocytes into DCs in the presence of GM-CSF and IL4 is influenced strongly by the cell type supporting virus replication. Virions that were produced in the Burkitt s lymphoma cell line Akata (B-EBV) exhibited significantly reduced pro-apoptotic activity on developing DCs, as compared to EBV virions that were derived from a cell line of epithelial origin (E-EBV). In addition, our data show that E-EBV efficiently enters the majority of monocytes and developing DCs, as demonstrated by the expression of the GFP gene, which was inserted into the virus genome. These results seem to contradict those of our previous publication, which suggested that the inhibitory activity of the virus is independent of viral gene expression (Li et al., 22). However, it should be taken into account that GFP expression in EBV-infected monocytes is driven by a heterologous CMV promoter and EBV promoters may remain silent in monocytes/dcs. In agreement with our previous data, short-uv inactivation of B-EBV or E-EBV abrogated induction of EBV nuclear antigens in B cells following virus infection, but did not affect the capacity of the virions to inhibit DC development (data not shown). Although we cannot exclude the possibility that not all open reading frames of the EBV genome were inactivated by UV treatment, in our previous study we failed to detect viral gene expression in infected monocytes or DCs by using RT-PCR analyses (Li et al., 22). Collectively, our results suggest that the effect of the virus is mediated by virion components during virus entry or post-entry events. In agreement with this hypothesis, the inhibitory activity of EBV virions correlated directly with the m.o.i. Therefore, accurate determination of virion content in different virus preparations was critical for estimating their relative activities. A good correlation was observed between the measurements of virion content, based either on the levels of gp35 in cell surface-associated virus or the amount of viral DNA in concentrated virus stocks, as assessed by DNA slot-blot analysis and according to previously published data (Neuhierl et al., 22). However, some dissociation between these parameters was revealed when the number of CD21-bound genomes was estimated by EBV-specific real-time PCR. After adjustment for the intensity of gp35-specific staining, preparations of B-EBV were shown to contain two- to threefold higher numbers of EBV genomes than preparations of E-EBV or E-II-EBV. Further analysis of protein composition of the virus envelope demonstrated that all EBV preparations had comparable amounts of gh and gb, whereas gp35 content was two- to threefold lower in B cell-derived virions (Fig. 2). The reasons for this decrease remain unclear, but it is conceivable that gp35 may be partially retained in B cells, due to an interaction with CD21, as has been described for the interaction of gp42 with MHC class II molecules (Borza & Hutt-Fletcher, 22). Nevertheless, it is unlikely that decreased amounts of gp35 contribute to the differences in virus tropism and activity described in this study. Firstly, we compared the biological activity and entry efficiency of different virions by using normalization of virus stocks based on gp35 content, which did not compensate for the lower activity of B-EBV. We failed to detect expression of CD21 on monocytes (Li et al., 2) and to block virus entry or its pro-apoptotic activity by using a gp35-specific blocking antibody (Li et al., 22). Finally, virions produced from AGS cells that constitutively express MHC class II molecules, E-II-EBV, had the same gp35 content, but were less efficient at entering monocytes and inhibiting DC development than E-EBV. The only molecular change detected that could explain the difference between E-EBV and E-II-EBV was the decreased gp42 content in the latter type of virions. Although E-II-EBV contained significantly higher amounts of gp42 in the virion envelope than B-EBV, its pro-apoptotic activity and tropism for monocytes were significantly lower than those of E-EBV. Notably, virions that were derived from the B95.8 EBV-producing cell line resemble E-EBV virions in their trimolecular complex composition and inhibitory activity on DC development [data not shown and Li et al., (22)], although the reason for high gp42 content in B95.8 virions remains unclear, as the virus is produced from monkey B cells. These results indicate that the trimolecular fusion complex, which is composed of gp42, gh and gl, plays a critical role in mediating EBV entry into monocytes and in the pro-apoptotic effects of the virus on DC development. The data suggest that, as in B-cell infection, the trimolecular gp42/gh/gl fusion complex mediates EBV infection of monocytes/immature DCs by involving the interaction of gp42 with MHC class II molecules. High expression levels of a putative binding partner for the gh/gl heterodimer, revealed by binding assays with purified proteins (Fig. 5), are