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We previously showed that the EBV glycoprotein BMRF-2 contains a functional integrin-binding Arg–Gly–Asp (RGD) domain that plays an important role in viral infection and cell-to-cell spread of progeny virions in oral epithelial cells. In this study, we found that EBV-seropositive human sera contain antibodies against BMRF-2. The inhibitory effect of EBV-positive sera on EBV infection of oral epithelial cells was substantially reduced by pre-incubation of serum samples with the BMRF-2 RGD peptide, suggesting that anti-BMRF-2 human antibodies possess neutralizing activity. EBV-specific sera reacted strongly with the BMRF-2 extracellular domain (170-213 aa) containing the RGD motif, whereas they reacted only weakly or not at all with a mutated form of the BMRF-2 extracellular domain containing AAA instead of RGD. These data indicate that that RGD motif of BMRF-2 is part of an immunodominant antigenic determinant within the extracellular domain of BMRF-2 that may contribute to EBV neutralization during EBV reactivation.
Epstein-Barr virus (EBV), a member of the herpes virus family and one of the most common human viruses, is associated with cancers of both lymphoid and epithelial origin. EBV-related diseases include Burkitt’s lymphoma, Hodgkin’s disease, some adult T-cell lymphomas, infectious mononucleosis, nasopharyngeal carcinoma (NPC), gastric carcinoma and oral hairy leukoplakia (HL)(Rickinson and Kieff, 2007). More than 90% of the adult human population are infected with EBV and are carriers of the virus, which exists in a latent state under normal immune surveillance (Rickinson and Kieff, 2007).
In addition to cytotoxic CD8+ cells, which are the major players in immune surveillance against EBV (Chapman et al., 2001; de Jong et al., 2000; McAulay et al., 2009; Rickinson and Moss, 1997; Savoldo et al., 2001; Savoldo et al., 2007), EBV-specific antibodies also play a role in controlling EBV infection (Beisel et al., 1985; Epstein and Morgan, 1983; Finerty et al., 1994; Finerty et al., 1992). The most abundant antibodies against EBV proteins target the viral capsid antigens (VCA) (Pearson et al., 1983; Ringborg et al., 1983), the early antigen diffuse (EA-D), and the early antigen restricted (EA-R) (Henle et al., 1971), which are expressed during the viral lytic cycle (Chapman et al., 2001). Antibody responses to VCAs and early antigens serve as important criteria for the diagnosis of EBV infection and understanding of its pathogenesis (Khanna, Burrows, and Moss, 1995). It has also been shown that antibodies directed against the EBV glycoproteins gp350/220 and gp85 have neutralizing activity, suggesting a role for these in the clearance of reactivated EBV (Khanna, Burrows, and Moss, 1995; Sashihara et al., 2009; Thorley-Lawson and Geilinger, 1980). EBVgp350 has been considered a candidate for a prophylactic EBV vaccine (Morgan, 1992), and immunization of seronegative children with recombinant gp350 has been shown to result in the development of neutralizing antibodies (Gu et al., 1995). Recently reported clinical trials have also shown that the EBV gp350 vaccine was immunogenic and safe, suggesting that it may be used for the prevention of EBV in seronegative individuals (Moutschen et al., 2007; Sokal et al., 2007). Antibody responses may also develop against EBV latent proteins, including Epstein-Barr nuclear antigen 1 (EBNA1) and EBNA2 (Rowe et al., 1988); however, these do not have significant neutralizing activity (Moss et al., 1992).
