Infection of Primary Human Monocytes by Epstein-Barr VirusMARTIN SAVARD,1 CAROLE BELANGER,1 MELANIE TARDIF,1 PIERRETTE GOURDE,1
LOUIS FLAMAND,2 AND JEAN GOSSELIN1*
Laboratory of Viral Immunology1 and Laboratory of Virology,2 Centre de Recherche en Rhumatologieet Immunologie, Centre de Recherche du CHUL, Universite Laval, Sainte-Foy, Quebec, Canada
Received 9 August 1999/Accepted 17 December 1999
Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and humanimmunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capabilityof Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electronmicroscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolatednuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This wassupported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two earlylytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with thereplicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were alsodetected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negativeresults. Viral replication was also confirmed by the release of newly synthesized infectious viral particles insupernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reducedphagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads.Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functionsmight represent a new mechanism to disrupt the immune response and promote viral propagation during theearly stages of infection.
Epstein-Barr virus (EBV), a member of the Herpesviridaefamily, has long demonstrated its capabilities to adapt andevade host defense mechanisms. While it was mainly believedthat EBV infects only B cells and epithelial cells of the oro-pharynx, there is growing evidence that EBV targeted cells arebroader than initially believed. In fact, recent studies havedemonstrated that EBV can infect thymocytes, as revealed bythe detection of BZLF-1 and EBV nuclear antigen (EBNA)-1transcripts (26). The presence of EBV genome was detected inT lymphocytes and in natural killer cells (21, 24). EBV-infectedfibroblasts obtained from the synovial tissue of a rheumatoidarthritis patient were also found to express EBNA-1, EBNA-2,and latent membrane protein 1 (LMP-1) and to spontaneouslytransform in vitro (29). The presence of EBV genome is alsofrequently detected in Reed-Sternberg cells found in Hodgkin’sdisease patients (11). More recently, it was reported that EBVinfects human neutrophils in vitro through a CD21 receptor-independent pathway and that such an infection leads to pre-mature cell death by apoptosis (5, 32). The clinical relevance ofthis study pertains to the observation that neutrophils frominfectious mononucleosis patients harbor EBV genome (32).
Mononuclear phagocytes play an active role in the defenseof the organism against viral invasion. Rapid recruitment ofmonocytes/macrophages at the site of infection provides animmediate immune response to limit the spread of the virusduring the early stages of infection. Direct elimination of in-fectious pathogens by monocytes/macrophages mostly occursby phagocytosis and the generation of degradative enzymesand reactive oxygen metabolites (31). Monocytic cells also con-
tribute to the generation of a specific antiviral immune responseby acting as antigen-presenting cells to activate cytotoxic andhumoral responses. Impairment in one of these monocyticfunctions could allow viral agents to evade immune response.
Human immunodeficiency virus type 1 (HIV-1) best illus-trates this situation, since several defective monocytic func-tions such as alteration of cell surface antigen expression,abnormal cytokines synthesis, and impaired accessory cellfunction were reported as a result of HIV-1 infection of mono-cytes/macrophages (41, 53). Influenza A virus, which is knownto infect human mononuclear phagocytes, selectively inducesmonocyte-attracting chemokine (46), such as macrophage in-flammatory protein 1a and monocyte chemotactic protein 1. Inthis case, the resulting influx of monocytic cells in infectedtissue may therefore represent a viral strategy to recruit newtarget cells. It was also demonstrated that hepatitis C virusinfects peripheral blood monocytes and suppresses secretion oftumor necrosis factor alpha (TNF-a) and interleukin-1b (IL-1b), two important proinflammatory cytokines playing activeroles in the regulation of the immune response (37).
Little is known about the interactions of EBV with humanmonocytes. First, it was reported that EBV specifically binds tomonocytes through a receptor distinct from CD21 (19). Sec-ond, such interactions were also found to result in the modu-lation of cytokine gene expression, e.g., induction of IL-1 andIL-6 production (18) and suppression of the synthesis ofTNF-a, a pleiotropic cytokine exhibiting antiviral activity (19).Finally, interactions of EBV with monocytes upregulate theformation of important lipid mediators of inflammation, suchas leukotrienes, by a mechanism involving the glycoproteingp350 of the viral envelope (16).
