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Journal of Virology, October 2005, p. 12408-12415, Vol. 79, No. 19
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.19.12408-12415.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Amino Terminus of Epstein-Barr Virus Glycoprotein gH Is Important for Fusion with Epithelial and B Cells

Jasmina Omerovi, Lori Lev, and Richard Longnecker^*

Department of Microbiology and Immunology, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received 30 April 2005/ Accepted 8 July 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epstein-Barr virus (EBV) is a gammaherpesvirus that has tropism for both B lymphocytes and epithelial cells (18, 42). Similar to other enveloped viruses, EBV entry into the target cells occurs in two steps: the initial attachment of the virus to the cell surface and the subsequent fusion of the viral envelope and the cell membrane (18, 51). EBV infection can occur by cell-free virus or through cell-cell spread, but fusion is required for both entry pathways (18, 42). The initiation of infection by EBV is driven by interaction of viral glycoproteins with cell-surface receptors (42). EBV encodes as many as 11 glycoproteins, but only a subset is required for efficient EBV entry (15, 42). In general, more is known about the mechanism of EBV entry into B lymphocytes than into epithelial cells. Infection of B lymphocytes is initiated by the attachment of glycoprotein gp350/220 to the CD21/CR2 receptor on the B cells (9, 43, 45). This interaction enhances infection efficiency of B cells, but it is not absolutely required for infection to occur (16). Subsequent to this binding, viral glycoprotein gp42 binds to the B-cell surface protein HLA class II, which triggers fusion mediated by a concerted action of three glycoproteins (gB, gH, and gL) (19, 43). The glycoprotein requirement for EBV entry differs between epithelial and B cells; gB, gH, and gL are essential for infection of both epithelial and B cells, while gp42 is required only for B-cell entry (12, 20, 24, 47). Even though gB, gH, and gL are conserved throughout the herpesvirus family, the structure and the exact mechanism of action of these three glycoproteins in the fusion process are still unclear.

EBV gH and gL are present on the viral envelope as a heterodimer complex, gH/gL. The formation of a gH/gL glycoprotein complex is a common theme in herpesviruses (31, 42). EBV gH is thought to function in virus cell fusion, while the role of gL is to serve as a chaperone, essential for the folding and transport of functional gH to the cell surface (34, 35, 48, 50). The depletion of gH from EBV virions abolishes the ability of virus to infect B cells and epithelial cells (13, 28). Additionally, virus lacking gH is unable to bind to epithelial cells, and soluble gH/gL protein binds to the surface of these cells, suggesting the existence of an epithelial cell receptor for gH/gL (3, 25, 28). In EBV, gH and gL form two different complexes, a bipartite complex that contains only gH and gL and a tripartite complex that also includes gp42 (2, 15, 48). These two complexes have a mutually exclusive ability to mediate infection of epithelial cells and B cells, respectively (2, 20).

As previously mentioned, very little is known about the functional role of gH/gL in EBV entry; the data from studies on other herpesviruses have been limited to the mutagenesis of gH from herpes simplex virus type 1 (HSV-1) (4, 10, 14). More recently, while this paper was in preparation, putative coiled-coil domains in human cytomegalovirus (HCMV) gH and in HSV-1 gH were identified; peptides from these regions inhibited HCMV and HSV-1 infection in vitro, respectively (11, 21). Alpha-helical coiled coils are important motifs found in a variety of viral and cellular fusion proteins, playing a pivotal role in membrane fusion (6, 41). The role of coiled coils in viral entry was demonstrated in studies of fusion glycoproteins for a number of viruses, of which influenza HA and human immunodeficiency virus (HIV) Env glycoproteins are classic examples (6, 8). Typically, these fusion proteins are organized in homotrimers, with each monomer possessing N-terminus and C-terminus heptad repeats. Although much progress has been made in understanding the mechanism of membrane fusion mediated by a single glycoprotein, the membrane fusion promoted by the action of multiple glycoproteins is not well understood. It is likely that in a more complex system, such as herpesvirus entry, cooperation and interaction between fusion glycoproteins are required for membrane fusion to occur.