EBV BMRF-2 is a multi-span transmembrane protein and is incorporated into the viral envelope (Johannsen et al., 2004; Tugizov, Berline, and Palefsky, 2003; Xiao et al., 2007). Its extracellular domain contains an RGD motif, which interacts with β1- and αv-family integrins of oral epithelial cells, thereby facilitating EBV entry and spread via basolateral membranes of polarized oral epithelial cells, where integrins are expressed (Xiao et al., 2008; Xiao et al., 2009; Xiao et al., 2007). The BMRF-2 open reading frame (ORF) is conserved throughout the gamma herpes virus family (Coulter and Reid, 2002; May et al., 2005a; May et al., 2005b). EBV BMRF-2 forms a protein complex with the viral protein BDLF-2 and facilitates the translocation of BDLF-2 to the cell surface (Gore and Hutt-Fletcher, 2008; Loesing et al., 2009). BMRF-2 and BDLF-2 are highly expressed in a benign lesion of the oral epithelium known as hairy leukoplakia (HL) (Hayes et al., 1999; Palefsky et al., 1997; Peñaranda et al., 1997; Xiao et al., 2007), which occurs primarily in persons with HIV-associated immunodeficiency (Greenspan and Greenspan, 1997; Greenspan et al., 1987; Greenspan et al., 1985). The presence of BMRF-2/BDLF-2 on cell membranes induces the formation of membrane protrusions and cellular processes that may facilitate cell-to-cell spread of virus (Gill et al., 2008; Loesing et al., 2009).
It has been shown that the RGD motifs of integrin-binding proteins in parechoviruses, coxsackievirus A9, foot-and-mouth disease virus and enteroviruses may generate immunodominant epitopes that substantially increase the immunogenicity of viral proteins (Joki-Korpela et al., 2000; Liebermann et al., 1991; Mason, Rieder, and Baxt, 1994). Furthermore, the immunogenicity of peptide antigens containing the RGD motifs were found to be ten times stronger than that of peptides without RGD motifs (Shimakage et al., 2003), indicating that RGD motifs may be components of immunodominant epitopes. Previously we showed that human sera from EBV-seropositive individuals significantly reduce BMRF-2 RGD-mediated EBV infection via the basolateral membranes of polarized epithelial cells (Tugizov, Berline, and Palefsky, 2003), suggesting that the BMRF-2 RGD domain serves as an immunodominant antigenic determinant for the humoral immune response and that antibodies to this domain may have neutralizing activity against EBV. Therefore, our goal in the current study was to determine whether EBV-seropositive humans have neutralizing antibodies against BMRF-2 and to examine the role of the BMRF-2 RGD motif in an anti-BMRF-2 immune response. We expressed BMRF-2 in B-lymphoblastoid and epithelial cells and measured the specific interaction of serum samples with BMRF-2 from three groups of EBV-seropositive people: HIV-negative EBV-asymptomatic individuals, HIV-negative NPC patients and HIV-positive individuals with HL. We found that all EBV-seropositive individuals have antibodies against BMRF-2 and that the immunodominant antigenic epitope (s) is located within the RGD-containing extracellular domain of BMRF-2.
We collected 15 serum samples from the following: 6 samples from HIV-negative, healthy individuals without any clinical symptoms of EBV-associated disease (asymptomatic serum samples, AS); 4 samples from HIV-negative patients with NPC (NPC samples); and 5 samples from HIV-infected patients with oral HL lesions (HL samples). To detect EBV-specific antibodies in human serum samples, we performed enzyme-linked immunosorbent assay (ELISA) using three different EBV proteins as test antigens: VCAs (p125 and p18), EA-D and EBNA-1. All serum samples were examined for the presence of the following antibody isotype and antigen specificity combinations: IgG anti-VCA, IgM anti-VCA, IgA anti-VCA, IgG anti-EAD and IgG anti-EBNA-1. As shown in Table 1, all serum samples were positive for both VCA p18 and EBNA-1, whereas none of the samples tested positive for IgM anti-VCA p125 antibodies. IgA anti-VCA p18 antibodies were found in only one of the six (17%) serum samples from asymptomatic individuals, three of the four (75%) NPC serum samples, and two of the five (40%) HL serum samples. IgG anti-VCA p18 antibodies were found in all of the serum samples, and IgG anti-EBNA-1 antibodies were present in all but two samples (Table 1). ). IgG anti–E-AD antibodies were detected in three of the NPC samples, one of the HL samples, and three of the samples from asymptomatic individuals. Thus, all serum samples were found to contain antibodies against EBV proteins, and the absence of IgM indicated that none of the donors was infected recently. However, detection of IgA anti-VCA p18 and IgG anti- E-AD in NPC and HL patients suggests the possible reactivation of virus in these donors.
