Infection of Primary Human Monocytes by Epstein-Barr Virus

Purification of monocytes.

Peripheral blood monocytes were isolated by centrifugation of heparinized venous blood obtained from healthy donors over a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). Monocytes were then further enriched by Percoll density centrifugation (12) followed by a cell-sorting procedure (Epics Elite ESP, Coulter Electronics Canada) which resulted in 99% pure monocyte suspension, as assessed by flow cytometry with an anti-CD14 monoclonal antibody (Becton Dickinson, Mississauga, Ontario, Canada). Cell viability was >99% as tested by the trypan blue dye exclusion procedure. Monocytes and the EBV-negative lymphoid cell lines YAC-1 and BJAB, which are of murine and human origin, respectively, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. The culture medium contained less than 10 pg/ml of endotoxins, as evaluated by the Limulus amoebocyte assay (Sigma, Oakville, Ontario, Canada).

Infection procedure.

Viral preparations of EBV strain B95-8 were produced as previously described (5). Samples of highly purified monocytes (5 × 10⁶ cells) were preincubated with infectious EBV (10⁵ transforming units) in 1 ml of culture medium for 1 h at 37°C in order to favor contacts between viral particles and cells. Cells were then washed three times in Hanks' balanced salt solution (HBSS) (pH 7.4) and further trypsinized with a solution containing 0.05% trypsin–0.5 mM EDTA in order to remove any remaining EBV particles adsorbed to the surface of the cells. Cells were subsequently resuspended (2 × 10⁷ cells/20 ml) in culture medium and cultured in a 75-cm² tissue culture flask (Falcon, Mississauga, Ontario, Canada) for varying periods of time. When indicated, monocytes were treated with phosphonoacetic acid (PAA) (200 μg/ml), an inhibitor of viral DNA polymerase, for 30 min prior to EBV infection.

Electron microscopy.

Purified monocytes were incubated with EBV for 5 min at 4°C to promote the binding of viral particles to the cell surface and then cultured at 37°C for time periods varying from 5 to 45 min. Cells were washed once in HBSS (pH 7.4) and processed for electron microscopy as described previously (32).

DNA isolation from nuclei of monocytes.

Monocytes (10⁷ cells) were pretreated with 10 μmol of cytochalasin B (Sigma, Oakville, Ontario, Canada) per liter, an inhibitor of phagocytosis, for 10 min prior to infection with EBV. Following infection, cells were washed in HBSS and resuspended in 100 μl of ice-cold buffer containing 0.25 M sucrose, 10 mM HEPES, 1 mM EGTA, and protease inhibitors (1 μM phenylmethylsulfonyl fluoride [PMSF] and 10 μg [each] of leupeptin and aprotinin per ml). Monocytes were then sonicated on ice (20 s, at a power setting of 2 and 60% duty cycle) in a Branson Sonifier 450 (VWR/Canlab, Montreal, Quebec, Canada), sonicates were centrifuged at 12,000 × g for 10 min at 4°C, and the pelleted nuclei were resuspended in HBSS. Genomic DNA was isolated as previously described (43), and any remaining RNA was eliminated by treatment with RNase (Promega, Madison, Wis.). The genomic DNA was digested with the restriction enzyme BamHI, and the presence of EBV genome was evaluated by PCR amplification with BamHI-W primers 5′-GCAGTAACAGGTAATCTCTG-3′ (position 20124 to 20143) and 5′-ACCAGAAATAGCTGCAGGAC-3′ (position 20523 to 20504), as deduced from the viral DNA sequence (3). Two micrograms of DNA was first denatured at 94°C for 2 min and then subjected to 35 amplification cycles as follows: denaturation for 1 min at 94°C, annealing for 1 min at 55°C, extension for 1 min at 72°C in the presence of 0.2 mM deoxynucleoside triphosphates (dNTPs), 2 mM of each primer, 1.7 mM MgCl₂, and 2.5 U of Taq DNA polymerase (Promega). PCR products were visualized by ethidium bromide staining on 2% agarose gels and confirmed by hybridization with a specific BamHI W probe (23). DNA integrity was confirmed by amplification and hybridization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primers and probe previously described (13).

Southern blot analysis.

