Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion

Abstract

Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.

Epstein-Barr virus (EBV) is a member of the lymphocryptovirus (LCV) subgroup of gammaherpesviruses and primarily infects epithelial and B lymphocytes, where it establishes productive and latent infections, respectively (8, 40). EBV is extremely prevalent in humans, with more than 90% of the population being infected. Following primary infection, EBV persists in a latent state in memory B lymphocytes, where it can remain indefinitely (3, 43). The expansion and proliferation of these cells can lead to the development of a number of EBV-related malignancies, including tumors of lymphoid tissues, such as Hodgkin's lymphoma, Burkitt's lymphoma, and some T-cell lymphomas, as well as tumors associated with epithelial tissues, such as nasopharyngeal carcinoma and gastric carcinoma (5, 32, 36, 48). EBV is also associated with lymphoproliferative disorders in immunocompromised patients, such as oral hairy leukoplakia and posttransplant lymphoproliferative disorders (12, 15, 43, 45).

Herpesvirus entry is a multistep process that includes the initial binding of the virus to the cell surface, interaction with a cellular entry receptor, membrane-virion fusion, and internalization of the virion (40). The enveloped membrane of EBV contains numerous glycoproteins that are necessary for mediating attachment and the subsequent fusion between viral and cell membranes, although the required glycoproteins differ for epithelial and B-cell infection (13, 22). Infection of B cells is initiated upon the binding of EBV glycoprotein gp350/220 to the CD21/CR2 cellular receptor expressed on target cells (9, 27, 41). Following the initial binding, viral glycoprotein gp42 binds to human leukocyte antigen class II (HLA class II) and triggers fusion, which is mediated by the concerted actions of glycoproteins gB, gH, and gL (21, 41). Glycoproteins gH and gL form a heteromeric complex that is conserved among the herpesvirus family despite a low degree of sequence homology in gH and gL across herpesviruses (33, 40). The gH/gL complex has an essential role in entry of herpesviruses, yet the exact role of each protein is not well understood. The role of gH is thought to be in cell fusion, while gL mediates processing and transport of gH to the cell surface (34, 35, 47, 51). Since gL is generally essential for the proper processing and transport of gH in all herpesviruses, it has been difficult to examine the role of gL separate from gH.

While little is known about the role of gL in EBV-mediated fusion, data from studies of other herpesviruses have suggested a more direct role of gL in fusion. Antibodies specific for the C terminus of herpes simplex virus (HSV) gL, mapped to residues 168 to 224, were found to inhibit fusion with some strains of HSV type 1 (HSV-1), suggesting a role of this region in fusion (28). Deletion analysis of HSV-1 gL determined that the first 161 amino acids of gL are sufficient for binding gH, and further deletion identified the region between amino acids 155 and 161 of HSV-1 gL as critical for gH transport and cell fusion (19, 20, 33). Together, the results of these studies suggest that the inhibition of fusion observed with gL antibodies (Abs) is not due to an inhibition of gH/gL complex formation. Another study found that mutations to the amino-terminal region of HSV-2 gH permitted transport of the glycoprotein to the cell surface independent of gL but that gL was still required for fusion, suggesting a role of gL in fusion after the complex has trafficked to the plasma membrane (6). Most recently, the results of an investigation of the functional homology of the primate rhesus LCV (Rh-LCV) gL (Rh gL) suggest that gL may have a functional role in EBV fusion with B cells. Rh gL shares a high degree of sequence similarity with EBV (81.6%), and yet, the glycoprotein was unable to mediate fusion with human B cells when expressed with either EBV gH or Rh gH, even though the gH/gL complex was detected on the cell surface and Rh gL could associate with gH and gp42 (31). The LCV that infects rhesus primates is biologically similar to EBV in that it shares a similar genome organization, repertoire of lytic and latent genes, and pathogenesis (7, 25, 37, 46).

To gain a better understanding of the role of gL in EBV-mediated fusion with B cells, we constructed chimeric gL proteins that included portions of Rh gL inserted into the sequence of EBV gL. We identified the amino terminus of EBV gL as being functionally necessary in mediating fusion with B cells, and this role is distinguishable from the role of gL in the processing and transport of gH to the cell surface. Single and double point mutations were constructed in both EBV and Rh gL, and our results indicated that residues 54 and 94 are functionally critical in mediating fusion with B cells. Interestingly, our studies also identified a species-specific reliance between gL and gB, suggesting a possible association between these two glycoproteins that is necessary in mediating fusion. Thus, our studies demonstrated that EBV gL has a more substantial role in mediating fusion with B cells that is distinct from its role in the processing and trafficking of gH.

MATERIALS AND METHODS

Cells and Abs.