Since EBV has a tropism for both B-lymphocytes and epithelial cells in vivo, we expressed the GFP-tagged BMRF-2 protein in both Akata 4E-3 B-lymphoblastoid cells and 293T kidney epithelial cells, hereafter referred to as Akata/BMRF-2 and 293T/BMRF-2, respectively. Expression of BMRF-2 was confirmed by detection of GFP fluorescence (Fig. 1A), as well as by Western blotting using rat anti-BMRF-2 serum (Fig. 1B). The BMRF-2 protein was detected in approximately 95% of Akata/BMRF-2 and 80% of 293T/BMRF-2 cells.
To determine whether EBV-seropositive humans have antibodies against BMRF-2, we performed immunofluorescence assays with all 15 serum samples from asymptomatic donors and individuals with NPC and HL using Akata/BMRF-2 and 293T/BMRF-2 cell lines. BMRF-2-specific immunostaining was confirmed by colocalization of antibody signal with BMRF-2 GFP signals (Fig. 2). All serum samples recognized BMRF-2 in both B-lymphoblastoid and epithelial cells (data not shown), consistent with the ELISA data (Table 1). Figure 2 shows immunostaining of BMRF-2 in Akata/BMRF-2 and 293T/BMRF-2 cell lines with the EBV-positive NPC-4 and EBV-negative serum samples. Cells immunostained with NPC-4 serum were strongly positive, whereas cells stained with negative serum did not have specific signals. All 15 EBV-positive serum samples were highly reactive to BMRF-2, i.e., 70-100% of BMRF-2 expressing cells of Akata/BMRF-2 and 293T/BMRF-2 cell lines were BMRF-2-positive. However, the intensity of BMRF-2 immunostaining levels in both Akata/BMRF-2 and 293T/BMRF-2 cell lines was variable among serum samples. Analysis of immunostained cells using secondary antibodies against human IgG, IgM and IgA showed that BMRF-2 was detected only with anti-human IgG antibodies, indicating that human antibodies against BMRF-2 is belong to the IgG class.
Next, to determine whether human anti-BMRF-2 antibodies react to the protein on the Akata/BMRF-2 and 293T/BMRF-2 cell surfaces, we performed flow cytometry assays on live cells using all of the serum samples from the asymptomatic, NPC and HL groups. The results from these assays are consistent with the findings from the ELISA and immunostaining experiments: BMRF-2 was detected on the surfaces of both Akata/BMRF-2 and 293T/BMRF-2 cells using all serum samples. The mean intensity (MN) of the BMRF-2-specific fluorescence signal with the positive serum samples in both cell lines was high and varied between 55 and 125 (data not shown). Fig. 3 shows the results of flow cytometry detection of BMRF-2 on cell surfaces of both cell lines by representative EBV-positive serum samples from asymptomatic, NPC and HL individuals.
The immunofluorescence and flow cytometry data clearly show that all EBV-positive human sera contain antibodies to BMRF-2 and that these antibodies recognize both intracellular and cell surface BMRF-2 in both B-lymphoblastoid and epithelial cells.
It has been shown that the RGD motifs of viral proteins are an essential part of immunodominant epitopes (Joki-Korpela et al., 2000; Liebermann et al., 1991; Mason, Rieder, and Baxt, 1994). Analysis of the hydrophobicity plot of BMRF-2 according to the Kyte-Doolittle scale shows that BMRF-2 is a highly hydrophobic multi-span trans-membrane protein, except for its RGD-containing extracellular domain (amino acids 171 to 218), suggesting that this domain might contain immunodominant epitopes. Therefore, we examined whether the RGD-containing domain can serve as an immunodominant region for the induction of the humoral immune response during lytic EBV infection. The BMRF-2 open reading frame encoding amino acids171 to 218 was subcloned into a pGEX vector and expressed as a 3 glutathione-S-transferase (GST) fusion protein. A control GST fusion protein in which RGD was replaced with alanines, was also prepared. We then examined the reactivity of four different human serum samples from asymptomatic, NPC and HL donors against the two GST fusion proteins using a Western blot assay. As shown in Fig. 4, all four EBV-positive human sera strongly recognized the BMRF-2-RGD peptide. However, three serum samples, AS-1, HL-1 and HL-2, only weakly recognized the mutant BMRF-2 AAA peptide, while the fourth (NPC-1) showed no detectable reactivity with the mutant peptide. This finding indicates that the BMRF-2 RGD motif plays an important role in the antibody recognition capacity to the extracellular domain (aa 171 to 218).