Enriched monocytes (10⁷ cells) were pretreated for 10 min with 10 μmol of cytochalasin B per liter prior to infection with EBV (as described above). Following infection, genomic DNA was isolated (43) and subjected to Southern blot analysis. Briefly, 10 μg of DNA was digested with BamHI, size fractionated by electrophoresis on a 0.8% agarose gel, and then transferred onto a GeneScreen Plus membrane (NEN Life Science Products, Boston, Mass.) for hybridization with the 400-bp BamHI-W PCR fragment (described above) labelled with [³²P]dCTP with the Prime-a-Gene labelling system (Promega). The hybridization was performed overnight at 42°C in a solution containing 50% formamide, 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10% dextran sulfate, 1× Denhardt's solution, and 1% sodium dodecyl sulfate (SDS). After hybridization, membranes were washed at 42°C in 2× SSC for 10 min, followed by two washes in 2× SSC–1% SDS for 20 min each and two stringent washes in 0.2× SSC–1% SDS for 20 min each. The signal was visualized by autoradiography. The YAC-1 cell line, which is nonpermissive to EBV infection, was used as a control.

RNA isolation and amplification of viral transcripts by reverse transcriptase (RT)-PCR.

Unstimulated and EBV-infected monocytes (10⁷ cells) were cultured for various periods of time before RNA extraction. Total RNA from monocytes was isolated with TRIzol reagent (Gibco BRL), according to the manufacturer's instructions. Two micrograms of DNase-treated RNA from unstimulated and EBV-infected monocytes was heated for 5 min at 72°C in the presence of random hexamers, rapidly cooled on ice, and then reverse transcribed to cDNA with 200 U of Moloney murine leukemia virus RT (Promega) in a 25-μl volume containing 0.5 mM dNTPs and 20 U of RNase inhibitor (Boehringer Mannheim, Laval, Quebec, Canada). After 60 min of incubation at 42°C, samples were boiled for 5 min at 94°C, and 5 μl of cDNA samples was subjected to PCR amplification in 50 μl of PCR mixture containing 0.2 mM of the appropriate primers (listed in Table 1), 0.2 mM dNTPs, 2.5 U of Taq polymerase, and 1.7 mM MgCl₂. PCR amplification conditions were as described above, and PCR products were separated by electrophoresis on a 2% agarose gel, transferred onto a Hybond-N nylon membrane, and hybridized with γ-³²P-5′-end-labeled internal oligonucleotide probes detailed in Table 1. PCR amplification of the GAPDH transcripts was also used as an internal control. To ensure the absence of contaminating genomic DNA, RNA samples were directly amplified under the PCR conditions described above.

Production of EBV particles by human monocytes.

Production of infectious EBV particles from enriched monocytes was performed by infecting 2 × 10⁷ cells with EBV (4 × 10⁵ transforming units) as described above and kept in culture (75-cm² flask) for 14 days. The YAC-1 cell line, which is nonpermissive to EBV infection, was used as a mock control. After the appropriate time of culture, cell supernatants containing EBV particles were harvested, and viral particles were purified by ultracentrifugation (25,000 ×g for 3 h at 4°C). Pelleted monocytes were also submitted to freeze-thaw cycles in order to liberate intracellular viral particles. Virus and mock preparations were resuspended in RPMI-1640 and used to infect permissive BJAB cells. The presence of EBNA in BJAB cells was monitored for 4 to 6 days postinfection by indirect immunofluorescence using EBV-positive reference antisera (38).

Phagocytosis assay.

The phagocytic activity of EBV-infected and uninfected purified monocytes was assessed by flow cytometry with carboxylated fluoresceinated microspheres, essentially as described previously (2, 9, 34, 47). For all experiments, 5 × 10⁵ cells were first washed with 1 ml of phosphate-buffered saline (pH 7.4) and resuspended in 350 μl of HBSS supplemented with 5% fetal calf serum in which 6 × 10⁶ carboxylated fluoresceinated microspheres (1.87 μm; Fluoresbrite, Polysciences, Warrington, Pa.) were added to give a ratio of 12 beads/cell. The fluorescent microspheres were examined both microscopically and by flow cytometry to insure that there was no agglomeration and membrane adsorption prior to use in uptake experiments. The phagocytosis proceeded at 37°C for 2 h with constant shaking (150 rpm). After the incubation, the mixture was centrifuged at 4°C (1,000 × g for 3 min) and washed twice with 1 ml of cold phosphate-buffered saline to separate cells from nonphagocytized microspheres. Cells were fixed in 500 μl of 0.5% paraformaldehyde, and 10⁴ cells were analyzed with an EPICS-XL flow cytometer (Coulter Electronics) at an excitation setting of 488 nm and emission setting of 540 nm. Since the microspheres are smaller in diameter than the monocytes, they can be easily discriminated by light scatter. The percentage of fluorescence-positive monocytes was determined by calculating the number of monocytes containing fluorescent beads per 10⁴ cells analyzed × 100. The fluorescence distribution was displayed in a histogram where each peak corresponded to a definite number of microspheres associated per cell. To confirm the results obtained by flow cytometry, each sample was also examined microscopically.