In the present study, we demonstrate that EBV infects andreplicates in human monocytes, a process which is accompa-nied by the suppression of phagocytosis by these cells. Thissuppressive effects caused by EBV may represent another
* Corresponding author. Mailing address: Laboratory of Viral Im-munology, Centre de Recherche en Rhumatologie et Immunologie,CHUQ, Pavillon CHUL, Room T 1-49, 2705 boul. Laurier, Sainte-Foy,Quebec G1V 4G2, Canada. Phone: (418) 654-2772. Fax: (418) 654-2127. E-mail: [email protected].
strategy to affect host defense and promote viral propagationin the early stages of infection.
MATERIALS AND METHODS
Purification of monocytes. Peripheral blood monocytes were isolated by cen-trifugation of heparinized venous blood obtained from healthy donors over aFicoll-Hypaque gradient (Pharmacia, Uppsala, Sweden). Monocytes were thenfurther enriched by Percoll density centrifugation (12) followed by a cell-sortingprocedure (Epics Elite ESP, Coulter Electronics Canada) which resulted in 99%pure monocyte suspension, as assessed by flow cytometry with an anti-CD14monoclonal antibody (Becton Dickinson, Mississauga, Ontario, Canada). Cellviability was .99% as tested by the trypan blue dye exclusion procedure. Mono-cytes and the EBV-negative lymphoid cell lines YAC-1 and BJAB, which are ofmurine and human origin, respectively, were cultured in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum. The culture mediumcontained less than 10 pg/ml of endotoxins, as evaluated by the Limulus amoe-bocyte assay (Sigma, Oakville, Ontario, Canada).
Infection procedure. Viral preparations of EBV strain B95-8 were produced aspreviously described (5). Samples of highly purified monocytes (5 3 106 cells)were preincubated with infectious EBV (105 transforming units) in 1 ml ofculture medium for 1 h at 37°C in order to favor contacts between viral particlesand 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% tryp-sin–0.5 mM EDTA in order to remove any remaining EBV particles adsorbed tothe surface of the cells. Cells were subsequently resuspended (2 3 107 cells/20ml) 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 mg/ml), an in-hibitor of viral DNA polymerase, for 30 min prior to EBV infection.
Electron microscopy. Purified monocytes were incubated with EBV for 5 minat 4°C to promote the binding of viral particles to the cell surface and thencultured at 37°C for time periods varying from 5 to 45 min. Cells were washedonce in HBSS (pH 7.4) and processed for electron microscopy as describedpreviously (32).
DNA isolation from nuclei of monocytes. Monocytes (107 cells) were pre-treated with 10 mmol of cytochalasin B (Sigma, Oakville, Ontario, Canada) perliter, an inhibitor of phagocytosis, for 10 min prior to infection with EBV.Following infection, cells were washed in HBSS and resuspended in 100 ml ofice-cold buffer containing 0.25 M sucrose, 10 mM HEPES, 1 mM EGTA, andprotease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF] and 10 mg[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 at12,000 3 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 remainingRNA was eliminated by treatment with RNase (Promega, Madison, Wis.). Thegenomic DNA was digested with the restriction enzyme BamHI, and the pres-ence of EBV genome was evaluated by PCR amplification with BamHI-Wprimers 59-GCAGTAACAGGTAATCTCTG-39 (position 20124 to 20143) and59-ACCAGAAATAGCTGCAGGAC-39 (position 20523 to 20504), as deducedfrom the viral DNA sequence (3). Two micrograms of DNA was first denaturedat 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 minat 72°C in the presence of 0.2 mM deoxynucleoside triphosphates (dNTPs), 2mM of each primer, 1.7 mM MgCl2, and 2.5 U of Taq DNA polymerase (Pro-mega). 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 glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) with primers and probe previouslydescribed (13).