Chinese hamster ovary cells (CHO-K1), kindly provided by Nanette Susmarski, were grown in Ham's F-12 medium (BioWhittaker) containing 10% FetalPlex animal serum complex (Gemini Bio-Products) and 1% penicillin-streptomycin (100 U penicillin/ml, 100 μg streptomycin/ml; BioWhittaker). Cells were grown in 75-cm² cell culture flasks and detached by using either trypsin-Versene (BioWhittaker) or Versene (phosphate-buffered saline [PBS]-1 mM EDTA). The Daudi 29 cell line stably expressing T7 RNA polymerase was grown in RPMI 1640 medium (BioWhittaker) containing 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin with 900 μg/ml G418 (Sigma) (39).

Monoclonal Ab E1D1 was a gift from L. Hutt-Fletcher and recognizes the gH/gL complex (4). The polyclonal gp42 Ab (PB11114) was generated by immunization of rabbits with soluble gp42 protein (Harlan Bioproducts for Sciences, WI) (26). Monoclonal Abs HA.11 and 12CA5 both recognize epitopes of the hemagglutinin (HA) tag that is present on all of the gL mutants (Covance).

Construction of EBV and Rh gL substitution mutants.

Point mutations in EBV and Rh gL were generated by using a QuikChange site-directed mutagenesis kit (Stratagene). The QuikChange kit was also used to insert a silent mutation in EBV and Rh gL in order to create the EBV/Rh gL chimeras. All mutants were sequenced by the Northwestern Biotech core sequencing facility, and DNA was isolated by using Qiagen Maxi kits (Qiagen).

Transfections.

All transfections were performed by using Lipofectamine 2000 transfection reagent (Invitrogen). For fusion assay and cell-based enzyme-linked immunosorbent assay (CELISA), cells were seeded into six-well plates 20 h before transfection in order to reach 80% confluence and transiently transfected with 0.5 μg each of EBV or Rh gB, gH, and gL (or gL mutant); 2 μg of gp42; and 0.8 μg of a luciferase reporter plasmid with a T7 promoter (13, 29). For Western blot experiments, CHO-K1 cells were plated in 25-cm² cell culture flasks to reach 70% confluence 1 day later. Cells were transiently transfected with 4.6 μg of gp42 and 1.15 μg each of gB, gH, and gL (or gL mutant). For all experiments, the vector pCAGGS is used as a negative control and to maintain DNA amounts.

Fusion assay.

The virus-free cell-based fusion assay was described previously (13, 24). Effector CHO-K1 cells were transfected with EBV or Rh-LCV glycoproteins and mutant proteins as stated above. Twelve hours posttranfection, cells were detached with Versene, counted with a Beckman Coulter Z1 particle counter, and mixed 1:1 with target B cells (0.25 × 10⁶ per sample) in a 24-well plate in 1 ml of Ham's F-12 medium. The target cells, Daudi 29 B cells, stably express T7 RNA polymerase and were under selection by G418 (30). Twenty-four hours later, the cells were washed with PBS and lysed with 100 μl of passive lysis buffer (Promega). Luciferase was quantified in duplicate by transferring 20 μl of lysed cells to a 96-well plate and adding 100 μl of luciferase assay reagent (Promega), and luminescence was measured on a Perkin-Elmer Victor plate reader. Luciferase activity was normalized to the EBV wild-type level, set to 100%.

CELISA.

CHO-K1 cells used in the fusion assay as described above were also used to detect surface expression of glycoproteins by CELISA, as described previously (24). The cells were incubated with either mouse E1D1 monoclonal Ab at 1:200 or mouse HA.11 Ab at 1:500. The cells were fixed in PBS with 2% formaldehyde and 0.2% glutaraldehyde and then incubated with secondary biotin-conjugated anti-mouse immunoglobulin at 1:500 (Sigma) and streptavidin-horseradish peroxidase (HRP) at 1:20,000 (GE Healthcare). TMB 1 component HRP microwell substrate (BioFX Laboratories) was added, and luminosity was measured at 370 nm on a Perkin-Elmer Victor plate reader.

Immunoprecipitation and Western blotting.