Detection of EBV particles in human monocytes.

We have previously reported that EBV binds to monocytes, exerts modulatory effects on inflammatory cytokine gene expression (18, 19), and primes monocytes for an increased synthesis of leukotrienes after stimulation with a second agonist (16). In the present study, our objectives were to determine if EBV could penetrate into freshly isolated monocytes and if viral transformation and/or replication would occur. First, we evaluated the presence of EBV particles in monocyte preparations by electron microscopy. Monocytes were incubated with viral particles for 45 min at 37°C and then processed for ultrathin sectioning and examination. As shown in Fig. 1, nucleocapsids of EBV were observed in the cytoplasm and nuclei of approximately 20% of monocytes, suggesting that EBV can indeed penetrate such cells in a phagocytosis-independent manner. EBV nucleocapsids observed were identical to those seen in the cytoplasm of B95-8 cells.

Detection of EBV genome in nuclei of monocytes.

To further confirm the presence of EBV in monocytes, we next attempted to detect EBV DNA by PCR amplification of a fragment located within the BamHI W region of the EBV genome (3). In order to prevent viral internalization by phagocytosis, we first pretreated monocytes with cytochalasin B, an inhibitor of phagocytosis prior to EBV infection, and at different periods of time postinfection, we next extracted viral DNA from isolated nuclei with an equal amount of DNA (1 μg). The presence of EBV genome could be detected at 5 h postinfection and increased from 20 to 40 h postinfection, suggesting that the EBV replicative cycle was initiated (Fig. 2A). The identity of the amplified PCR products was demonstrated by hybridization with an EBV-specific BamHI W probe (data not shown). As controls, nuclei from EBV- and cytochalasin B-treated p815 cells (a cell line capable of phagocytosis) were isolated and submitted to PCR amplification. No EBV DNA could be amplified from such cells (data not shown).

The presence of EBV DNA in infected monocytes was further confirmed by Southern blot analysis of genomic DNA isolated at indicated times postinfection with the 400-bp PCR fragment contained in the BamHI-W internal repeat region as a probe (Fig. 2B). As with the PCR results (Fig. 2A), the 3-kb BamHI-W signal was detectable at 20 and 40 h postinfection, thereby confirming the presence of viral DNA in monocytes. The decrease in the signal intensity is likely due to the low sensitivity of the Southern blot technique compared to that of PCR. Importantly, the presence of EBV genome was not detected in YAC-1 control cells treated under the same experimental conditions as monocytes.

Detection of immediate-early and early lytic transcripts in EBV-infected monocytes.

EBV infection may result in the production and release of new virions or may lead to the immortalization and/or transformation of B lymphocytes. As EBV genome was clearly detected in monocytes and as no signs of cellular transformation were observed, we decided to evaluate the presence of specific viral mRNA transcripts associated with the replicative cycle of EBV by RT-PCR analysis. We first looked for the presence of three mRNAs: the BZLF-1 transcript, which is a key immediate-early transactivator of early EBV lytic gene expression, and two early replicative cycle transcripts, BALF-2 and BHRF-1. Monocytes were infected with EBV, and total RNA was extracted at indicated times and submitted to RT-PCR analysis. As shown in Fig. 3A, BZLF-1 transcripts were detectable as early as 2 h postinfection, reached a maximum at 20 h, and declined thereafter. Two other transcripts, BALF-2 (Fig. 3B) and BHRF-1 (Fig. 3C), were detected at 5 h postinfection and also reached a maximum at 20 h. However, the presence of the early antigen (EA) protein could not be detected by immunofluorescence. Taken together, these results suggest that the EBV replicative cycle is initiated in infected monocytes.

Detection of EBNA-1 and late lytic transcripts in EBV-infected monocytes.