To gain a better understanding of the role of gH in EBV-mediated fusion with epithelial and B cells, we performed mutagenesis studies with a putative coiled-coil domain of EBV gH detected by a coiled-coil prediction software. Interestingly, this region maps very closely to the coiled-coil region recently identified in HCMV gH (21). We demonstrate gH mutants containing either a single leucine to alanine substitution or a combination of two residues mutated in this domain have an altered ability to mediate fusion with B cells and epithelial cells. Our results thus indicate that the N-terminus region of gH between amino acids 54 and 74 is important for EBV-mediated fusion with target cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and antibodies (Abs). All cells were grown in medium containing 10% FetalPlex animal serum complex (Gemini Bio-Products) and 1% penicillin-streptomycin (100 U penicillin/ml, 100 µg streptomycin/ml; BioWhittaker). Chinese hamster ovary cells (CHO-K1) kindly provided by Nanette Susmarski were grown in Ham's F-12 medium (BioWhittaker). EBV-positive HLA class II- and CD21-expressing Daudi B lymphocytes were obtained from the American Type Culture Collection (ATCC), Manassas, VA, and were grown in RPMI 1640 medium (BioWhittaker). To more easily monitor membrane fusion, the Daudi 29 cell line stably expressing T7 RNA polymerase was used (40). Human embryonic kidney 293 cells were passaged in Dulbecco's modified Eagle medium (BioWhittaker). Two types of 293-derived cell lines were used: 293-P cells, selected for high transfection frequency (Edge BioSystems, Gaithersburg, MD) and 293-T cells, expressing the simian virus 40 large T antigen (ATCC). Construction of a 293-T cell line stably expressing T7 RNA polymerase is described in "Transfection," below. Cells were grown in 75-cm² cell culture flasks (Corning), and adherent cells were detached by using either trypsin-Versene (BioWhittaker) or Versene (phosphate-buffered saline [PBS]-1 mM EDTA).

Monoclonal antibodies E1D1 and F-2-1 were gifts from L. Hutt-Fletcher (Louisana State University Health Sciences Center, Shreveport, La.) and recognize the gH/gL complex and gp42, respectively (1, 44). A large-scale preparation of the E1D1 and the F-2-1 antibodies was made at the Northwestern University Monoclonal Antibody Facility. The HL-800 Ab, a polyclonal antibody that recognizes gH and gL, was obtained through genetic immunization by immunizing rabbits with EBV gH and gL expression vectors (Aldevron, North Dakota) (12). gp42 polyclonal antibody (PB1114) was generated by immunization of rabbits with soluble gp42 protein (Harlan Bioproducts for Sciences, Wisconsin) (27, 40).

Construction of mutants. Point mutations in EBV gH were introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The QuikChange Kit uses PCR to introduce a specific mutation via primers designed with a silent mutation for diagnostic purposes. PCR was performed as suggested by the manufacturer to generate mutant clones, which were then diagnostically digested and sequenced to confirm their authenticity. Mutant DNA plasmids were isolated by cesium chloride density gradients.

Transfection. All of the transfections were performed by a standard protocol using Lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four hours before transfection, CHO-K1 cells were seeded in six-well plates and the next day were transiently transfected with 0.5 µg each of EBV gB, gL, and gH (or gH mutant); 2 µg of gp42; and 0.8 µg of a luciferase-containing reporter plasmid with a T7 promoter (12, 29). For Western blot experiments, CHO-K1 cells were plated in T-25 cell culture flasks and 1 day later were transfected with either all four EBV glycoproteins or gH and gL alone. 293 cells seeded in 10-cm² dishes were either transiently or stably transfected to express T7 polymerase. For transient transfection, 293-P cells were transfected with 10 µg of pCAGT7 (29). 293-T cells were stably transfected to express T7 polymerase and green fluorescent protein. Briefly, 293-T cells were cotransfected with 16 µg pCAGT7 containing T7 RNA polymerase and 4 µg of pczCFG5 IEGZ containing green fluorescent protein and zeocin resistance (kindly provided by Dirk Lindemann). Three days posttransfection, the cells were plated at 0.1, 1, 10, 100, 1,000, and 10,000 cells per 96-well plate and selected with zeocin (100 µg/ml). For the 96-well plates with 0.1 to 100 cells, a feeder layer of 5,000 irradiated 293-T cells per well was added. Clones emerged 3 weeks postplating. Ten clones were expanded from the 96-well plates and tested in the fusion assay as described below. Of the tested clones, cell line 14 was the only line that showed luciferase expression in the fusion assay. Line 14 was maintained in Dulbecco's modified Eagle medium with 100 µg/ml zeocin.