CHO-K1 cells were transfected as stated above, and fresh medium was added 12 h after transfection. The cells were lysed 24 h later with 1% Triton X-100 lysis buffer, and insoluble material was removed by centrifugation at 13,000 rpm at 4°C. Lysates were immunoprecipitated overnight at 4°C with the E1D1 or HA.11 Ab and protein G-Sepharose (GE Healthcare). Samples were washed multiple times with lysis buffer, resuspended in sodium dodecyl sulfate sample buffer, boiled at 95°C for 10 min, and pelleted by centrifugation. Sample supernatants were run on Bio-Rad 15% Criterion sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to Immobilon membranes (Millipore). Membranes were blocked in Tris-buffered saline plus 1% Tween 20 with 5% milk overnight at 4°C and probed for 1 h at room temperature with primary Ab. To confirm an association between gL or gL mutants and gH, membranes were probed with the monoclonal anti-HA Ab (12CA5) diluted 1:1,000 in blocking solution. To confirm association of the gH/gL complex with gp42, membranes were probed with the polyclonal anti-gp42 Ab (PB1114) diluted 1:2,000 in blocking solution. Membranes were washed in Tris-buffered saline plus Tween 20, and secondary HRP-conjugated anti-mouse or anti-rabbit Ab was applied for 0.5 h at room temperature. Membranes were then washed, and blots were incubated in ECL Western blotting detection reagents (GE Healthcare) and exposed to Hyperfilm (GE Healthcare).

RESULTS

Rh gB restores B-cell fusion in Rh gL-, EBV gp42-, and EBV gH-transfected cells.

EBV and Rh gL share a high degree of sequence homology, and yet, we previously found that Rh gL does not mediate fusion with B cells when expressed with EBV glycoproteins gp42, gB, and gH (31). This reduction of fusion was not due to an inability of Rh gL to be expressed at the cell surface or to bind gH and gp42, as was confirmed by the results of CELISA and immunoprecipitation. It is also unlikely to be due to an absence of a Rh-LCV-specific receptor on human B cells, since Rh-LCV has been shown to infect human B cells in vitro (25) and Rh glycoproteins gp42, gB, gH, and gL can mediate fusion with human B cells (data not shown).

To examine the properties of Rh gL further, we tested different combinations of EBV and Rh-LCV glycoproteins in our cell-based fusion assay. Since a change in fusion could be attributed to a difference in the ability of the gH/gL complex to traffic to the cell surface, we first performed CELISA using the E1D1 monoclonal Ab that recognizes gH/gL and the HA.11 monoclonal Ab that recognizes the HA tag present on EBV and Rh gL. The results of previous studies, as well as the results presented below, demonstrate that a tag on gL at the carboxy terminus does not alter gL function (31). We verified that there was no change in the level of cell surface expression of Rh gL whether it was expressed with EBV gB or Rh gB (Fig. 1A). The fusion assay was done by transfecting CHO-K1 effector cells with EBV or Rh gp42, gB, gH, and gL along with a plasmid containing a luciferase reporter (13, 23, 24). These cells were then overlaid with the human B cell Daudi 29 target cells that constitutively express T7 RNA polymerase (39). Fusion of the effector and target cells led to the expression of luciferase, which was quantified by measuring relative luciferase activity and normalized to the EBV wild-type level, set at 100%. We examined fusion with EBV gp42, gB, and gH and Rh gL and found, similar to our previous finding, that fusion was reduced to near background levels (Fig. 1B). However, in the current experiment, we also tested if the inclusion of Rh gB with Rh gL might restore fusion, which it did to levels slightly higher than the fusion activity of wild-type EBV (Fig. 1B). This result suggests that the reduction of fusion observed when Rh gL is expressed with EBV glycoproteins is specifically due to an inability of Rh gL and EBV gB to function together. Interestingly, we also found that EBV gB could not mediate fusion with B cells when expressed with Rh gp42, gH, and gL (data not shown). This demonstrates that there is species specificity for fusion, since gL and gB must both be from either EBV or Rh-LCV to function. The reduction in fusion that is seen when EBV gB is expressed with Rh gL can be resolved by expressing either EBV gB and gL together or Rh gL and gB together. Overall, these data suggest that gL plays a direct role in fusion with B cells by interacting with gB or promoting the recruitment of gB to the gH/gL complex.

FIG. 1.

Rh gB restores fusion of Rh gL with human B cells. CHO-K1 cells were transiently transfected with EBV gp42 and gH and either EBV or Rh gB and gL. (A) Posttransfection, cells were transferred to 96-well plates and CELISA was performed with either anti-gH/gL Ab (black bars) or anti-gL-HA Ab (gray bars). (B) Transfected CHO-K1 cells were overlaid with Daudi B cells, and luciferase activity was measured. Luciferase activity was normalized to the wild-type EBV level, which was set to 100%. Parenthetical ellipses (… .) indicate transfection with the glycoproteins indicated below each bar of the graph. Error bars show standard errors.

Construction of EBV/Rh chimeric gL proteins.

