Evidence for a multiprotein gamma-2 herpesvirus entry complex

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV-68) gB, gH, gL, and gp150 all participate in entry. gB and gL are involved in binding, gB and gH are conserved fusion proteins, and gp150 inhibits cell binding until glycosaminoglycans are engaged. Here we show that a gH-specific antibody coprecipitates gB and thus that gH and gB are associated in the virion membrane. A gH/gL-specific antibody also coprecipitated gB, implying a tripartite complex of gL/gH/gB, although the gH/gB association did not require gL. The association was also independent of gp150, and gp150 was not demonstrably bound to gB or gH. However, gp150 incorporation into virions was partly gL dependent, suggesting that it too contributes to a single entry complex. gp150⁻ and gL⁻ gp150⁻ mutants bound better than the wild type to B cells and readily colonized B cells in vivo. Thus, gp150 and gL appear to be epithelial cell-adapted accessories of a core gB/gH entry complex. The cell binding revealed by gp150 disruption did not require gL and therefore seemed most likely to involve gB.     

Epstein-Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells

Entry of Epstein-Barr virus (EBV) into B cells is initiated by attachment of glycoprotein gp350 to the complement receptor type 2 (CR2). A complex of three glycoproteins, gH, gL, and gp42, is subsequently required for penetration. Gp42 binds to HLA class II, which functions as an entry mediator or coreceptor and, by analogy with other herpesviruses, gH is then thought to be involved virus-cell fusion. However, entry of virus into epithelial cells is thought to be different. It can be initiated by attachment by an unknown glycoprotein in the absence of CR2. There is no interaction between gp42 and HLA class II and instead a distinct complex of only the two glycoproteins gH and gL interacts with a novel entry mediator. Again, by analogy with other viruses gH is thought to be critical to fusion. To investigate further the different roles of gH in infection of the two cell types and to examine its influence on the assembly of the gH-gL-gp42 complex, we constructed two viruses, one in which the gH open reading frame was interrupted by a cassette expressing a neomycin resistance gene and the gene for green fluorescent protein and one as a control in which the neighboring nonessential thymidine kinase gene was interrupted with the same cassette. Virus lacking gH exited from cells normally, although loss of gH resulted in rapid turnover of gL and gp42 as well. The virus bound normally to B lymphocytes but could not infect them unless cells and bound virus were treated with polyethylene glycol to induce fusion. In contrast, virus that lacked the gH complex was impaired in attachment to epithelial cells and the effects of monoclonal antibodies to gH implied that this resulted from loss of gH rather than other members of the complex. These results suggest a role for gH in both attachment and penetration into epithelial cells.     

Binding-site interactions between Epstein-Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-cell membrane fusion

Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.     

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.     

Bovine herpesvirus type 4 glycoprotein L is nonessential for infectivity but triggers virion endocytosis during entry

The core entry machinery of mammalian herpesviruses comprises glycoprotein B (gB), gH, and gL. gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. However, the gL of the rhadinovirus murid herpesvirus 4 (MuHV-4) is nonessential for infection. In order to define more generally what role gL plays in rhadinovirus infections, we disrupted its coding sequence in bovine herpesvirus 4 (BoHV-4). BoHV-4 lacking gL showed altered gH glycosylation and incorporated somewhat less gH into virions but remained infectious. However, gL(-) virions showed poor growth associated with an entry deficit. Moreover, a major part of their entry defect appeared to reflect impaired endocytosis, which occurs upstream of membrane fusion itself. Thus, the rhadinovirus gL may be more important for driving virion endocytosis than for incorporating gH into virions, and it is nonessential for membrane fusion.     

Glycosaminoglycan interactions in murine gammaherpesvirus-68 infection

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces.     

Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.     

Antibody evasion by a gammaherpesvirus O-glycan shield

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog--gp180--contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target.     

Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays

Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.Epstein-Barr virus (EBV) is a large DNA virus belonging to the family of gammaherpesviruses. The virus is transmitted through saliva, and it can infect epithelial cells, as well as B cells, which provide the host latency reservoir (1, 22). Reactivation of the virus can occur intermittently, allowing virus infection of new hosts (1). Viral reactivation from latency is quickly controlled by the immune system. Primary infection with EBV can lead to the development of infectious mononucleosis. In addition, EBV infection is associated with a variety of human cancers, such as nasopharyngeal carcinoma, Hodgkin''s lymphoma, and Burkitt''s lymphoma (4, 8, 9, 27, 30). EBV is an enveloped virus which contains a number of membrane glycoproteins required for membrane fusion and viral entry into the host cell. EBV-mediated entry into epithelial cells requires the three viral glycoproteins gB, gH, and gL, which are conserved among herpesviruses, and entry into B cells additionally requires the viral glycoprotein gp42 (7, 16, 17). EBV lacking gp42 can attach to B cells but cannot enter them (29). However, EBV lacking gp42 can still efficiently infect epithelial cells. In fact, gp42 acts as an inhibitor of epithelial cell infection, and recent studies suggest that the level of gp42 in the virion regulates whether EBV preferentially infects epithelial cells or B cells (2). EBV gp42 has been shown to play an essential role in membrane fusion with B cells (7, 16, 17). It binds to human leukocyte antigen class II (HLA class II) proteins expressed on B cells to trigger virus-cell membrane fusion (6, 7, 10, 16, 25).Interestingly, EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain (21). Both the full-length form and the secreted gp42 form bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is not completely clear. In a virus-free cell-cell fusion assay, enhanced secretion of gp42 promotes fusion with B lymphocytes. Cleavage and secretion of gp42 are necessary for membrane fusion with B lymphocytes (24). However, membrane fusion with epithelial cells is inhibited by the presence of gp42 for both virus infection and cell-cell fusion (14, 29). This is likely due to the formation of a heterotrimeric gH/gL/gp42 complex that is unable to mediate membrane fusion with epithelial cells, possibly due to steric hindrance of gH/gL receptor binding (3, 11).The interaction of gH/gL and gp42 plays a key role in membrane fusion, but it has not yet been fully understood. The crystal structures of a gH/gL/gp42 complex and gH/gL alone have not been available. Although the crystal structures of gp42 alone and gp42/HLA class II complex have been solved (15, 19), the N-terminal region of gp42 (bound to gH/gL) is not visible in the structures, most likely due to its flexibility. Previous studies have shown that the N-terminal region of gp42 contains multiple functional regions, including a cleavage site that results in the secretion of gp42, a potential homodimerization region, and two segments (15 residues each) required for gH/gL binding (Fig. ​(Fig.11 A) (13, 14). Extensive gp42 N-terminal deletion analysis demonstrated that residues 37 to 56 and 72 to 96 include functional regions of the N terminus of gp42 required to trigger fusion and suggested that some of the residues within residues 67 to 71 are also important. Additional experiments showed that amino acids within segments from residues 47 to 61 and 67 to 81 are critical for binding gH/gL (13). Those two segments are spaced by five residues, which appear to act as a linker. Previous studies also showed that a 46-mer peptide spanning residues 36 to 81, mimicking the full-length gp42, binds to gH/gL and inhibits the formation of gH/gL/gp42 complex, thus blocking membrane fusion with epithelial cells and fusion with B cells (13, 14).Open in a separate windowFIG. 1.Schematic representation of EBV gp42. (A) Representation of wild-type gp42 showing the relative locations of known functional domains. The transmembrane domain is predicted to span residues 9 to 22 and is shown as a gray box. The site of gp42 cleavage is between residues 40 and 42 and is indicated by a black bar. The two gH/gL binding regions, spanning residues 47 to 61 and 67 to 87, are indicted with hatched boxes, flanking the five-residue linker. The C-terminal C-type lectin domain, including the hydrophobic pocket and HLA class II-binding region, is indicated by cross-hatched boxes. The putative dimerization region is indicated by a dotted box. (B) Amino acid sequence of gp42 peptide spanning residues 36 to 81 of the gp42 protein.In order to obtain a better understanding of the interaction of gp42 with gH/gL, and the role of this interaction in membrane fusion, we probed the gH/gL binding site by using 27 synthetic peptide analogs spanning residues 36 to 81 of gp42. Peptides were tested for binding affinity to soluble gH/gL by using a fluorescence polarization (FP) assay, probed for inhibition of B-cell and epithelial cell fusion in cell-based assays, and finally investigated for their ability to block epithelial cell infection. The data from the FP assay agreed very well with cell-cell fusion data and infection data, providing correlative data for peptide binding affinity and inhibition of cell-cell fusion and infection in an apparent competitive manner. We have defined the minimal length requirements for high-affinity binding to gH/gL and obtained a more detailed map of the key amino acids of the gp42 N terminus that are necessary for optimal gH/gL binding and inhibition of epithelial cell and B-cell membrane fusion.     

The bovine herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1. In this study, we characterized the positional homologous glycoprotein of bovine herpesvirus 4 (BoHV-4), encoded by the Bo10 gene. We identified a 180-kDa gene product, gp180, that was incorporated into the virion envelope. A Bo10 deletion virus was viable but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since compared to the wild-type virus, the Bo10 mutant virus was both less infectious for GAG-positive (GAG(+)) cells and more infectious for GAG-negative (GAG(-)) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the murid herpesvirus 4 gp150 and also to that of the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus, they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor.     

Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion

Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4) entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.     

Glycoprotein L disruption reveals two functional forms of the murine gammaherpesvirus 68 glycoprotein H

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells.     

Human herpesvirus‐6A gQ1 and gQ2 are critical for human CD46 usage

Based on genetic and antigenic differences and on their cell tropism, human herpes virus‐6 (HHV‐6) has been classified into two variants, HHV‐6A and HHV‐6B. Recently, these variants were re‐classified as two different species. The HHV‐6A glycoprotein complex, gH/gL/gQ1/gQ2 binds to its cellular receptor, CD46; however, the corresponding complex in HHV‐6B rarely binds to CD46. To determine which viral molecules in the glycoprotein complex determine HHV‐6A‐CD46 binding, each molecule of the HHV‐6A complex (i.e., gH, gL, gQ1, or gQ2) was replaced with the corresponding HHV‐6B molecule, and the ability of the replaced protein to be incorporated into the complex and the ability of the complex to bind CD46 were examined. It was found that when all four glycoproteins were expressed, they were able to form a tetrameric complex. However, a complex formed by HHV‐6A gH/gL/gQ1/gQ2 complexes replaced with HHV‐6B gQ1 or gQ2 scarcely bind CD46, whereas HHV‐6A complexes in which gH or gL was replaced with the HHV‐6B molecules did bind it. These results indicate that HHV‐6A gQ1 and gQ2 play an important role in CD46 binding.     