The above data indicate that the BMRF-2 RGD domain might serve as an important antigenic determinant for the stimulation of the anti-EBV humoral immune response. Short synthetic peptides have been used successfully to test the neutralizing effect of antibodies directed against viral proteins (Hosein et al., 1991; Wang, 1988). Here we adapted this method to determine whether anti-BMRF-2 antibodies in EBV-positive human sera specifically interact with the BMRF-2 RGD domain. Six serum samples from asymptomatic, NPC and HL individuals were pre-incubated with 47 amino acid peptides cleaved from GST-fusion protein containing either the wild-type BMRF-2 RGD or the mutant BMRF-2 AAA sequence to deplete sera of the RGD-specific antibodies. Subsequently, polarized oral epithelial cells were infected with the EBV B95-8 strain in the presence of the pre-incubate serum samples. At 3 days post-infection, infected cells were examined by immunostaining using monoclonal anti-BZLF-2 (Fig. 5A) antibodies, respectively. Incubation of virus with a pool of 15 EBV-positive serum samples without pre-incubation with BMRF-2 peptides led to approximately 80% inhibition of infection. Pre-incubation of serum samples with either the RGD- or AAA-containing peptides both reduced the neutralizing activity of the serum samples. However, the degree of inactivation of BMRF-2- specific antibodies was significantly different between the two peptides (Fig. 5B). Pre-incubation of all six serum samples with the BMRF-2 RGD peptide led to a significant (approximately 3-5 fold) increase in EBV infection of oral epithelial cells. In contrast, pre-incubation of these serum samples with the BMRF-2 AAA peptide had a far more modest effect: four of six serum samples from asymptomatic and HL donors resulted only moderate (approximately 2-fold) increases in EBV infection rates, and 2 serum samples from NPC donors did not increase EBV infection rates at all.
In parallel experiments we examined the neutralizing activity of a 17 amino acid synthetic peptide encompassing the BMRF-2 RGD domain (aa 192 to 209), as well as that of a BMRF-2 unrelated peptide. Preincubation of the 11 human sera with the RGD-containing peptide reduced the inhibitory effect of serum samples in EBV infection of oral epithelial cells by about 2-4 fold (Fig. 5C). Pre-incubation of the serum samples with the BMRF-2-unrelated control peptide did not affect the inhibitory activity of human sera against EBV infection. These findings indicate that the neutralizing effect of the human sera on EBV infection of epithelial cells can be abrogated specifically by the BMRF-2 RGD-containing extracellular domain, but far less effectively or not at all by peptides lacking the RGD motif.
In this study, we examined the human antibody response to EBV BMRF-2 in asymptomatic healthy individuals, NPC patients and HIV-infected patients with HL. All serum samples from the three groups were positive for EBV and contained IgG anti-VCA p18 and anti-EBNA-1 antibodies. However, none of the serum samples was positive for IgM anti-VCA p125 antibodies, indicating that the sera came from individuals with long-standing EBV infections and not recent, primary infections (Rickinson and Kieff, 2007). IgA anti-VCA and IgG anti–EA-D antibodies were found mainly in HL and NPC patients, suggesting reactivated EBV infection in these individuals (Rickinson and Kieff, 2007). All EBV-positive sera recognized BMRF-2 in B-lymphoblastoid and epithelial cells, indicating that BMRF-2 is a target of the humoral immune response. However, recognition of BMRF-2 by all serum samples, including those from latently-infected (IgG anti-VCA p18 - and IgG EBVA-1-positive samples) and EBV-reactivated individuals (IgA anti-VCA p18- and IgG EA-D– positive samples) indicates that the antibody response to BMRF-2 may not serve as a marker for the reactivation of virus. More likely, antibodies to BMRF-2 appear soon after primary infection and may serve as markers for seroconversion.