EBV and Rh gL are extremely homologous proteins; excluding the signal sequence, there are only 17 amino acids that differ between the two (Fig. 2A). In order to further study the role of gL in fusion and identify regions necessary for mediating fusion with B cells, chimeric proteins were constructed using regions of Rh gL inserted into EBV gL (Fig. 2B). Using the C-terminally HA-tagged variants of EBV and Rh gL, chimeric gL proteins were constructed. The chimeras were generated by introducing a unique silent mutation at amino acid residue 97 of both glycoproteins using the QuikChange site-directed mutagenesis kit (Stratagene), and the location of the mutation was verified by sequence analysis. EBV/Rh gL chimera 1 (ERh gL-1) was constructed with the amino-terminal region of EBV gL to amino acid residue 97 and the carboxy terminus of Rh gL. ERh gL-2 is the reciprocal chimera, containing the amino-terminal region of Rh gL to amino acid residue 97 and the carboxy terminus of EBV gL. A third chimeric gL protein was constructed to further limit the possible residues important for gL fusion by introducing a second unique silent mutation at amino acid residue 52 of EBV and Rh gL. This chimera, ERh gL-3, was generated by introducing the region from amino acid residue 52 to amino acid residue 97 of Rh gL into EBV gL.

FIG. 2.

Schematic diagram of EBV and Rh gL chimeric proteins and substitution mutants. (A) Diagram of EBV and Rh gL proteins with the positions of amino acid differences (excluding signal sequence) indicated by black bars. (B) Chimeric gL proteins were constructed using regions from EBV gL (light gray) and Rh gL (dark gray). ERh gL-1 includes the N-terminal region of EBV gL to amino acid 97 and the C-terminal region of Rh gL. ERh gL-2 includes the N-terminal region of Rh gL to amino acid 97 and the C-terminal region of EBV gL. ERh gL-3 includes the region of Rh gL from amino acid 52 to amino acid 97 within EBV gL. A sequence alignment of EBV and Rh gL is shown below, with the nonhomologous residues in this region displayed in bold. Point mutants containing either single or double substitutions were generated by site-directed mutagenesis using the QuikChange kit (Stratagene). Point mutants of EBV gL that contain substitutions of the corresponding Rh gL amino acids were generated. Point mutants generated from Rh gL contain substitutions of the corresponding EBV gL amino acids.

gL chimeras maintain their ability to associate with EBV gH and gp42.

The association of the gH/gL complex with gp42 is necessary to mediate EBV entry into B cells (17, 22, 47). Therefore, we examined whether the insertion of Rh gL sequences into EBV gL had an effect on binding EBV gH and gp42. CHO-K1 cells were transfected with EBV glycoproteins gp42, gB, and gH and either EBV, Rh, or chimeric gL as described in Materials and Methods. Lysates were immunoprecipitated for gH by using monoclonal Ab E1D1 that recognizes the gH/gL complex. The membranes were probed with a monoclonal Ab against the HA tag on the gL proteins (12CA5) and the polyclonal gp42 Ab (PB1114). As shown in Fig. 3A, equal levels of EBV and Rh gL and each of the three EBV/Rh gL chimeric proteins were immunoprecipitated. Association with gp42 was also detected at equal levels for the three EBV/Rh gL chimeric proteins, although slightly less gp42 was detected with Rh gL. These data indicate that the insertion of Rh gL residues into EBV gL does not affect the gH/gL/gp42 tripartite complex formation.

FIG. 3.

Association of EBV/Rh gL chimeras with EBV gH and gp42. CHO-K1 cells were transiently transfected with EBV glycoproteins gp42, gH, and gB and either EBV gL, Rh gL, or one of three EBV/Rh gL chimeras. Proteins were immunoprecipitated with an Ab against gH/gL (mouse monoclonal Ab E1D1) and probed with an Ab against gL-HA (mouse monoclonal Ab 12CA5) or gp42 (rabbit polyclonal Ab PB1114). Molecular size markers (kDa) are shown on the left. IP, immunoprecipitation.

Amino terminus of EBV gL is necessary for mediating B-cell fusion.