Fusion assay. Effector CHO-K1 cells were transfected with plasmids encoding the glycoproteins as stated above. After 12 h, CHO-K1 and 293 cells were washed with PBS and detached with Versene. The cells were counted with a Beckman Coulter Z1 particle counter, and then the effector and the target cells were mixed in equal amounts (0.2 x 10⁶ per sample) and plated into a 24-well plate in Ham's F-12 medium (12, 24). Twenty-four hours later, the cells were washed with PBS and lysed, and luciferase was quantified by using the Promega Reporter Assay system. Relative luciferase activity was measured on a Perkin-Elmer Victor plate reader.

CELISA. CHO-K1 cells used for the fusion assay as described above were also used to detect surface expression of the glycoproteins via cell enzyme-linked immunosorbent assay (CELISA) as described previously (24). Briefly, the cells were incubated with either the mouse E1D1 monoclonal Ab diluted at 1:200 or the rabbit HL-800 polyclonal Ab diluted at 1:500. The cells were fixed and then incubated sequentially with secondary biotin-conjugated anti-mouse immunoglobulin G or anti-rabbit immunoglobulin G (Sigma) and tertiary antibodies. The plates were read as previously described (24).

Immunoprecipitation of biotinylated cells and Western blotting. CHO-K1 cells were transfected as stated above. After 12 h, the cells were washed with PBS, and fresh Ham's F-12 medium was added. The cells were harvested 24 h later and washed three times with ice-cold PBS. Following washes, cells were incubated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) by rotation for 0.5 h at 4°C. Biotin was inactivated by washing cells three times with ice-cold 100 mM glycine-PBS. Cytoplasmic lysates were prepared by lysing the cells with 1% Triton X-100 lysis buffer (40), and the insoluble material was removed by centrifugation at 4°C. Cleared lysates were immunoprecipitated overnight at 4°C with either E1D1 Ab, HL-800 Ab, or F-2-1 Ab, depending on the experiment, and captured with protein G-Sepharose (Amersham). Samples were then washed two times in the lysis buffer, resuspended in sodium dodecyl sulfate sample buffer, boiled at 95^°C for 10 min, and pelleted by centrifugation. The supernatants were separated on Bio-Rad 12.5% Criterion sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred to Immobilon-P membranes, and blocked in Tris-buffered saline plus Tween with 5% milk for 1 h at room temperature (RT) or overnight at 4°C. The membranes were probed for 0.5 h at RT with a horseradish peroxidase (HRP)-conjugated avidin (Bio-Rad) diluted 1:2,000 in blocking solution. For studies examining the association of the gH/gL complex with gp42, membranes were incubated for 1 h at RT with a rabbit polyclonal anti-gp42 antibody (PB1114) diluted 1:1,000 in blocking solution. Membranes were washed in Tris-buffered saline plus Tween, and a HRP-conjugated-protein A (Amersham) was applied for 0.5 h at RT. Following five washes, blots were mixed in equal volumes of ECL solutions and exposed to hyperfilm (Amersham Biosciences).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Location of a putative coiled-coil domain in EBV gH and construction of gH mutants. Since very little is known of the role of EBV gH in fusion and the domains that are important, instead of making random insertion mutations that could significantly alter the structure and binding to gL, we decided on a more systematic strategy for EBV gH mutagenesis. We analyzed gH protein sequences from EBV and two primate gammaherpesviruses (rhesus and marmoset) for their probability to form coiled-coils. The COILS program at EMBnet designed for prediction of coiled coils from protein sequences was used for this purpose (23). A region at the N terminus of gH proteins from all three viruses was predicted with some probability to form an alpha-helical coiled coil. In EBV gH, this region was found between amino acids 54 and 74, in a location similar to that of a potential coiled-coil domain recently reported for HCMV gH (Fig. 1A) (21). The probability of this region forming a coiled-coil in both EBV and rhesus primate gH proteins was relatively low (score, about 0.15), while the probability score for the marmoset gH was about 0.5. Since examples of viral fusion glycoproteins carrying functional coiled-coil regions with low probability scores have been described (i.e., F protein of Newcastle disease virus) and since this region was conserved in all three gammaherpesviruses analyzed, we decided to examine the importance of this region in EBV gH fusion with epithelial and B cells (11, 26).