Southern blot analysis. Enriched monocytes (107 cells) were pretreated for 10min with 10 mmol of cytochalasin B per liter prior to infection with EBV (asdescribed above). Following infection, genomic DNA was isolated (43) andsubjected to Southern blot analysis. Briefly, 10 mg of DNA was digested withBamHI, size fractionated by electrophoresis on a 0.8% agarose gel, and thentransferred 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 labellingsystem (Promega). The hybridization was performed overnight at 42°C in asolution containing 50% formamide, 23 SSC (13 SSC is 0.15 M NaCl plus 0.015M sodium citrate), 10% dextran sulfate, 13 Denhardt’s solution, and 1% sodiumdodecyl sulfate (SDS). After hybridization, membranes were washed at 42°C in23 SSC for 10 min, followed by two washes in 23 SSC–1% SDS for 20 min eachand two stringent washes in 0.23 SSC–1% SDS for 20 min each. The signal wasvisualized by autoradiography. The YAC-1 cell line, which is nonpermissive toEBV 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 culturedfor various periods of time before RNA extraction. Total RNA from monocyteswas isolated with TRIzol reagent (Gibco BRL), according to the manufacturer’sinstructions. Two micrograms of DNase-treated RNA from unstimulated and
EBV-infected monocytes was heated for 5 min at 72°C in the presence of randomhexamers, rapidly cooled on ice, and then reverse transcribed to cDNA with 200U of Moloney murine leukemia virus RT (Promega) in a 25-ml volume contain-ing 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 5min at 94°C, and 5 ml of cDNA samples was subjected to PCR amplification in50 ml of PCR mixture containing 0.2 mM of the appropriate primers (listed inTable 1), 0.2 mM dNTPs, 2.5 U of Taq polymerase, and 1.7 mM MgCl2. PCRamplification conditions were as described above, and PCR products were sep-arated by electrophoresis on a 2% agarose gel, transferred onto a Hybond-Nnylon membrane, and hybridized with g-32P-59-end-labeled internal oligonucle-otide probes detailed in Table 1. PCR amplification of the GAPDH transcriptswas also used as an internal control. To ensure the absence of contaminatinggenomic DNA, RNA samples were directly amplified under the PCR conditionsdescribed above.
Production of EBV particles by human monocytes. Production of infectiousEBV particles from enriched monocytes was performed by infecting 2 3 107 cellswith EBV (4 3 105 transforming units) as described above and kept in culture(75-cm2 flask) for 14 days. The YAC-1 cell line, which is nonpermissive to EBVinfection, was used as a mock control. After the appropriate time of culture, cellsupernatants containing EBV particles were harvested, and viral particles werepurified by ultracentrifugation (25,000 3g for 3 h at 4°C). Pelleted monocyteswere also submitted to freeze-thaw cycles in order to liberate intracellular viralparticles. Virus and mock preparations were resuspended in RPMI-1640 andused to infect permissive BJAB cells. The presence of EBNA in BJAB cells wasmonitored for 4 to 6 days postinfection by indirect immunofluorescence usingEBV-positive reference antisera (38).
Phagocytosis assay. The phagocytic activity of EBV-infected and uninfectedpurified monocytes was assessed by flow cytometry with carboxylated fluores-ceinated microspheres, essentially as described previously (2, 9, 34, 47). For allexperiments, 5 3 105 cells were first washed with 1 ml of phosphate-bufferedsaline (pH 7.4) and resuspended in 350 ml of HBSS supplemented with 5% fetalcalf serum in which 6 3 106 carboxylated fluoresceinated microspheres (1.87 mm;Fluoresbrite, Polysciences, Warrington, Pa.) were added to give a ratio of 12beads/cell. The fluorescent microspheres were examined both microscopicallyand by flow cytometry to insure that there was no agglomeration and membraneadsorption prior to use in uptake experiments. The phagocytosis proceeded at37°C for 2 h with constant shaking (150 rpm). After the incubation, the mixturewas centrifuged at 4°C (1,000 3 g for 3 min) and washed twice with 1 ml of coldphosphate-buffered saline to separate cells from nonphagocytized microspheres.Cells were fixed in 500 ml of 0.5% paraformaldehyde, and 104 cells were analyzedwith an EPICS-XL flow cytometer (Coulter Electronics) at an excitation settingof 488 nm and emission setting of 540 nm. Since the microspheres are smaller indiameter than the monocytes, they can be easily discriminated by light scatter.The percentage of fluorescence-positive monocytes was determined by calculat-ing the number of monocytes containing fluorescent beads per 104 cells ana-lyzed 3 100. The fluorescence distribution was displayed in a histogram whereeach peak corresponded to a definite number of microspheres associated percell. To confirm the results obtained by flow cytometry, each sample was alsoexamined microscopically.