Previous studies have shown that an important role of gL of the herpesvirus family is to mediate the proper processing and trafficking of gH to the cell surface. Since gL is necessary for the trafficking of gH, it has been difficult to examine the role of gL in fusion without also affecting gH. The observation that the highly homologous Rh gL cannot substitute for EBV gL, even though it can process and transport gH to the cell surface, makes it a useful tool in studying the role of gL in mediating fusion with B cells and identifying gL functional regions. Since chimeric EBV/Rh gL proteins were expressed and bind gH and the resulting gH/gL complexes bind gp42, we determined whether the gH/gL complex could be transported to the cell surface by performing CELISA. The levels of cell surface expression of all the chimeras were similar to that of wild-type gL with both the E1D1 Ab that recognizes the gH/gL complex and the HA.11 Ab that recognizes the HA tag on gL (Fig. 4A). Since cell surface expression was not altered in any of the chimeras, they were next tested for function in fusion with B cells (Fig. 4B). We first performed fusion assays using chimeras ERh gL-1 and ERh gL-2 to determine the effect on B-cell fusion of inserting portions of Rh gL into the EBV gL sequence. Figure 4B shows the percentage of fusion for each chimera, normalized to that of wild-type EBV gL with Daudi B cells set to 100%. ERh gL-1 was able to mediate fusion at levels higher than the fusion activity of wild-type EBV gL, indicating that mutating the carboxy terminus of gL does not alter the fusion function with B cells. Interestingly, ERh gL-2 did not mediate fusion at levels above background, demonstrating that the amino-terminal 97 residues of EBV gL are necessary for mediating fusion with B cells. This reduction in fusion was not a result of decreased cell surface expression, since ERh gL-1 and ERh gL-2 are both expressed equally (Fig. 4A). We next determined if replacing a smaller region of the amino terminus of EBV gL would alter fusion function by testing the ERh gL-3 chimera, which has residues 52 to 97 of EBV gL replaced with those residues of Rh gL. While fusion with ERh gL-3 was not reduced to background levels like ERh gL-2, fusion was significantly decreased, to about 45% compared to the level for wild-type gL, and as with all our chimeric mutants, there was no change in cell surface expression (Fig. 4A). Overall, replacing regions in the amino terminus of EBV gL had a drastic impact on fusion function, indicating an important role of this region in EBV-induced B-cell fusion.

FIG. 4.

Ability of EBV/Rh gL chimeras to mediate fusion and gH/gL trafficking. CHO-K1 cells were transfected with EBV glycoproteins gp42 and gH and different combinations of EBV or Rh gB and either wild-type gL or one of three EBV/Rh chimeras. (A) Posttransfection, cells were transferred to 96-well plates and CELISA was performed with an Ab against gH/gL (black bars) or gL-HA (gray bars). (B) Transfected CHO-K1 cells were overlaid with Daudi B cells, and fusion activity assayed 24 h later. Luciferase activity was normalized to the wild-type EBV level, which was set to 100%. Parenthetical ellipses (… .) indicate transfection with the glycoproteins indicated below each bar of the graph. Error bars show standard errors.

Since fusion with Rh gL could be restored by expressing Rh gB in place of EBV gB, we determined if Rh gB could restore fusion of the EBV/Rh gL chimeras. We tested each of the three chimeras in fusion with EBV gp42 and gH and Rh gB. Interestingly, as shown by the last three bars of Fig. 4B, fusion was restored to levels above that for wild-type gL with Rh gB, and this is not due to an increase in cell surface expression (Fig. 4A).

Association of EBV and Rh gL point mutants with EBV gH and gp42.

Our results from the EBV/Rh gL chimeras demonstrated that the residues between amino acids 52 and 97 are important for mediating fusion with B cells. To investigate the functional role of single amino acid residues in this region, mutants with point mutations were created. As shown in Fig. 2C, point mutations were constructed in both EBV and Rh gL at amino acid residues 54, 67, 71, 72, 80, 92, and 94. Rather than making random or alanine substitution mutants, the residues in EBV gL were mutated to the corresponding amino acid that is present in Rh gL to determine if single residues would result in a decrease in fusion activity. The same approach was used to construct single point mutations in Rh gL that replaced amino acid residues with those of EBV gL to establish if single residues would restore fusion function.

To determine if the amino acid substitutions had an effect on the ability to bind EBV gH, coimmunoprecipitations were performed using Ab E1D1 that recognizes the gH/gL complex. All EBV and Rh gL substitution mutants were able to bind gH (Fig. 5A and B), as is shown by the Western blots that were probed with Ab HA.11 to detect the HA tag on wild-type gL and each of the single point mutants. We then confirmed if the EBV and Rh gL point mutants could bind gp42 in coimmunoprecipitations using Ab HA.11. Association with gp42 is shown for all EBV and Rh gL single point mutants (Fig. 5A and B) in the Western blots probed with the polyclonal Ab gp42 (PB1114). Similar to what was seen for the EBV/Rh gL chimeras, mutations of single amino acid residues in EBV and Rh gL do not impede the complex formation of gH/gL and gp42.

FIG. 5.

Association of EBV and Rh gL point mutants with EBV gH and gp42. CHO-K1 cells were transiently transfected with EBV glycoproteins gp42, gH, and gB and EBV gL, or one of seven EBV gL mutants (A) or Rh gL mutants (B). To confirm binding with gH, proteins were immunoprecipitated with an Ab against gH/gL (mouse monoclonal Ab E1D1) and blots were probed with an Ab against gL-HA (mouse monoclonal Ab 12CA5). To confirm binding with gp42, proteins were immunoprecipitated with an Ab against gL-HA (mouse monoclonal Ab HA.11) and blots were probed with an Ab against gp42 (rabbit polyclonal Ab PB1114). Molecular size markers (kDa) are shown on the left. IP, immunoprecipitation.