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FIG. 1. Schematic diagram of a putative coiled-coil domain in gH of EBV and primate gammaherpesviruses. (A) gH protein sequences from EBV, rhesus, and marmoset (CalHV-3) gammaherpesviruses were analyzed by the COILS program (EMBnet) for the probability to form coiled-coils (http://www.ch.embnet.org/software/COILS_form.html) (23). A region at the N terminus was detected as a potential coiled-coil domain for all three proteins, which in EBV gH is located between amino acids 54 and 74. Conserved leucine residues are highlighted in boldface type. (B) Point mutants containing either a single or a double leucine-to-alanine substitution were generated by site-directed mutagenesis with the QuikChange kit (Stratagene). Mutations were verified by restriction digestion and DNA sequencing.

Studies of HIV and other enveloped viruses have shown that hydrophobic residues, especially leucines, located in the coiled coils are important for the proper function of these regions in fusion (5, 7, 22, 36). The putative coiled-coil domain of EBV gH contains four leucines; three of these are also conserved in rhesus and marmoset gH proteins. To examine the role of leucines in this domain in fusion, point mutants containing either single or double leucine-to-alanine substitutions were generated (Fig. 1B). Since slight amino acid changes in viral glycoproteins can inhibit protein folding and transport to the cell surface, we chose to introduce alanine substitution to minimize the effect of mutations on the overall protein structure. Alanine substitution mutagenesis was used successfully to analyze the role of leucines in membrane fusion and virus entry mediated by the murine coronavirus spike protein (22). The mutations in EBV gH were introduced with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). For each site-specific mutation, a unique restriction site was silently incorporated into the reading frame to allow easy identification of each mutant. The presence of the mutation was also verified by DNA sequence analysis, and plasmid DNA was isolated by cesium chloride density gradient.

Residue L65 is important for the integrity of the E1D1 epitope. Prior to testing the mutants in a functional assay, we verified the expression of each mutant at the cell surface. Cell surface expression of wild-type and mutant gH/gL complexes was measured by CELISA at 36 h posttransfection as described in Materials and Methods. Interestingly, when the E1D1 Ab (a gH/gL conformational Ab) was used, the L65A mutants were reduced in expression when compared to wild-type gH (Fig. 2A). The E1D1 Ab recognizes the native gH/gL complex and blocks epithelial cell entry and fusion, while it has no effect on entry of EBV into B cells and B cell fusion (20, 24). The cell surface expression of the single L65A mutant was about 50% of that of the wild type, while the expression of both L55/65A and L65/74A mutants was reduced by about 25%. The L65/69A mutant showed the most dramatic reduction in expression, with an approximate 75% decrease compared to that of wild-type gH. To determine if the expression of gH was reduced or if the reduction in expression was a result of failure of the E1D1 Ab to react with the mutant forms of gH, cell surface expression was examined by a rabbit polyclonal gH/gL Ab (HL-800). The HL-800 antibody was confirmed to specifically recognize EBV gH and gL and transport of the gH/gL complex to the cell surface by CELISA (Fig. 2B) and Western blotting (Fig. 2E). When a CELISA with the HL-800 Ab was performed to analyze the cell surface expression of all the mutants, including the ones with the L65A mutation, the cell surface expression was similar to that of wild-type gH (Fig. 2C). These results suggested that the decrease in the expression of L65A-containing mutants was not due to reduced expression of gH/gL on the plasma membrane but rather to a loss of reactivity with the E1D1 Ab.

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FIG. 2. Residue L65 is important for the integrity of the E1D1 epitope. (A and C) CHO-K1 cells were transiently transfected to express gp42, gB, gL, and either a wild-type gH or a mutant gH. (B) CHO-K1 cells were transiently transfected with either gH or gL alone or both proteins together. Posttransfection, cells were transferred to 96 wells in triplicate and CELISA was performed with either E1D1 Ab (A) or a rabbit gH/gL Ab (HL-800) (B and C). Data are averages of three independent experiments with the standard deviations indicated by vertical lines. (D and E) CHO-K1 cells were transfected with gL and either a wild-type or mutant gH plasmid. Cells were harvested 36 h posttransfection and the cell surface proteins were labeled with biotin at 4°C. Biotinylated lysates were immunoprecipitated with either the E1D1 Ab (D) or the HL-800 Ab (E) and probed with avidin-HRP in a Western blot. Data for some mutants are shown in panel E, but all analyses were performed. gH and gL bands are indicated with an arrow and a bracket, respectively. Asterisks indicate the location of a background band that somewhat overlaps with gL. Molecular masses (in kilodaltons) are noted to the left.