RESULTS
Detection of EBV particles in human monocytes. We havepreviously reported that EBV binds to monocytes, exerts mod-ulatory effects on inflammatory cytokine gene expression (18,19), and primes monocytes for an increased synthesis of leu-kotrienes after stimulation with a second agonist (16). In thepresent study, our objectives were to determine if EBV couldpenetrate into freshly isolated monocytes and if viral transfor-mation and/or replication would occur. First, we evaluated thepresence of EBV particles in monocyte preparations by elec-tron microscopy. Monocytes were incubated with viral particlesfor 45 min at 37°C and then processed for ultrathin sectioningand examination. As shown in Fig. 1, nucleocapsids of EBVwere observed in the cytoplasm and nuclei of approximately20% of monocytes, suggesting that EBV can indeed penetratesuch cells in a phagocytosis-independent manner. EBV nucleo-capsids observed were identical to those seen in the cytoplasmof B95-8 cells.
Detection of EBV genome in nuclei of monocytes. To furtherconfirm the presence of EBV in monocytes, we next attemptedto detect EBV DNA by PCR amplification of a fragmentlocated within the BamHI W region of the EBV genome (3). Inorder to prevent viral internalization by phagocytosis, we firstpretreated monocytes with cytochalasin B, an inhibitor of
phagocytosis prior to EBV infection, and at different periods oftime postinfection, we next extracted viral DNA from isolatednuclei with an equal amount of DNA (1 mg). The presence ofEBV genome could be detected at 5 h postinfection and in-creased from 20 to 40 h postinfection, suggesting that the EBVreplicative cycle was initiated (Fig. 2A). The identity of theamplified PCR products was demonstrated by hybridizationwith an EBV-specific BamHI W probe (data not shown). Ascontrols, nuclei from EBV- and cytochalasin B-treated p815cells (a cell line capable of phagocytosis) were isolated andsubmitted to PCR amplification. No EBV DNA could be am-plified from such cells (data not shown).
The presence of EBV DNA in infected monocytes was fur-ther confirmed by Southern blot analysis of genomic DNAisolated at indicated times postinfection with the 400-bp PCRfragment contained in the BamHI-W internal repeat region asa probe (Fig. 2B). As with the PCR results (Fig. 2A), the 3-kbBamHI-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 lowsensitivity of the Southern blot technique compared to that ofPCR. Importantly, the presence of EBV genome was not de-tected in YAC-1 control cells treated under the same experi-mental conditions as monocytes.
Detection of immediate-early and early lytic transcripts inEBV-infected monocytes. EBV infection may result in the pro-duction and release of new virions or may lead to the immor-talization and/or transformation of B lymphocytes. As EBVgenome was clearly detected in monocytes and as no signs ofcellular transformation were observed, we decided to evaluatethe presence of specific viral mRNA transcripts associated withthe replicative cycle of EBV by RT-PCR analysis. We firstlooked for the presence of three mRNAs: the BZLF-1 tran-script, which is a key immediate-early transactivator of earlyEBV lytic gene expression, and two early replicative cycletranscripts, BALF-2 and BHRF-1. Monocytes were infectedwith EBV, and total RNA was extracted at indicated times andsubmitted 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. Twoother transcripts, BALF-2 (Fig. 3B) and BHRF-1 (Fig. 3C),were detected at 5 h postinfection and also reached a maxi-mum at 20 h. However, the presence of the early antigen (EA)protein could not be detected by immunofluorescence. Takentogether, these results suggest that the EBV replicative cycle isinitiated in infected monocytes.
Detection of EBNA-1 and late lytic transcripts in EBV-in-fected monocytes. Although EBNA proteins are known to haveDNA-binding activity, EBNA-1 and EBNA-2 have very differ-ent biological properties. In fact, EBNA-1 is associated withepisome persistence, acts as a transactivator of latent genes,activates initiation of DNA replication, and has no effect ontumor cell growth (14, 15, 35, 49). In contrast, EBNA-2 isdirectly involved in cellular transformation (39, 45, 52). InEBV-infected monocytes, only EBNA-1 transcripts were de-tected (Fig. 4A) while EBNA-2 transcripts were found to beabsent even after 14 days of infection. The presence of EBNAprotein was also detected (,5%) in EBV-infected monocytesby immunofluorescence. This is in perfect agreement with thefact that no signs of monocyte transformation were observedfollowing EBV infection.