The amino terminus of Epstein-Barr virus glycoprotein gH is important for fusion with epithelial and B cells

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.     

The Murid Herpesvirus-4 gL regulates an entry-associated conformation change in gH

The glycoprotein H (gH)/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion.     

Murine gammaherpesvirus 68 lacking gp150 shows defective virion release but establishes normal latency in vivo

All gammaherpesviruses encode a virion glycoprotein positionally homologous to Epstein-Barr virus gp350. These glycoproteins are thought to be involved in cell binding, but little is known of the roles they might play in the whole viral replication cycle. We have analyzed the contribution of murine gammaherpesvirus 68 (MHV-68) gp150 to viral propagation in vitro and host colonization in vivo. MHV-68 lacking gp150 was viable and showed normal binding to fibroblasts and normal single-cycle lytic replication. Its capacity to infect glycosaminoglycan (GAG)-deficient CHO-K1 cells and NS0 and RAW264.7 cells, which express only low levels of GAGs, was paradoxically increased. However, gp150-deficient MHV-68 spread poorly through fibroblast monolayers, with reduced cell-free infectivity, consistent with a deficit in virus release. Electron microscopy showed gp150-deficient virions clustered on infected-cell plasma membranes. MHV-68-infected cells showed reduced surface GAG expression, suggesting that gp150 prevented virions from rebinding to infected cells after release by making MHV-68 infection GAG dependent. Surprisingly, gp150-deficient viruses showed only a transient lag in lytic replication in vivo and established normal levels of latency. Cell-to-cell virus spread and the proliferation of latently infected cells, for which gp150 was dispensable, therefore appeared to be the major route of virus propagation in an infected host.     

Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells

The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.     

Capturing the herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment

Herpes simplex virus (HSV) entry requires the core fusion machinery of gH/gL and gB as well as gD and a gD receptor. When gD binds receptor, it undergoes conformational changes that presumably activate gH/gL, which then activates gB to carry out fusion. gB is a class III viral fusion protein, while gH/gL does not resemble any known viral fusion protein. One hallmark of fusion proteins is their ability to bind lipid membranes. We previously used a liposome coflotation assay to show that truncated soluble gB, but not gH/gL or gD, can associate with liposomes at neutral pH. Here, we show that gH/gL cofloats with liposomes but only when it is incubated with gB at pH 5. When gB mutants with single amino acid changes in the fusion loops (known to inhibit the binding of soluble gB to liposomes) were mixed with gH/gL and liposomes at pH 5, gH/gL failed to cofloat with liposomes. These data suggest that gH/gL does not directly associate with liposomes but instead binds to gB, which then binds to liposomes via its fusion loops. Using monoclonal antibodies, we found that many gH and gL epitopes were altered by low pH, whereas the effect on gB epitopes was more limited. Our liposome data support the concept that low pH triggers conformational changes to both proteins that allow gH/gL to physically interact with gB.     

The Epstein-Barr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells.

Glycoprotein gp85, the product of the BXLF2 open reading frame (ORF), is the gH homolog of Epstein-Barr virus (EBV) and has been implicated in penetration of virus into B cells. Like its counterparts in other herpesviruses, it associates with a gL homolog, gp25, which is the product of the BKRF2 ORF. Unlike the gH homologs of other herpesviruses, however, gp85 also complexes with two additional glycoproteins of 42 and 38 kDa. Glycoproteins gp42 and gp38 were determined to be alternatively processed forms of the BZLF2 gene product. Coexpression of EBV gH and gL facilitated transport of gH to the cell surface and resulted in formation of a stable complex of gH and gL. It also restored expression of an epitope recognized by monoclonal antibody E1D1, which immunoprecipitates the native gH complex but not recombinant gH expressed in isolation. Coexpression of gH, gL, and the BZLF2 ORF restored expression of an epitope recognized by a second monoclonal antibody, F-2-1, which immunoprecipitates the native gH-gL-gp42/38 complex but not the complex of recombinant gH and gL alone. The epitope recognized by antibody F-2-1 was mapped to the BZLF2 gene product itself. Antibody F-2-1 inhibited the ability of EBV to infect B lymphocytes but had no effect on the ability of the virus to infect the epithelial cell line SVK-CR2. In contrast, antibody E1D1 had no effect on infection of the B-cell line but inhibited infection of the epithelial cell line. These results indicate that penetration of the two cell types by EBV involves differential use of the gH-gL-gp42/38 complex and suggest the hypothesis that the BZLF2 gene product has evolved as a unique adaptation to infection of B lymphocytes by EBV.