Verification of cell surface expression by biotinylation and Western blotting. To further verify the cell surface expression of gH mutants, CHO-K1 cells transfected with EBV gL and either a wild-type or mutant EBV gH plasmid were biotinylated at the cell surface. Wild-type and mutant gH/gL complexes were immunoprecipitated with either the E1D1 Ab (Fig. 2D) or the HL-800 Ab (Fig. 2E). Consistent with the CELISA results, the mutants containing the L65A mutation did not immunoprecipitate well with the E1D1 Ab, as their cell surface expression, as detected by Western blotting, was reduced. However, the cell surface expression of all mutants immunoprecipitated with the HL-800 Ab was similar to that of wild-type gH (Fig. 2E and data not shown). Therefore, the data from the Western blots confirmed that the gH N-terminus mutants with leucine-to-alanine substitutions were expressed at the cell surface similar to wild-type gH, although the mutants containing the L65A mutation had reduced ability to bind the E1D1 antibody. Additionally, these results also showed that the interaction between gH and gL was not altered by leucine-to-alanine substitutions.

gH mutants retain their ability to associate with gp42. For the gH/gL complex to mediate EBV entry into B cells, association with gp42 is also required (20, 47). Therefore, we examined whether leucine mutations in the N terminus of gH had any effect on the binding of the gH/gL complex to gp42. CHO-K1 cells transfected with EBV glycoproteins gp42, gB, gH, and gL were biotinylated as described in Materials and Methods. Lysates were immunoprecipitated for gp42 and membranes were probed with avidin-HRP to detect cell surface proteins associating with gp42. As shown in Fig. 3A, gH and gL were equally immunoprecipitated for the wild type and each of the gH mutants when the gp42 monoclonal Ab F-2-1 was used. Moreover, the specificity of this interaction was confirmed by immunoprecipitation for gH and gL. For these experiments, the lysates were immunoprecipitated with either the E1D1 Ab (data not shown) or the HL-800 Ab (Fig. 3B), both of which recognize gH/gL. Figure 3B shows that immunoprecipitation of gH/gL with the HL-800 Ab resulted in the coimmunoprecipitation of gp42, as detected by a rabbit polyclonal gp42 antibody (PB1114). Data for some mutants are shown, but all were tested and coimmunoprecipitated similarly. Comparable results were obtained with the E1D1 Ab with the exception of the L65A mutants. As might be expected, less gp42 coimmunoprecipitated with the E1D1 Ab, due to the reduced reactivity of the Ab with the L65A mutants (data not shown). These data indicated that substitution mutants of gH are still able to form tripartite complexes of gH/gL/gp42.

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FIG. 3. gH mutants retain their ability to associate with gp42. CHO-K1 cells were transfected with gp42, gB, gL, and either a wild-type or mutant gH plasmid and biotinylated at 4°C. (A) Biotinylated lysates were immunoprecipitated for gp42 with the F-2-1 Ab and probed with avidin-HRP in a Western blot. Cells transfected with an empty vector were used as a negative control (labeled as vector). The asterisks indicate the locations of two background bands with the lower one overlapping with gL. (B) Lysates that were used in the experiment shown in panel A were immunoprecipitated for gH/gL with the HL-800 Ab and probed with a rabbit gp42 Ab (PB1114) in a Western blot. For a control, CHO-K1 cells transfected with gp42 were immunoprecipitated with the F-2-1 Ab and probed with the PB1114 Ab (lane 1). The two bands observed for gp42 were reported previously (27). Molecular masses (in kilodaltons) are noted to the left of the blots.