More importantly, human antibodies recognized BMRF-2 on the surface of both lymphocytes and epithelial cells, indicating that the immunodominant epitopes of BMRF-2 were localized to its extracellular domain. The predicted structure of BMRF-2 suggests that it might have nine or ten hydrophobic trans-membrane domains and one major hydrophilic domain (aa 170 to 213 aa) that contains the integrin-binding RGD motif. We previously showed that this major hydrophilic domain is exposed at the cell surface and that its RGD motif is functional for integrin binding (Tugizov, Berline, and Palefsky, 2003). In this study, we show that mutation of the RGD motif substantially reduces or eliminates EBV-specific, human antibody binding to the RGD-containing extracellular domain of BMRF-2. These findings indicate that the RGD motif may serve as a key component of immunogenic epitopes within the extracellular loop of BMRF-2. Our findings are consistent with published data showing that the RGD motif is essential not only for integrin binding, but also for antigenic determination (Joki-Korpela et al., 2000; Liebermann et al., 1991; Mason, Rieder, and Baxt, 1994). In human parechoviruses and the foot-and-mouth disease virus, the RGD motif of viral capsid protein VP1 has been shown to be critical for virus attachment, infection and stimulation of the antibody response (Joki-Korpela et al., 2000; Liebermann et al., 1991; Mason, Rieder, and Baxt, 1994). It is possible that exposure of the BMRF-2 loop between 171 and 218 aa to the extracellular environment, and the presence of the RGD motif in this region, creates a favorable condition for generation of a highly immunogenic region, i.e., is easily accessible by the humoral immune system for a strong antibody response.
Experiments with blocking or inactivation of BMRF-2-specific antibodies from EBV-positive serum samples with BMRF-2 RGD peptides (aa 171 to 218) yielded interesting data. These sera failed to inhibit EBV infection of polarized oral epithelial cells from their basolateral membranes, indicating that human antibodies against the BMRF-2 RGD domain may have neutralizing activity against EBV. Mutation of the RGD motif in the extracellular domain (aa 171 to 218) of BMRF-2 did not completely abolish the ability of this peptide to inactivate BMRF-2-specific antibodies in human sera, suggesting that this extracellular region may contain other immunogenic epitopes. However, shorter peptides (aa 192-209) also exhibited a similar degree of inactivation on EBV positive serum samples, suggesting that the RGD motif and its flanking amino acid residues may play an important role in the formation of immunogenic epitopes. These data are consistent with our previously published data showing that rat immune serum raised against the BMRF-2 RGD domain blocks the attachment and entry of EBV in polarized oral epithelial cells (Tugizov, Berline, and Palefsky, 2003). The immunodominant role of the BMRF-2 RGD domain in human antibody response is well-supported by our previous finding that the generation of highly reactive rat anti-BMRF-2 immune sera was possible only against RGD-containing extracellular domain (aa 171 to 218) of BMRF-2 (Tugizov, Berline, and Palefsky, 2003). In contrast, immunization of rats with the N-terminus (aa 2 to 73) or the C-terminus (aa 315 to 354) peptides of BMRF-2 did not induce any antibody response or generated low titer antibodies to BMRF-2, respectively.
Detection of EBV BMRF-2-specific antibodies in patients with NPC and HIV-positive individuals with EBV-infected HL lesions suggest that during ongoing EBV infection these antibodies may not prevent EBV spread within the oro-nasopharyngeal mucosal epithelium. It has been shown that naturally occurring neutralizing antibodies against HSV-1 also do not prevent or reduce the development of epithelial lesions by reactivated virus (Corey and Spear, 1986), i.e., cell-to-cell spread of virus within the epithelium.