During the replicative cycle, the activation of EBV late lyticgenes is also initiated. These genes code mostly for structuralviral proteins or for proteins that modify the phenotype ofinfected cells. Among the late genes, the major nucleocapsidprotein is encoded by BcLF-1. The presence of BcLF-1 tran-scripts was evaluated at different times post-EBV infection. Asseen in Fig. 4B, these late transcripts were detected after 20 hof infection and were found to increase by 40 h postinfection.As controls, monocytes treated with the viral DNA polymeraseinhibitor 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 immunofluorescenceeven after 2 weeks of culture. On the other hand, flow cytom-etry with anti-gp350 72A1 monoclonal antibodies showed that
TABLE 1. Primers and probes used in RT-PCR analyses
Transcript (protein) Primers and probe Genome coordinates Sequence
BKRF1 (EBNA-1) 59 Primer (U exon) 67483–67502 59-TTAGGAAGCGTTTCTTTGAGC-3939 Primer (K exon) 107986–107967 59-CATTTCCAGGTCCTGTACCT-39Probe (U exon) 67544–67563 59-AGAGAGTAGTCTCAGGGCAT-39
BZLF-1 (ZEBRA) 59 Primer (exon 1) 102719–102700 59-TTCCACAGCCTGCACCAGTG-3939 Primer (exon 2/3 splice) 102330–102341 and
approximately 1% of EBV-infected monocytes expressed EBVglycoprotein gp350 after 14 days of infection (data not shown),suggesting that some monocytes are fully permissive to EBVinfection and replication. EBV glycoprotein gp350 is found on theviral envelope and on the cellular membrane of lytically in-fected cells (40).
The results reported above strongly suggest that EBV rep-licates in human monocytes. To further confirm this observa-tion, we performed an additional experiment. Monocytes wereinfected with EBV and cultured for 14 days to allow viralreplication. The YAC-1 mouse cell line, which is not permis-sive to EBV infection, was also treated with the same cultureconditions and used as mock preparation. Cell supernatantswere then harvested, and viral particles were isolated by ultra-centrifugation. EBV and mock preparations were then used toinfect BJAB cells, and after 4 days of culture, the presence ofEBNA was evaluated by immunofluorescence. The presence ofEBNA-positive cells ('7%) was only detected in BJAB cellcultures treated with viral preparation produced in monocytes.
Effect of EBV on phagocytic activity of monocytes. In addi-tion to producing soluble immunoregulatory molecules, mono-cytes play an active role in the phagocytosis of foreign organ-isms, which is a crucial step towards the presentation ofparticulate antigens in the context of major histocompatibilitycomplex class II. Thus, to investigate further the immunosup-pressive effects caused by EBV, we tested if the phagocyticactivity of monocytes was altered by infection with EBV. Thephagocytic activity was evaluated by measuring the uptake of
fluoresceinated beads by flow cytometry as described in Mate-rials and Methods. This simple, but highly sensitive, methodwas used to determine both the percentage of cells with phago-cytic activity (fluorescence-positive cells) and the average num-ber of beads associated with each positive cell. Figure 5Ashows a kinetic analysis demonstrating that EBV-infectedmonocytes have a reduced capacity to phagocytose fluorescentbeads at 24, 48, and 72 h postinfection with reductions of 40,52, and 73%, respectively. An average reduction of 50% inphagocytic activity was routinely observed with monocytes iso-lated from different healthy donors, and the effect was ob-served for up to 6 days postinfection, after which time both cellviability and their phagocytic ability declined rapidly. Repre-sentative histograms obtained with uninfected and EBV-in-fected monocytes are shown in Fig. 5B and C, respectively. Thesuppressive effect of EBV on phagocytic activity was seen at allfluorescence levels, each peak corresponding to a definitenumber of associated fluorescent microspheres. In this partic-ular example, there was a 36, 55, 66, 80, or 77% reduction influorescent positive cells associated with 1, 2, 3, 4, or more than5 beads, respectively (similar results were obtained with a dif-ferent donor). The probability of coincidence of free beadswith cells as they pass through the flow cytometer was kept ata minimum by following these two conditions: (i) a low ratio ofbeads/cell (12 beads/cells) was used for the assay, and (ii)several washings were performed following the incubation pe-riod to remove any nonphagocytosed beads (2). The suppres-sive effect of EBV was confirmed by another phagocytosis
FIG. 1. EBV infection of human monocytes. Monocytes were incubated on ice in the presence or absence of infectious EBV for 5 min and then cultured at 37°Cfor 45 min. The preparation and examination of samples was performed as described in Materials and Methods. Virions are indicated by arrows (magnification of345,000). Black bar 5 200 nm.
assay which used opsonized zymosan and albumin-fluoresceinisothiocyanate. Although less sensitive, there was at least a50% decrease of fluorescence-positive cells in EBV-treatedmonocytes (data not shown).