Residues L65 and L69 are important for fusion with B cells and epithelial cells. Studies of HIV Env glycoprotein and other viral fusion proteins have shown that mutations in the conserved coiled-coil domains can have dramatic effects on the ability of the glycoprotein to mediate fusion (7, 33, 49). A single amino acid change at a leucine or isoleucine residue completely abrogates envelope-mediated cell fusion and viral infectivity, but the mutation does not interfere with protein transport and expression at the cell surface. Since the cell surface expression of gH mutants was unaltered by leucine-to-alanine substitutions, we screened the mutants for their ability to mediate membrane fusion in a virus-free cell fusion assay (Fig. 4). As previously described, effector CHO-K1 cells were transfected with gp42, gB, gL, and either a wild type or one of the mutant gH plasmids along with a plasmid containing the T7 promoter upstream of a luciferase reporter (12). For B-cell entry, gp42, gB, gH, and gL are required, while gp42 is not necessary for epithelial cell entry (20, 47). The presence of gp42 during epithelial cell fusion can be inhibitory, but as shown previously this inhibition is only slight and epithelial cells are still able to mediate fusion in the presence of gp42 (24). Therefore, the same CHO-K1 cells transfected with all four EBV glycoproteins were used in examining B-cell and epithelial cell fusion. Following transfection, the effector CHO-K1 cells were mixed with the target cells, either Daudi cells for B-cell fusion or 293 cells for epithelial cell fusion. The target cells express T7 RNA polymerase, so that upon fusion of the two cell types, luciferase is expressed and can be measured to quantitate the extent of fusion. The target Daudi cells constitutively express T7 RNA polymerase, while 293 cells were either stably or transiently transfected to express T7. The data from 293 cells transfected by these two different methods were virtually identical. Previous reports from our laboratory showed that 293 cells behave similarly to other epithelial cell lines in the fusion assay, such as human carcinoma cell lines AGS and SCC68, and provide a model of epithelial cell fusion in vitro (24). Because of their high transfection efficiency and low cytotoxicity to most transfection reagents, 293 cells are easier to work with and were thus used in this study.

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FIG. 4. EBV gH mutants have altered fusion with B cells and epithelial cells. CHO-K1 cells were transfected with EBV glycoproteins gp42, gB, and gL and either a wild-type or mutant gH. The transfected CHO-K1 cells were mixed 1:1 with either a B-cell line, Daudi cells (black bars), or an epithelial cell line, 293 cells (gray bars); 24 h later, the relative luciferase activity was measured. Luciferase activity was normalized to wild-type levels, which was set to 100% for both cell types. (A) Fusion results from mutants containing a single leucine-to-alanine change in residue 55, 65, 69, or 74; (B) data from mutants with a combination of two leucine residues mutated. Data are averages of three independent experiments with the standard deviations indicated by vertical lines.

We first performed fusion assays using the four mutants containing single leucine-to-alanine substitutions at positions 55, 65, 69, and 74 in EBV gH (Fig. 4A). As described in Materials and Methods, at 24-h postoverlay of the effector and the target cells, fusion was measured by assessing the levels of relative luciferase activity. Figure 4A shows the percentage of fusion for each of the mutants calculated by setting the fusion for the wild type with either Daudi or 293 cells at 100%. The reduction in fusion was observed for mutants L65A and L69A with both B cells and epithelial cells, although the effect was more dramatic for epithelial cell fusion. Mutant L65A was reduced about 60% in fusion with B cells and about 80% in fusion with epithelial cells when compared to wild-type gH. A somewhat lower reduction in fusion was observed for the L69A mutant. This reduction was about 30% in B cells and 65% in 293 cells when compared to wild-type gH. Interestingly, unlike the mutations in the two middle leucines, mutants L55A and L74A had slightly increased fusion levels for both cell types compared to the wild type. The increase in fusion is not a result of higher gH cell surface expression, since the data from the previous figures showed that the expression of these mutants at the cell surface was similar to wild-type gH. Overall, the single point mutations in the N terminus of EBV gH altered the ability of the mutant gH/gL glycoprotein complexes to mediate fusion with B cells and epithelial cells, pointing to an important role of this region in EBV fusion and subsequent entry.