likely to contribute to the efficient entry of E-EBV into monocytes. Previous studies have demonstrated that EBV infection of monocytes results in a number of biological effects (Gosselin et al., 1991, 1992; Roberge et al., 1997; Shimakage et al., 1999; Savard et al., 2a, b; Masy et al., 22; Tardif et al., 22) that have been attributed either to virus entryindependent effects or infection of a small proportion of cells. In this respect, our finding that E-EBV can infect virtually all cells in monocyte cultures is quite remarkable. The relatively high capacity of EBV from epithelial cells to inhibit DC development from monocyte precursors efficiently may play an important role in the regulation of virus host interactions. EBV spreads in the human population through the oral route of infection and cells of the oral or nasopharyngeal epithelium represent its most likely entry site. This hypothesis is supported by a recent demonstration of EBV replication in polarized in vitro cultures of non-transformed human epithelial cells (Tugizov et al., 23). In vivo, replication of the virus in epithelial cells is observed in hairy leukoplakia [reviewed by Rickinson & Kieff (1996)], as well as in histologically normal epithelium of severely immunocompromised patients (Herrmann et al., 22), and is likely to occur during primary EBV On: Tue, 13 Nov 218 :42:19

10 A. O. Guerreiro-Cacais and others infection in individuals lacking virus-specific immunity. One arbitrary unit of EBV used in our experiments corresponded to 2 virions per cell on average, an m.o.i. that seems to be easily achievable at a site of EBV replication in vivo. Although strong humoral and cellular immune responses are eventually elicited against the virus, EBV infection is asymptomatic in the majority of infected individuals. Even in rare cases of infectious mononucleosis, a lag period of several weeks is frequently observed between the infection episode and appearance of clinical symptoms of the disease, which may be caused by the pathological side effects of extensive anti-virus immunity (Peter & Ray, 1998). DCs are critical for efficient induction of primary immune responses and inhibition of their function is likely to delay the development of both cellular and humoral specific immunity in EBV-infected individuals. In agreement with this model, sites of hairy leukoplakia lack morphological and histological signs of inflammation and have a decreased content of Langerhans cells (Daniels et al., 1987; Walling et al., 24); this may be explained by the ability of EBV virions, which are produced abundantly in leukoplakia lesions, to exert an inhibitory effect on DCs. In contrast, reactivation of the virus in latently infected B cells of immune, chronic virus carriers should result in the production of EBV virions with a significantly decreased capacity to infect and modulate the function of monocytes and DCs, which would be more compatible with asymptomatic and non-pathogenic EBV persistence. DCs that develop from monocyte precursors in the presence of IL4 and GM-CSF gradually lose their sensitivity to the pro-apoptotic effects of EBV infection. Whether this reflects decreased infectivity of immature DCs by EBV, their decreased general sensitivity to apoptosis or changes in the regulation of signalling pathways mediated by EBV infection was unclear. Here, we show that immature DCs that become resistant to EBV-induced apoptosis still support virus entry, as demonstrated by GFP expression conferred by infection with recombinant EBV strains (Fig. 4). Therefore, resistance to EBV-induced apoptosis can be accounted for by differentiation-associated changes in developing DCs. However, the ability of EBV to infect immature DCs raises the possibility that the virus can influence phenotypic and functional characteristics of these cells without affecting their lifespan. In addition to a number of EBV-associated malignancies and infectious mononucleosis, EBV has been suggested to play a role in the pathogenesis of many other diseases, such as rheumatoid arthritis, histiocytosis, multiple sclerosis, meningitis, encephalitis and peripheral neuropathia, based primarily on the results of epidemiological studies and analysis of EBV serology (Scotet et al., 1996; Rubin & Daube, 1999; Takeda et al., 2; Ascherio et al., 21; Kleinschmidt-DeMasters & Gilden, 21). Involvement of the virus in some of these pathological conditions has been difficult to prove, as EBV replication or latent infection is often not detected in the tissues or cell types that are affected in a given disease. Our results suggest that EBV replication can be involved in disease pathogenesis indirectly through effects that are mediated by EBV virions independently of viral gene expression. 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M., Granelli-Piperno, A., Pope, M., Trumpfheller, C., Ignatius, R., Arrode, G., Racz, P. & Tenner-Racz, K. (23). The On: Tue, 13 Nov 218 :42:19