Although EBNA proteins are known to have DNA-binding activity, EBNA-1 and EBNA-2 have very different biological properties. In fact, EBNA-1 is associated with episome persistence, acts as a transactivator of latent genes, activates initiation of DNA replication, and has no effect on tumor cell growth (14, 15, 35, 49). In contrast, EBNA-2 is directly involved in cellular transformation (39, 45, 52). In EBV-infected monocytes, only EBNA-1 transcripts were detected (Fig. 4A) while EBNA-2 transcripts were found to be absent even after 14 days of infection. The presence of EBNA protein was also detected (<5%) in EBV-infected monocytes by immunofluorescence. This is in perfect agreement with the fact that no signs of monocyte transformation were observed following EBV infection.

During the replicative cycle, the activation of EBV late lytic genes is also initiated. These genes code mostly for structural viral proteins or for proteins that modify the phenotype of infected cells. Among the late genes, the major nucleocapsid protein is encoded by BcLF-1. The presence of BcLF-1 transcripts was evaluated at different times post-EBV infection. As seen in Fig. 4B, these late transcripts were detected after 20 h of infection and were found to increase by 40 h postinfection. As controls, monocytes treated with the viral DNA polymerase inhibitor PAA were tested for BcLF-1 expression. As expected (Fig. 4B), no BcLF-1 transcripts could be detected in PAA-treated cells. However, the presence of viral capsid antigen (VCA) protein could not be detected by immunofluorescence even after 2 weeks of culture. On the other hand, flow cytometry with anti-gp350 72A1 monoclonal antibodies showed that approximately 1% of EBV-infected monocytes expressed EBV glycoprotein gp350 after 14 days of infection (data not shown), suggesting that some monocytes are fully permissive to EBV infection and replication. EBV glycoprotein gp350 is found on the viral envelope and on the cellular membrane of lytically infected cells (40).

The results reported above strongly suggest that EBV replicates in human monocytes. To further confirm this observation, we performed an additional experiment. Monocytes were infected with EBV and cultured for 14 days to allow viral replication. The YAC-1 mouse cell line, which is not permissive to EBV infection, was also treated with the same culture conditions and used as mock preparation. Cell supernatants were then harvested, and viral particles were isolated by ultracentrifugation. EBV and mock preparations were then used to infect BJAB cells, and after 4 days of culture, the presence of EBNA was evaluated by immunofluorescence. The presence of EBNA-positive cells (≈7%) was only detected in BJAB cell cultures treated with viral preparation produced in monocytes.

Effect of EBV on phagocytic activity of monocytes.

In addition to producing soluble immunoregulatory molecules, monocytes play an active role in the phagocytosis of foreign organisms, which is a crucial step towards the presentation of particulate antigens in the context of major histocompatibility complex class II. Thus, to investigate further the immunosuppressive effects caused by EBV, we tested if the phagocytic activity of monocytes was altered by infection with EBV. The phagocytic activity was evaluated by measuring the uptake of fluoresceinated beads by flow cytometry as described in Materials and Methods. This simple, but highly sensitive, method was used to determine both the percentage of cells with phagocytic activity (fluorescence-positive cells) and the average number of beads associated with each positive cell. Figure 5A shows a kinetic analysis demonstrating that EBV-infected monocytes have a reduced capacity to phagocytose fluorescent beads at 24, 48, and 72 h postinfection with reductions of 40, 52, and 73%, respectively. An average reduction of 50% in phagocytic activity was routinely observed with monocytes isolated from different healthy donors, and the effect was observed for up to 6 days postinfection, after which time both cell viability and their phagocytic ability declined rapidly. Representative histograms obtained with uninfected and EBV-infected monocytes are shown in Fig. 5B and C, respectively. The suppressive effect of EBV on phagocytic activity was seen at all fluorescence levels, each peak corresponding to a definite number of associated fluorescent microspheres. In this particular example, there was a 36, 55, 66, 80, or 77% reduction in fluorescent positive cells associated with 1, 2, 3, 4, or more than 5 beads, respectively (similar results were obtained with a different donor). The probability of coincidence of free beads with cells as they pass through the flow cytometer was kept at a minimum by following these two conditions: (i) a low ratio of beads/cell (12 beads/cells) was used for the assay, and (ii) several washings were performed following the incubation period to remove any nonphagocytosed beads (2). The suppressive effect of EBV was confirmed by another phagocytosis assay which used opsonized zymosan and albumin-fluorescein isothiocyanate. Although less sensitive, there was at least a 50% decrease of fluorescence-positive cells in EBV-treated monocytes (data not shown).