Mutants with either L65A or L69A mutation fuse better in combination with L55A or L74A mutants. To further assess the importance of leucine residues in the putative coiled-coil region of EBV gH in fusion, we examined mutants containing a combination of two leucines changed to alanines. For the murine coronavirus fusion protein, single alanine substitution mutations had minimal effect on cell-to-cell fusion, but when two leucine or isoleucine residues were replaced by alanines, a significant reduction in fusion activity was observed (22). Additionally, mutation of a single conserved leucine into alanine in the F1 subunit of the Newcastle disease virus fusion protein had little effect on fusion, while replacement of two or three leucine residues abolished the fusogenic activity of the protein (36). In our study, double EBV gH mutants containing either the L65A or L69A mutations exhibited overall lower fusion activity levels with both cell types than with wild-type gH. Interestingly, the combination of L65A or L69A with the single mutation in L55 or L74 resulted in higher levels of fusion than when L65A or L69A mutants were present alone (Fig. 4B). For B cells, the presence of enhancing mutations in combination with either L65A or L69A brought the fusion activity to almost wild-type levels or about 80% of the levels observed for wild-type gH. Since the L65A and L69A mutants had a more prominent effect of fusion with the epithelial cells, enhanced fusion of these mutations seen with either L55A or L74A mutants was not as great. However, epithelial cell fusion with both L55/69A and L69/74A was above 50% of the levels observed for wild-type gH. Moreover, the double L55/74A mutant enhanced fusion with both B cells and epithelial cells more than either mutant alone, which further confirmed the fusion enhancing phenotype of these mutations. The L65/69A mutant, which had the greatest reduction in reactivity with the E1D1 Ab, had a 40% reduction in fusion with B cells, while fusion with 293 cells was only about 25% of wild-type gH. These results indicate that double-substitution changes of leucines to alanines in the N-terminus region of gH do not accentuate the single leucine-to-alanine mutants when EBV fusion is tested. Additionally, the double mutants confirmed the enhancing phenotype of mutations L55A and L74A, as the fusion activity of L65A and L69A mutants was higher in their presence.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gH/gL glycoprotein complex is critical for entry of all herpesviruses, yet the exact role of this complex or individual members of the complex in entry is not understood (42). In this study, we demonstrated that an N-terminal region of EBV gH is important for efficient membrane fusion with B cells and epithelial cells. This region was predicted to form a coiled coil, and a similar motif was conserved in EBV-related gamamherpesviruses that infect rhesus and marmoset primates. We analyzed the effect of single and double leucine-to-alanine substitutions in this region on cell-cell fusion. All of the mutants generated were expressed at the cell surface at wild-type levels and retained their ability to bind gL and gp42. Reduced fusion activity was observed with mutants containing either the L65A and/or L69A mutations. The L65A mutants had a more dramatic decrease in fusion than the L69A mutants. In contrast, the L55A and L74A mutants resulted in increased fusion. This enhanced fusion phenotype of gH mutants L55A and L74A was observed with both B cells and epithelial cells and was more prominent when both of these mutations were present together. Interestingly, gH mutants with either the L65A or L69A mutation in combination with either the L55A or L74A mutants had higher fusion activity than the L65A or L69A mutants alone.

When the E1D1 Ab was used to analyze expression of the gH mutants, the four mutants that contained the L65A mutation were detected at somewhat lower levels at the cell surface. The most dramatic reduction in expression was seen in the combined L65/69A mutant. The E1D1 Ab recognizes the native gH/gL complex. Interestingly, these same mutants were expressed equally well when compared to wild-type gH or any of the other gH mutants when a rabbit polyclonal gH/gL antibody was used. This reduction in reactivity indicates that L65 is an important determinant of the E1D1 epitope and that L69 may contribute to the overall epitope. As previously published, the E1D1 antibody can neutralize EBV infection and attachment to epithelial cells, while it has no effect on B-cell entry (20). Interestingly, the L65A, L69A, and L65/69A mutants all had a greater reduction in epithelial cell fusion than B-cell fusion, compatible with a more important role of the E1D1 epitope in epithelial cell entry than B-cell entry.

Simplistically, there may be two putative roles of L65 and L69 in gH function in fusion. First, compatible with the greater reduction in epithelial fusion when compared to B cell fusion, L65 and L69 may be an important determinant for the binding of gH to epithelial cells. Suggestive of an epithelial cell receptor for gH/gL, EBV lacking gH is unable to bind to epithelial cells; soluble gH/gL binds to the surface of these cells (3, 25, 28). This binding can be blocked by the E1D1 Ab (3). Although our current results provide important data in regard to the epitope on gH/gL that the E1D1 Ab binds, it is not clear whether the antibody binds directly to a receptor binding site on gH or whether binding to a distal site of gH may alter receptor binding. Recently, the gH/gL receptors for HCMV, HSV-1 and human herpesvirus 6 were identified. It was shown that both HCMV and HSV-1 gH bind to vß3 integrins, while human herpesvirus 6 gH/gL interacts with a membrane cofactor protein, CD46 (30, 38, 39, 46). Unlike HSV-1 gH, HCMV gH does not have the RGD integrin binding motif; the domain responsible for binding of HCMV gH to integrins is yet to be determined. The ability of HCMV gH to bind integrins in the absence of the RGD motif raises the possibility that EBV gH, which also lacks this motif, might bind integrins. The receptor for EBV gH is yet to be identified. In light of a potential defect in EBV gH receptor binding, it will be of interest to examine whether the L65A or L69A mutants are impaired in binding to epithelial cells.