EBV gL amino acid residues 54 and 94 are important for B-cell fusion.

The substitution mutants of EBV gL were examined by CELISA, and all were expressed at levels similar to the level of wild-type gL, as shown by using both Ab E1D1 and Ab HA.11 (Fig. 6A). The insertion of Rh gL amino acid residues at positions 67, 71, 72, 80, and 92 had no effect on fusion with B cells (Fig. 6B). Fusion levels varied with these substitutions from about 95% of that of the wild type for the mutant with an alanine substitution at residue 91 to about 125% of that of the wild type for the mutants with an alanine substitution at residue 67 or a threonine substitution at residue 71. Interestingly, a significant reduction in fusion was observed for mutants with substitutions at residues 54 and 94 of EBV gL. The substitution of lysine for glutamine at residue 54 of EBV gL resulted in a reduction of fusion to 58% of the level with wild-type gL. Also, the substitution of glutamine for lysine at residue 94 of EBV gL resulted in a reduction of fusion to 62% of the level for the wild type. This decrease in fusion is not due to a decrease in the expression of the gH/gL complex at the cell surface, since the data show that cell surface expression for mutants with substitutions at positions 54 and 94 are at or slightly above the wild-type gL level (Fig. 6A). Overall, we identified two amino acids that are specifically involved in the role of gL in mediating fusion with B cells.

FIG. 6.

Ability of EBV gL mutants to mediate fusion and gH/gL trafficking. CHO-K1 cells were transfected with EBV glycoproteins gp42, gH, and gB and either wild-type gL or one of seven EBV gL mutants. (A) Posttransfection, cells were transferred to 96-well plates and CELISA was performed with an Ab against gH/gL (black bars) or gL-HA (gray bars). (B) Transfected CHO-K1 cells were overlaid with Daudi B cells, and fusion activity assayed 24 h later. Luciferase activity was normalized to the wild-type EBV level, which was set to 100%. Parenthetical ellipses (… .) indicate transfection with the glycoproteins indicated below each bar of the graph. Error bars show standard errors.

Substitution of EBV gL residue 94 for that of Rh gL increases B-cell fusion.

By substituting Rh gL amino acid residues for the corresponding residues of EBV gL, we were able to determine that residues 54 and 94 are important for mediating fusion with B cells. We next wanted to determine if fusion with Rh gL could be enhanced by substituting amino acids from EBV gL. The same panel of amino acid substitution mutants were constructed for Rh gL and were expressed at the cell surface at levels close to that of the wild type (Fig. 7A). As expected, substitution mutations at residues 67, 71, 72, 80, and 91 did not increase fusion activity to levels above that of the vector control (Fig. 7B). The substitution of glutamine for lysine at amino acid residue 54 of Rh gL increased the fusion activity to 20% of that of the wild type, but this increase was not statistically significant. However, when the glutamine at residue 94 was mutated to a lysine, a significant increase in fusion activity was observed. This single amino acid substitution was able to increase fusion with B cells to more than 60% of the fusion activity with wild-type gL, while Rh gL and the other substitution mutants consistently display less than 10% fusion activity. The ability to reduce or increase fusion activity by switching the amino acid at residue 94 would suggest that this amino acid is critical for mediating B-cell fusion.

FIG. 7.

Ability of Rh gL mutants to mediate fusion and gH/gL trafficking. CHO-K1 cells were transfected with EBV glycoproteins gp42, gH, and gB and either wild-type gL or one of seven Rh gL mutants. (A) Posttransfection, cells were transferred to 96-well plates and CELISA was performed with an Ab against gH/gL (black bars) or gL-HA (gray bars). (B) Transfected CHO-K1 cells were overlaid with Daudi B cells, and fusion activity assayed 24 h later. Luciferase activity was normalized to the wild-type EBV level, which was set to 100%. Parenthetical ellipses (… .) indicate transfection with the glycoproteins indicated below each bar of the graph. Error bars show standard errors.

Mutants with gL amino acid substitutions at residues 54 and 94 in either Rh gL or EBV gL switch species specificity for gB in regard to fusion phenotype.