DISCUSSION
Despite the fact that monocytes/macrophages constitute thekey elements in nonspecific and specific immune defensesagainst viral infection, very little is known about the interac-tions of EBV with these cell types. In the present study, weclearly demonstrated that EBV penetrates and replicates inmonocytes. First, by using electron microscopy, nucleocapsidsof EBV were observed in the cytoplasm and nuclei of mono-cytes. Second, the presence of EBV genome in nuclei fromcytochalasin B (an inhibitor of phagocytosis)-treated mono-cytes is also of interest. These results indicate that followingviral adsorption, EBV penetrates into monocytes without be-ing phagocytosed. Since CD21 receptor is not expressed on
monocytes, these results reinforce our previous observationssuggesting that EBV recognizes on monocytes a molecule dis-tinct from this receptor.
It is well known that EBV penetration into human B lym-phocytes results in latent or lytic infection in vivo or in cellulartransformation in vitro. The fact that no signs of cellular trans-formation were observed in EBV-enriched monocyte culturessuggests that viral replication may occur. This is in perfectagreement with the presence of immediate-early (BZLF-1)and early lytic (BALF-2 and BHRF-1) transcripts. BZLF-1transcripts appeared within 2 h after viral infection to reach a
FIG. 2. Detection of EBV genome in infected monocytes. Monocytes (107
cells) were treated with the phagocytosis inhibitor cytochalasin B (10 mM) for 10min and infected with EBV for the indicated times. (A) The presence of EBVgenome in purified cell nuclei was evaluated by PCR amplification of the BamHIW fragment, and the resulting 400-bp PCR product was visualized by ethidiumbromide staining on a 2% agarose gel. In some samples, monocytes were alsopretreated with PAA (200 mg/ml) before EBV infection. The Raji cell line wasused as a positive control, and noninfected monocytes were used as negativecontrols. The first lane on the left represents a 100-bp DNA ladder. (B) GenomicDNA isolated from noninfected or EBV-infected monocytes (10 mg) or the Rajicell line (2 mg) was digested with BamHI and subjected to Southern blot analysiswith a 32P-labelled BamHI-W probe. The 3-kb hybridization signal (BamHI-Wfragment) obtained from the Raji cell line and monocytes previously infectedwith EBV for 20 and 40 h (EBV 20h, EBV 40h) are shown. Results are repre-sentative of three different experiments. YAC-1 cells were used as negativecontrols.
FIG. 3. Detection of immediate-early and early transcripts by RT-PCR anal-ysis. Total RNA was isolated from enriched monocytes (107 cells) exposed toEBV for 2 h and cultured for the indicated time periods. Following treatmentwith DNase I, RNA was reverse transcribed and amplified with sets of PCRprimers specific for each gene (see Table 1). The size of the amplified fragmentswas 182 bp for BZLF-1 (A), 285 bp for BALF-2 (B), and 211 bp for BHRF-1 (C).PCR products were hybridized by Southern blot analysis using specific probes.GAPDH cDNA was used as an internal control. Tetradecanoyl phorbol acetate-treated B95-8 cells were used as positive controls, and noninfected monocyteswere used as negative controls. The results presented are from one experimentand are representative of three separate experiments.
maximal level at 20 h postinfection and declined thereafter.Expression of BZLF-1 is transient and encodes the key imme-diate-early Zebra protein essential to activate the lytic cycle.This is in perfect agreement with other studies with EBV-infected Burkitt’s lymphoma tumor cells (Akata cells) in whichthe lytic cycle was induced with anti-immunoglobulin M treat-ment and a transient expression of BZLF-1 was observed (50).Later in the lytic cycle, the BALF-2 gene, which encodes for amajor DNA-binding protein involved in viral DNA replication,and the BHRF-1 gene, which encodes for an early proteinvirtually absent in latently infected B lymphocytes, were alsodetected 5 h postinfection (for a review, see reference 28).