In regard to a second role of gH in EBV entry, the reduction of B-cell fusion observed with the L65 and L69 mutants is suggestive of gH contributing mechanistically for fusion besides receptor binding, since the L65 and L69 mutants reduced both B-cell and epithelial cell fusion. Studies of other enveloped nonherpesviruses have shown that a drastic refolding of a fusion protein between prefusion and postfusion forms is required for entry to occur (17). The transition to the postfusion state is associated with an irreversible conformational change, suggested to provide energy required for membrane fusion to take place. As previously mentioned, very little is known about the mechanism of entry mediated by herpesvirus glycoproteins, but based on data from other viruses it could be speculated that upon triggering of fusion by receptor binding a conformation change in gH/gL and gB is required to allow the entry of virus into target cells. The L65 and L69 mutants may be altered in their ability to undergo the conformation change required for membrane fusion. If L65 and L69 are part of a coiled coil, the mutations might block the interaction with other components of the fusion complex on gH or other glycoproteins involved in fusion, notably glycoprotein gB. Interestingly, an alanine in residue 65 reduces the probability that EBV gH will form a coiled coil from 0.15 to 0.05. Furthermore, the failure to form an appropriate fusion complex would result in a reduction of fusion, as seen with the L65 and L69 mutants. Although EBV gH has not been previously shown to bind proteins other than gL and gp42, any potential interaction could be transient or unstable and difficult to detect. Interaction between the gH/gL complex and gB would appear to be required, since the gH/gL complex and gB are all that is necessary for epithelial fusion. Recent reports on HSV have shown that gD and gH are able to interact with each other only after gD binds to the herpes simplex virus entry mediator receptor and likely after attaining a new conformation (32). Similarly, although EBV gB and gH are not known to interact, a conformation change such as receptor binding may be required to allow gB and gH interaction and subsequent fusion to occur.

Along with mutants that reduced both B-cell and epithelial cell fusion (L65 and L69), we also obtained mutants with enhanced fusion activity (L55 and L74). The increase in fusion observed with these mutants may result from a decrease in energy required to activate the gH/gL complex, resulting in a fusion complex that is in a more favorable fusion conformation. This may be similar to mutations of conserved glycines in the paramyxovirus F fusion protein that cause a decrease in energy required to activate F in the fusion cascade, resulting in increased cell-cell fusion (37). Alternatively, these mutants may form a better interaction with a viral or cellular protein, resulting in higher fusion activity. Interestingly, as mentioned above, fusion-defective mutants L65A and L69A in combination with mutations L55A and L74A had an improved ability to mediate fusion when compared to the L65A and L69A mutants alone. In addition, loss of E1D1 Ab binding to the L55/65A and L65/74A mutants was not as great as that seen with the L65A and L65/69A mutants. These data suggest that L55A and L74A weakly complement the L65A and L69A mutations, since the fusion activity and reactivity with the E1D1 Ab were still below wild-type levels.

Although the current study does not necessarily prove that the region of EBV gH between amino acids 54 and 74 forms a coiled coil and functions as such in EBV-mediated fusion, it indicates that this region is important for fusion with both epithelial and B cells. The potential presence of a putative coiled coil in EBV gH is somewhat consistent with recent reports of gH of HCMV and HSV-1 herpesviruses and suggests an evolutionary conservation of this domain in herpesvirus fusion (11, 21). Thus, delineating the role of this gH region in herpesvirus entry may be a key to the overall herpesvirus fusion process, a process that mechanistically has been elusive. A better understanding of the mechanism of EBV and herpesvirus entry in general will be of significance in developing new therapeutics that would specifically inhibit the entry step of infection.

 
We thank Lindsey Hutt-Fletcher for providing the E1D1 and the F-2-1 antibodies, Nanette Susmarski for cell line expertise, and the members of the Longnecker laboratory for help and support.

R.L. is supported by U.S. Public Health Service grants CA62234, CA73507, and CA93444 from the National Cancer Institute and grant DE13127 from the National Institute of Dental and Craniofacial Research. R.L. is a Stohlman Scholar of the Leukemia and Lymphoma Society of America. This work is supported in part by a predoctoral fellowship from the American Heart Association, Midwest Affiliate (J.O.).

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, The Feinberg School of Medicine, Northwestern University, Ward 6-231, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0467. Fax: (312) 503-1339. E-mail: r-longnecker{at}northwestern.edu.

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Journal of Virology, October 2005, p. 12408-12415, Vol. 79, No. 19
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