To further analyze the role of residues 54 and 94 of gL in B-cell fusion, we examined mutants that contained substitutions at both residues. The 54K/94Q EBV gL double mutant exhibited a further reduction in fusion in combination with EBV gB compared to the levels of fusion of the single EBV 54K gL and 94Q gL mutants, with the double and single mutants having approximately 45% and 60% of the fusion activity of the wild type, respectively (compare Fig. 6B and 8B). Similar to the experiments whose results are shown in Fig. 4, in which the reduced fusion of the chimeric gL proteins could be corrected by using Rh gB in place of EBV gB, we found that fusion with the EBV 54K/94Q gL double point mutant was significantly restored when EBV gB was replaced with Rh gB. In contrast, good fusion levels were only observed for the Rh gL 94K single substitution mutant, whereas the Rh gL 54Q mutant did not increase fusion by a significant amount in combination with EBV gB (Fig. 7B). However, when both residues were replaced, the Rh gL 54Q/94K mutant exhibited enhanced fusion, with levels of more than approximately 125% of the activity of wild-type gL (Fig. 8B) with EBV gB, demonstrating that both residues are critical. These results indicate that double substitution changes at residues 54 and 94 can enhance the effects that were seen with the single substitution mutants, further demonstrating a critical role of these residues in mediating B-cell fusion.

FIG. 8.

Ability of EBV and Rh gL double point mutants to mediate fusion and gH/gL trafficking with EBV gB and Rh gB. CHO-K1 cells were transfected with EBV glycoproteins gp42 and gH, wild-type gL or the EBV 54K/94Q or Rh 54Q/94K gL mutant, and EBV or Rh gB. (A) Posttransfection, cells were transferred to 96-well plates and CELISA was performed with anti-gH/gL Ab. (B) Transfected CHO-K1 cells were overlaid with Daudi B cells, and fusion activity assayed 24 h later. Luciferase activity was normalized to the wild-type EBV level, which was set to 100%. Parenthetical ellipses (… .) indicate transfection with the glycoproteins indicated below each bar of the graph. Error bars show standard errors.

DISCUSSION

In the current study, we provide evidence for a direct role of gL in the recruitment of gB in EBV- and Rh-LCV-induced B-cell membrane fusion by identifying an essential gL functional domain required for virally induced membrane fusion. The ability of gL to influence the recruitment of gB is likely either through a direct interaction of the two glycoproteins or by alteration of the conformation of gH to allow association of gB with the gH/gL complex. Specifically, amino acids 54 and 94 of EBV gL are critical in mediating fusion. If either or both residues in EBV gL are mutated, a significant reduction in fusion is observed. For these studies, we used the interesting observation that the closely related gL encoded by Rh-LCV was not able to replace EBV gL in our cell-based fusion assay.

Specifically, we demonstrated that Rh gL can substitute for EBV gL in mediating fusion with B cells, but only when Rh gL is expressed with Rh gB. The restoration of fusion when Rh gB was expressed was not due to a change in the cell surface expression of Rh gL or the EBV gH/Rh gL complex, as both were detected at levels similar to those of the wild-type controls, indicating that the transport function of gL was not altered. Interestingly, we also found that EBV gB could not substitute for Rh gB in Rh-LCV-mediated fusion with B cells, even though our fusion assay system is amenable to fusion with the Rh-LCV glycoproteins (gp42, gB, gH, and gL) and Rh-LCV has been previously shown to infect human B cells in vitro (25). The data from this study suggest that Rh gL does not mediate fusion with B cells because it cannot mediate the recruitment of EBV gB, indicating that there is a species-specific association between gB and gL or the gH/gL complex that is essential for B-cell fusion. Previous studies with HSV have demonstrated that the gH/gL complex associates with gB during membrane fusion (1, 2), and a recent study of human cytomegalovirus found that a small amount of gB coprecipitates with gH/gL (44). An interaction between EBV gB and gH or gL has not been detected using standard immunoprecipitation techniques, possibly because the association is a transient interaction that occurs during the fusion process, similar to what is observed for HSV in that gB and gH/gL interact after gD binds the receptor (1, 2, 42).

The function of the gH/gL complex during entry and fusion events appears to vary significantly among the herpesvirus family and subfamilies, as the necessity of gL for transport and processing of gH varies among the viruses. The expression of gL is generally thought to be necessary for the efficient processing and transport of gH for HSV and EBV (16, 33-35, 38, 51). HSV-1 virions lacking gL do not incorporate gH and are nonfunctional, making it difficult to examine the functions of the glycoproteins independently (38). Entry of murid herpesvirus 4 can occur independent of gL, which results in an altered conformation of gH that still traffics to the cell surface and can function in entry (10, 11). The human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) gH is also expressed at the cell surface independent of gL expression, but the ability of gH to mediate fusion or infection in the absence of gL has not been examined (14). It is interesting that these two viruses show similar characteristics in regard to the function of the gH/gL complex, as both murid herpesvirus 4 and Kaposi's sarcoma-associated herpesvirus are members of the rhadinovirus subfamily of gammaherpesviruses. Pseudorabies virus gH is incorporated into virions independent of gL, but infectivity and cell-to-cell spread are dependent on the expression of gL, providing some evidence for a functional role for pseudorabies virus gL that is separate from gH processing and transport (18). The work in the current study shows a similar distinction between the chaperone role of gL and a functional role in fusion with B cells. We found that while all of the chimeric EBV/Rh gL glycoproteins were able to bind gH and gp42 and were expressed at the cell surface at wild-type levels, only the EBV/Rh gL chimera expressing the amino-terminal 97 amino acids of EBV gL was able to mediate fusion with B cells at levels comparable to the fusion activity of the wild type. Similarly, reduced fusion was observed with EBV gL mutants containing the 54K and/or 94Q mutations, even though there was no change in cell surface expression. This is the first mutational analysis of EBV gL to provide evidence that EBV gL is involved in membrane fusion, but this result is consistent with data for HSV showing that the replacement of residue 156 of HSV-1 gL leads to a modest decrease in fusion and virus entry without interrupting gH trafficking (19).