Cell-to-cell spread of HSV-1 and other alpha herpes viruses occurs across the lateral junctions of epithelial cells, and a complex of two viral glycoproteins, gE and gI, plays a key role in this process (Alconada et al., 1998; Balan et al., 1994; Brack et al., 2000; Dingwell et al., 1994; Dingwell and Johnson, 1998; Johnson et al., 2001). The gE/gI complex first accumulates in the trans-Golgi network (TGN) and is then delivered to the cell junction area by basolateral sorting vesicles (Farnsworth and Johnson, 2006; Johnson et al., 2001; McMillan and Johnson, 2001). The basolateral sorting of gE/gI leads to the accumulation of nascent virions at cell junctions and their spread via neighboring membranes (Farnsworth and Johnson, 2006; Johnson et al., 2001; McMillan and Johnson, 2001; Polcicova et al., 2005). We have shown that EBV BMRF-2 also accumulates in the trans-Golgi network (TGN) and is then transported to the basolateral membranes of oral epithelial cells and that it plays a critical role in cell-to-cell spread of virus via the junctional areas of oral epithelial cells (Xiao et al., 2009). The presence of extensive cell junctions (desmosomes and both tight and adherens junctions) may prevent penetration of neutralizing antibodies into these cell-to-cell contact areas and therefore protect the infectious virions from host immune surveillance (Johnson and Huber, 2002; Sattentau, 2008). Therefore, it is possible that the presence of anti-BMRF-2 antibodies in EBV-infected individuals may not have a significant effect on the cell-to-cell spread of EBV within the oral or nasopharyngeal epithelium. However, human anti-BMRF-2 neutralizing antibodies may contribute to the control of EBV infection of epithelial cells by cell-free virus. Indeed, our previous data have shown that BMRF-2-mediated cell-free EBV infection of oral epithelial cells is reduced by human sera from EBV-infected individuals and rat immune sera against the BMRF-2 RGD-containing extracellular domain.
In summary, in this study we provide the first evidence that EBV-infected human sera contains BMRF-2-specific antibodies, indicating that the EBV BMRF-2 protein, an important component of virions and lytic viral infections, may serve as a significant target for the humoral immune response. The immunodominant epitope (s) of BMRF-2 was/were localized within the BMRF-2 RGD-containing extracellular domain, between 170 and 213 aa, and seroreactivity to peptides corresponding to this region may serve as an immunodiagnostic marker for EBV infection.
Serum samples from HIV-negative asymptomatic individuals and HIV-infected HL patients were provided by the AIDS Specimen Bank (Department of Oral Biology, University of California, San Francisco, California). EBV-specific immune sera from HIV-negative NPC patients were provided by E. Lennette (Viralab Inc, Berkeley, California). All human sera were previously collected from volunteers after providing informed consent. Use of human sera was approved by the Committee on Human Research Review Board of the University of California, San Francisco (UCSF) (CHR approval #RS00908). EBV-negative human serum was purchased from Blackhawk BioSystem, Inc and Wampole Laboratories.
Commercial ELISA kits were used to determine antibody levels in human serum samples. ELISA assays were performed to detect anti-EBV antibodies according to protocols provided by the manufacturers. The ELISA kits to detect IgG, IgM and IgA antibodies specific for EBV viral capsid antigens (VCA p18 for IgG and IgA, and VCA p125 for IgM), IgG against nuclear antigen-1 (EBNA-1) and IgG against BMRF-1 (early antigen-D (EA-D) were obtained from Wampole Laboratories (Princeton, New Jersey). The VCA IgA ELISA kit was purchased from Bio-Quant (San Diego, California). ELISA data were evaluated according to protocols provided by the manufacturers as follow: negative “-”, an OD readings of 0-0.9; negative or weakly positive “+/-”, an OD readings 0.91-1.0; moderately positive “+”, an OD readings of 1.1-3.0; positive “++”, an OD readings of 3.1-5.0; strongly positive “+++”, an OD readings of 5.1 and above.