Another interesting result supporting our findings that EBVreplicates in human monocytes is the successful detection oflate lytic BcLF-1 transcripts which encode for the major nu-cleocapsid protein and the detection of gp350 expressed on thesurface membranes of infected monocytes. These genes areexpressed later in the replicative cycle and therefore after theactivation of EBV DNA polymerase. This was nicely supportedby results from the treatment of monocytes with PAA, aninhibitor of viral DNA polymerase, prior to EBV infection. InPAA-treated cultures, BcLF-1 transcripts were not detected,supporting the idea that EBV initiates a complete lytic cycle inmonocytes. While only 1% of EBV-infected monocytes werefound to express gp350, we believe that this percentage under-estimates the reality. Two main reasons may explain this result:first, viral replication may not occur simultaneously in all EBV-
FIG. 4. Detection of EBNA-1 and late lytic transcripts by RT-PCR analysis.Total RNA was isolated from enriched monocytes (107 cells), infected with EBVfor 2 h, and cultured for the indicated periods of time. Following treatment withDNase I, RNA was reverse transcribed and amplified with sets of PCR primersspecific for each gene. The size of the amplified fragments was 212 bp forEBNA-1 (A) and 332 bp for BcLF-1 (B). PCR products were hybridized withspecific probes. GAPDH cDNA was used as an internal control. Tetradecanoylphorbol acetate-treated B95-8 cells were used as positive controls, and nonin-fected monocytes were used as negative controls. Results are from one experi-ment and are representative of three separate experiments.
FIG. 5. Suppression of phagocytosis in EBV-infected monocytes. EBV-in-fected or uninfected monocytes (5 3 105 cells) were incubated with carboxylatedfluoresceinated microspheres (at a ratio of 12 particles/cell), and the uptake offluorescent particles was measured by flow cytometry, as described in Materialsand Methods. (A) The percentage of fluorescence-positive cells (cells associatedwith at least one fluorescent microsphere) for EBV-infected and uninfectedmonocytes was measured at 24, 48, and 72 h postinfection. This time courseanalysis is representative of two experiments performed with two differenthealthy donors. Panels B and C are typical histograms showing the percentage offluorescence-positive cells at each level of fluorescence intensity for uninfectedand infected monocytes, respectively (this experiment was done at 60 h postin-fection). Each peak is related to a definite number of fluorescent microspheres,and the percentage of positive cells contained in each peak is shown in the insert.A total of 104 cells was analyzed for each histogram. Results are representativeof four other experiments.
infected monocytes, and second, in contrast to B lymphocyteswhich express large amount of gp350, infected monocytescould weakly express gp350 on their cellular membrane, thusmaking it hard to detect gp350 by flow cytometric analysis. Thefact that gp350 was never detected in PAA-treated EBV-in-fected monocytes at any time during the experimental proce-dures reinforces the validity of the results obtained. Anotherinteresting result is the presence of EBNA-1 transcripts inEBV-infected monocytes. EBNA-1 is known to bind to the oriPdomain to initiate viral DNA replication or to govern the EBVepisomes in infected cells (14, 15, 35, 49). The absence ofEBNA-2 also reinforces the fact that a lytic cycle is activated inEBV-infected monocytes. EBNA-2 is required for the initia-tion of cell transformation, is readily detectable in the first 24 hof B-cell infection, and reaches maximal levels before EBNA-1can be detected (1, 22, 32, 42). While EBNA-1 transcripts arepresent, EBNA-2 transcripts were never detected in EBV-in-fected monocytes, nor was cellular transformation observed. Inaddition, LMP-1 and -2 transcripts, or protein expression, alsoassociated with latent infection, were not detected by RT-PCR,flow cytometry, or immunoblotting analysis (data not shown).These results, together with the production and the isolation ofinfectious viral particles from EBV-infected monocytes, clearlyconfirm that EBV infects and replicates in human monocytes.