The observations from the current study are consistent with results from Omerovic and Longnecker, who found that Rh gL and Rh gH/gL had almost undetectable levels of fusion with B cells (31). These findings contrast with the results from a study by Wu and Hutt-Fletcher, who found that Rh gL could mediate fusion in combination with EBV gH at various levels (49). The same study found low levels of fusion for Rh gH/gL, similar to our results, but also found that Rh gH could mediate fusion in the absence of Rh gL, which we have not found in our studies (data not shown). Possible explanations for the observed differences include variations in the cell-based fusion assays performed in the different studies and observations made using gH and gL homologs from other herpesviruses. As highlighted in the preceding paragraph, the absolute requirements for the chaperone function of gL and its function in fusion can vary between different human and animal herpesviruses, but in general, the most efficient fusion is observed when gH and gL are present. As a result, the absolute requirement for gL may have been overcome by differences in the way the fusion assays were performed, which could have resulted in more “permissive” fusion in the Wu and Hutt-Fletcher (49) studies than in the current studies. For the Omerovic and Longnecker (31) studies and the experiments that we are reporting, approximately 1/5 the number of effector cells and 1/10 the number of target cells were used compared to the numbers used by Wu and Hutt-Fletcher (49). In addition, the effector and target cells were mixed in approximately equal numbers in the current study, whereas in the Wu and Hutt-Fletcher (49) studies, twice the number of target cells in relationship to the number of effector cells were added.

Herpesvirus entry generally involves a number of glycoproteins working in concert to mediate binding and subsequent fusion of the two membranes, but the exact mechanism diverges based on the specific herpesvirus and target host cell. This is a result of herpesviruses targeting diverse cell types in animal and human hosts and the requirement for the acquisition of specific viral glycoproteins that allow this targeting. Thus, it is possible that the role of gL is determined by conformational changes based on whether gH is interacting with gp42, gL, or a cellular receptor. The crystal structures of gL and the gH/gL complex are still unknown, but a recent protein structure prediction study proposed that herpesvirus gL is distantly related to chemokine receptor ligands (50). For EBV gL, the amino-terminal region between amino acids 23 and 95 was specifically predicted to adopt a CC-type chemokine fold with conserved cysteines, hydrophilic and hydrophobic residues, and secondary structural elements (50). It is interesting that loss-of-function mutations in this region did not affect binding of gH or gp42, suggesting that the predicted chemokine-like fold region of gL may not function in binding these proteins but could be important in binding another glycoprotein or cellular ligand. Also of note is the interesting observation that the changes at amino acids 54 and 94 interchange a lysine or glutamine, implying that the two regions may form part of an interactive domain with another protein or key interaction in the domain structure of gL itself that requires these specific amino acids. In the predicted structure of gL based on the CC-type chemokine fold, amino acids 54 and 94 could be in close proximity.

Subramanian and Geraghty developed an assay to distinguish between hemifusion and complete fusion of the outer membranes and determined that for HSV-1, gD and gH/gL were sufficient for hemifusion while gB was necessary for full fusion (42). Based on the results from the current study, we would hypothesize that EBV-mediated fusion of B cells occurs through a similar mechanism that utilizes gp42, gH, gL, and gB in a sequential manner in which binding of gp42 to HLA class II induces a conformational change in the gp42/gH/gL tripartite complex that allows gL and, possibly, other members of the complex to allow recruitment or activation of gB to complete fusion. Rh gL can associate with EBV gH and gp42 at the cell surface but cannot engage EBV gB due to diversity in amino acids essential for the recruitment of gB, and the combination is therefore unable to mediate fusion. We found that fusion could be restored by either expressing Rh gL and gB together or by mutating the critical amino acids to make Rh gL more like EBV gL.