The EBV-producing marmoset B lymphoblastoid cell line B95-8 and EBV-negative human B-lymphocytic cell line Akata 4E-3 cells (a gift from Dr. Hutt-Fletcher at Louisiana State University Health Sciences Center) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. The human kidney epithelial cell line 293T cells (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT). Rat anti-BMRF-2 serum was previously generated in our laboratory by immunizing rats with a GST fusion protein containing the hydrophilic extracellular domain of the BMRF-2 protein (aa 178-218). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania).
The BMRF-2 ORF from EBV B95-8 strain was previously cloned into a retrovirus vector as described previously (Xiao et al., 2007). The plasmid expressing the BMRF-2 protein tagged with the green fluorescent protein and designed for stable expression of BMRF-2 GFP in B-lymphoblastoid cell line Akata-4E-3 cells was transfected with BMRF-2-GFP using LipofectAmine 2000 (Invitrogen) according to the manufacturer’s the protocol. At 48 h after transfection, cells were plated into 96-well plates at 104 cells/well in growth medium containing G418 (500 mg/ml). One half of the culture medium was replaced with fresh growth medium containing G418 every five days. After 3-4 weeks of selection, G418-resistant and BMRF-2-expressing clones were pooled and expanded. The cell line was designated Akata/BMRF-2 and was maintained in growth medium containing G418 (400 mg/ml). Transient transfection was used to express BMRF-2 in 293T cells (human kidney epithelial cells) since they are highly transfectable with more than 80% transfection efficiency. Cells were seeded in 8-well chamber slides or 10 cm Petri-dishes 12-16 h before transfection so that they would reach 80-90% confluence at the time of transfection. The BMRF-2 gene was introduced into the cells using LipofectAmine 2000 (Invitrogen) according to the manufacturer’s protocols. 24-36 h post-transfection, cells were used for experiments. 293T cells transfected with BMRF-2 were designated 293T/BMRF-2.
For flow cytometry analysis Akata/BMRF-2 cells were collected and washed once with ice-cold wash buffer (3% bovine serum album (BSA) in PBS) by centrifugation (800 rpm for 3 min). 293T/BMRF-2 cells were dissociated with cell dissociation buffer (Invitrogen, Carlsbad, CA) for 5 min at room temperature and then washed once with ice-cold wash buffer. The cell pellets were then re-suspended in wash buffer and each human serum sample (primary antibodies) was incubated with 106 cells at 1:50 dilution in PBS (pH 7.2) at 4-8 °C with gentle rocking for 45 min. Cells were washed three times with ice-cold PBS and reacted with R-Phycoerythrin (RPE)-labeled goat anti-human IgG as secondary antibodies. Surface expression of BMRF-2 was measured by FACS in a FACSCAN (Becton-Dickinson, San Jose, CA).
For immunofluorescence staining, 293T/BMRF-2 cells grown on chamber slides were washed once with PBS and air-dried. Akata/BMRF-2 cells were washed once with PBS, re-suspended in PBS (106cells/ml), and 10 μl of cells were dropped onto a glass slides and air semi-dried. The cells were then rinsed once with PBS and fixed with methanol/acetone (50/50) for 30 min at -20 °C. EBV-positive human sera were then applied to the slides for 45 min and then washed 3 times with PBS. Texas-red labeled goat anti human IgG was used as secondary antibody incubated at room temperature for 45 min. Cell nuclei were stained with TO-PRO-3 (blue fluorescence) or propidium iodide (red fluorescence) for 10 min. Immunostained cells were analyzed using a krypton-argon laser coupled with a Bio-Rad MRC2400 confocal head. The data were analyzed using Laser Sharp software.