EBV has developed strategies to escape elimination by theimmune system, such as induction of latency, capture of hostgenes, or inhibition of proinflammatory cytokines. One of themain function of monocytes is their ability to internalize for-eign organisms. We show here that EBV-infected monocytesare significantly impaired in their ability to phagocytose. Thesuppressive effect of EBV was observed as early as 24 h postin-fection, at which time a decrease of at least 40% in phagocyticactivity was noted. In contrast, at 2 h postinfection, monocyteswere not functionally compromised in their ability to phago-cytose, suggesting that viral genes expressed at later stages ofthe infection are necessary to cause a defect in phagocytosis.Such impairment of the phagocytosis machinery is expected tobe advantageous for the viral outcome. First, it may directlyfavor the spread of the virus, since phagocytosis is involved inthe elimination of foreign organisms. Second, a downregula-tion of the phagocytic process is likely to interfere with theantigen-presenting capacity of monocytes, which in turn willaffect the immune response of the effector T cells. Concomi-tant alterations of both phagocytosis and antigen-presentingprocesses have already been reported with monocytes infectedwith HIV or bovine respiratory syncytial virus (4, 6, 7, 25). Inaddition, human herpesviruses, including cytomegalovirus, hu-man herpesvirus 6, and human herpesvirus 8, have been shownto infect primary human monocytes/macrophages.
The mechanisms by which EBV affects phagocytosis remainto be elucidated. Possible alterations in the expression of Fcgand complement receptors, as demonstrated for HIV-1 (27),are currently being investigated. Perhaps deregulation of ty-rosine activation motif-mediated phagocytosis by Fcg recep-tors is a potential means by which EBV could disrupt phago-cytic activity (for a review, see reference 20). As well, there aremultiple transmembrane signals aside from protein-tyrosinephosphorylation which could be involved in phagocytosis, in-cluding protein kinase C, protein kinase A, casein kinase II,and as-yet-unidentified serine-threonine protein kinases (20,51). Thomas et al. (51) reported that the impairment of Fcreceptor-mediated phagocytosis in HIV-1 infected promono-cytic cells was associated with an increased accumulation ofcyclic AMP which could be relieved by the addition of aninhibitor of cyclic AMP-dependent protein kinase A. Whether
EBV uses similar mechanisms to deregulate phagocytosis re-mains to be established.
In previous studies, we have demonstrated that EBV inter-acts with premyelomonocytic cell lines, such as U937 and HL-60, as well as with human monocytes and was able to modulatecytokine synthesis (17, 19). Indeed, upon EBV interaction withthese cells, we observed that IL-1 and IL-6 gene transcriptionwas activated, whereas that of TNF-a was inhibited. SinceTNF-a is known to exert antiviral activities, we postulated thatthe suppression of TNF-a release may favor the spread ofinfection. Knowing that the protein coded by the BARF-1 geneof EBV can neutralize the activity of colony-stimulating factor1 and block alpha interferon secretion in mononuclear cells(10, 48), we tested for BARF-1 expression in EBV-infectedmonocytes. No BARF-1 transcripts could be detected in EBV-infected monocytes which might have accounted for the pre-viously reported TNF-a suppression (19). Taken together, wecan postulate that targeting monocytes/macrophages may rep-resent an evolutionary advantage for ensuring propagation andpersistence of EBV and other herpesviruses within the host (8,30, 33, 36). This was reinforced by another study which pre-sented evidence of EBV replication in macrophages (44). Cul-tured macrophages obtained from patients with benign or ma-lignant neoplasms and from healthy donors were kept inculture for several weeks. The authors found EBV genome anddetected latent gene expression (EBNA-2 and LMP-1) in thosecultures. The presence of EBNA-2 might have facilitated thenumber of passages performed in vitro and delayed the de-crease of cell viability. In our case, EBNA-2 transcripts werealways absent in all cultures, which could explain why no cel-lular growth was observed. The most surprising results fromthe study by Shimakage et al. (44) is the presence of such latentgenes in macrophages from normal tissues and the induction ofreplicative-associated proteins after treatment with tumor pro-moter in vitro, indicating that macrophages could be a sourcefor latent infection. Whether macrophages express a higherlevel of EBV-specific genes than monocytes and whether EBVexists in a different replicative state in macrophages versusmonocytes remain to be elucidated. However, this study pro-vides additional indications that monocytes/macrophages mayserve as reservoirs of EBV infection.
We have demonstrated that EBV infects, replicates in hu-man monocytes, and significantly reduces the ability of thesecells to phagocytose. It was long established that immunosup-pression is a key factor for the persistence of EBV within thehost. Such effects on monocytes may then contribute to thespread of the virus but also may affect the antigen presentationby reducing incorporation of foreign antigens.
ACKNOWLEDGMENTS
This work was supported by a Medical Research Council of Canadagrant to J.G. J.G. and L.F. are recipients of FRSQ and MRC schol-arships, respectively.
We thank Pierrette Cote for excellent secretarial assistance.
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