For the peptide neutralization assay we used the BMRF-2 extracellular domain containing the RGD motif (BMRF-2 RGD, amino acids 171 to 218), which was previously constructed as a glutathione-S-transferase (GST) –BMRF-2 RGD fusion protein. The QuikChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, California) was used to mutate the RGD sequence in the BMRF-2 RGD peptide using the GST–BMRF-2 RGD fusion gene as a template and the following HPLC-purified primers (Invitrogen). Primers for the BMRF-2 AAA mutant were forward primer 5’-cattttctgcgccgccgcagctcattcggtggcatc-3’ and reverse primer 5’-gatgccaccgaatgagctgcggcggcgcagaaaatg. GST fusion proteins were propagated in the Escherichia coli Bl-21 strain (Xiao et al., 2007) and purified using Sepharose-4 beads. BMRF-2 peptides were cleaved by thrombin (Amersham, Piscataway, New Jersey) according to protocols provided by the manufacturer. After thrombin cleavage, peptide fragments were washed three times and concentrated in PBS using Amicon Ultra-10 centrifugal filter devices (Millipore). Endotoxin levels were measured using the Limulus Amebocyte Lysate assay kit E-TOXATE (Sigma) and were found to be less than 0.005 EU/mg in purified protein preps. We also used synthetic peptides containing the RGD motif of BMRF-2 (RRRSIFCARGDHSVASL) and a control, unrelated peptide (GARRNQIYTSGLERRR) purchased from Biopeptide Co (San Diego, California).
Peptide neutralization assays were performed according to Wang et al., (Wang, 1988) with some modifications. Polarization of oral epithelial cells and their infection with EBV were described previously (Tugizov, Berline, and Palefsky, 2003; Xiao et al., 2008). Briefly, primary tongue epithelial cells were cultured on 24-mm diameter Transwell filters (Costar) to form a polarized monolayer. To test the ability of various RGD domain-related peptides to abrogate the neutralizing activity of BMRF-2-specific antibodies in human sera, the sera were first diluted 1:50 with PBS and then mixed with BMRF-2 RGD, BMRF-2 AAA, or the BMRF-2-unrelated control peptide at 100μg/ml for one h at 37°C. To examine the inhibitory effect of the treated sera against EBV infection, EBV virions of the B95-8 strain were first incubated with the peptide-treated or un-treated human sera at 37°C for 1 h with gentle shaking. The above-treated virions were then added to the epithelial cells from the bottom chamber at 100 virions/cell, and the filters were incubated at 37°C with gentle shaking for 1 h. The medium was then removed and cells were maintained in growth medium in a 5% CO2 incubator. Three days after infection, cells were fixed with 3% paraformaldehyde in PBS at 4°C for 30 min and immunostained with mouse monoclonal anti-BZLF-1 antibodies, respectively. EBV protein expression was visualized by staining the cells with Texas-red labeled goat-anti-mouse secondary antibodies. EBV-infected (BZLF-1 positive) cells were analyzed and counted by confocal microscopy.
Membrane protein extraction of Akata/BMRF-2 and 293T/BMRF-2 cells was performed as described previously (Xiao et al., 2007). Briefly, membrane fractions of BMRF-2 expressing cells were solubilized in urea sample buffer (7 M urea, 2 M thioreurea, 1% TX100, 1% DTT, 4% chaps, and 10 mM Tris, pH 9.5) at room temperature for 1 h. Before loading, samples were mixed with one-tenth volume of 1 M DTT and denatured at 70 °C for 10 min. Proteins were then separated on 7 M urea-SDS PAGE gels. The BMRF-2 protein was detected using rat anti-BMRF-2 serum. To determine the reactivity of sera samples to BMRF-2 RGD domain the GST-BMRF-2-RGD and GST-BMRF-2-AAA fusion proteins were separated in Tris-glycine SDS PAGE gel, and proteins were detected using the various human serum samples.
Data were compared using the Student’s t test; P values of <0.05 were regarded as significant.
We thank Dr. L. Hutt-Fletcher (University of Louisiana, Shreveport) for providing Akata 4E-3 cells, E. Lennette for providing sera from nasopharyngeal carcinoma patients, Drs Deborah and John Greenspan for providing sera from HIV-positive patients with HL lesion, J Berline for production and purification of BMRF-2 RGD and AAA GST proteins, and Dr. M. Petitt for editorial assistance. This project was supported by National Institute of Health grants R01 DE14894 and R21 DE016009 (to S.T).
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