Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain

Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. gD serves as a receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in an N-terminal domain carrying the receptor-binding sites and a C-terminal domain carrying the profusion domain, required for fusion but not receptor binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other, we searched for biochemical defined complexes in infected and transfected cells and in virions. Previously, interactions were detected in transfected whole cells by split green fluorescent protein complementation (Atanasiu, D., Whitbeck, J. C., Cairns, T. M., Reilly, B., Cohen, G. H., and Eisenberg, R. J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18718–18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532–11537); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540–4544). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240–260 portion and downstream with Thr³⁰⁴-Pro³⁰⁵ as critical residues and of the binding site to gH/gL at the amino acids 260–310 portion with Pro²⁹¹-Pro²⁹² as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain.     

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.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD,gH/gL,and gB

Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.Herpesviruses enter cells by fusing their envelopes with host cell membranes either by direct fusion at the plasma membrane or by pH-dependent or -independent endocytosis, depending on the target cell (27, 29, 39). Although the entry pathways of other enveloped viruses are similarly diverse (8), all systems for which molecular details have been obtained rely on a single fusion protein (43); herpesviruses are unique in their use of gB and the gH/gL heterodimer as their core fusion machinery (17, 37). Some herpesviruses employ additional receptor-binding glycoproteins, e.g., herpex simplex virus (HSV) gD, and others require gH/gL-associated proteins, e.g., UL128-131 of cytomegalovirus (CMV) (34) or gp42 of Epstein-Barr virus (EBV) (42). This complexity has made it difficult to unravel the mechanism of herpesvirus entry.Ultrastructural and biochemical studies have shown that for HSV entry, binding of gD to one of its receptors, either HVEM or nectin-1 (36), activates the downstream events that drive gB- and gH/gL-dependent fusion (17). The structure of the gB ectodomain (18) bears striking structural homology to the postfusion form of the single fusion protein G of vesicular stomatitis virus (VSV) (33). However, unlike the other class III viral fusion proteins, VSV G and baculovirus gp64 (5), gB requires gH/gL to function in virus-cell and cell-cell fusion (17). A number of investigations support the concept that gH/gL might also be fusogenic (13, 41). Some have suggested that a multiprotein complex comprised of gD, gH/gL, and gB might be assembled to cause fusion (14). Using bimolecular complementation (BiMC), we and others showed that interactions can occur between half enhanced yellow fluorescent protein (EYFP)-tagged gB (e.g., gBn) and tagged gD (e.g., gDc) or between tagged gD and tagged gH (1, 3). However, because these occur in the absence of one of the other essential components, e.g., a receptor, we could not assess their functional significance. Importantly, gH/gL and gB interact with each other only in response to receptor binding by gD (1-3, 12). We subsequently showed that this interaction precedes fusion and is required for it to occur (2). Thus, we were able to conclude that gH/gL must interact with gB, whether transiently or stably, in order for fusion to occur. Whether gD was indeed involved in a multiprotein complex was not clear, nor was the role of gH/gL in promoting fusion initiated by gD-receptor binding. The lack of structural data for gH/gL left its potential role as a fusogen unresolved.However, in 2010, the structure of gH/gL of HSV-2 was solved in collaboration with Chowdary et al. (12). Structurally, gH/gL does not resemble any known viral fusogen, thereby forcing a reconsideration of its function in promoting virus-cell and cell-cell fusion. We hypothesized that gH/gL does not likely act as a cofusogen with gB but rather regulates fusion by gB (12).In this report, we argue that as a regulator of fusion, gH/gL might not have to be in the same membrane as gB in order to regulate its activity, i.e., gH/gL on one cell might promote fusion of gB expressed by another cell, as long as gD and a gD receptor are also present. In support of this, it was recently shown that gH/gL and gB of human cytomegalovirus (HCMV) can cause cell-cell fusion when expressed by distinct cells (in trans) (41). We present evidence that HSV gB and gH/gL can cause cell-cell fusion when they are expressed in trans, a process that requires both gD and a gD receptor. Although the efficiency of fusion in trans is low compared with that of fusion when gB and gH/gL are in cis (as they would be when in the virus), separation of these proteins onto two different cells enabled us to dissect the order in which each protein acts along the pathway to fusion. Moreover, we found that a combination of soluble gD (not membrane bound) and soluble gH/gL (also not membrane bound) could trigger fusion of receptor-bearing cells that had been transfected with the gene for gB. Our data show that gD, gH/gL, and gB act in a series of steps whereby gD is first activated by binding its cell receptor. Previous studies showed that receptor binding causes gD to undergo conformational changes (17). Based on the data in this paper, we propose that these changes then enable gD to activate gH/gL into a form that in turn binds to and activates the fusogenic activity of gB. Although we do not know whether any of these reactions result in the formation of a stable complex, our data suggest that gB is the sole HSV fusogen and that gD and gH/gL act to upregulate cell-cell fusion and most likely virus-cell fusion, leading to HSV entry.     

Coiled-coil domains in glycoproteins B and H are involved in human cytomegalovirus membrane fusion

Human cytomegalovirus (CMV) utilizes a complex route of entry into cells that involves multiple interactions between viral envelope proteins and cellular receptors. Three conserved viral glycoproteins, gB, gH, and gL, are required for CMV-mediated membrane fusion, but little is known of how these proteins cooperate during entry (E. R. Kinzler and T. Compton, submitted for publication). The goal of this study was to begin defining the molecular mechanisms that underlie membrane fusion mediated by herpesviruses. We identified heptad repeat sequences predicted to form alpha-helical coiled coils in two glycoproteins required for fusion, gB and gH. Peptides derived from gB and gH containing the heptad repeat sequences inhibited virus entry when introduced coincident with virus inoculation onto cells or when mixed with virus prior to inoculation. Neither peptide affected binding of CMV to fibroblasts, suggesting that the peptides inhibit membrane fusion. Both gB and gH coiled-coil peptides blocked entry of several laboratory-adapted and clinical strains of human CMV, but neither peptide affected entry of murine CMV or herpes simplex virus type 1 (HSV-1). Although murine CMV and HSV-1 gB and gH have heptad repeat regions, the ability of human CMV gB and gH peptides to inhibit virus entry correlates with the specific residues that comprise the heptad repeat region. The ability of gB and gH coiled-coil peptides to inhibit virus entry independently of cell contact suggests that the coiled-coil regions of gB and gH function differently from those of class I, single-component fusion proteins. Taken together, these data support a critical role for alpha-helical coiled coils in gB and gH in the entry pathway of CMV.     

Reevaluating Herpes Simplex Virus Hemifusion

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.Membrane fusion is an essential step during the entry process of enveloped viruses, such as herpes simplex virus (HSV), into target cells. The general pathway by which enveloped viruses fuse with target membranes through the action of fusion proteins is fairly well understood. Viral fusion proteins use the free energy liberated during their own protein conformational changes to draw the two membranes—viral and target—together. Fusion is thought to proceed through a “hemifusion” intermediate, in which the proximal leaflets of the two bilayers have merged but a viral pore has not yet formed and viral contents have not yet mixed with the cell cytoplasm (10, 38). Fusion proteins then drive the completion of fusion, which includes fusion pore formation, pore enlargement, and complete content mixing.HSV, an enveloped neurotropic virus, requires four glycoproteins—glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL)—to execute fusion (9, 57, 60). gB, gD, and gH are membrane bound; gL is a soluble protein which complexes with gH to form a heterodimer (gH/gL). HSV-1 gH is not trafficked to the cell or virion surface in the absence of gL (32, 52). The requirement of four entry glycoproteins sets HSV apart from other enveloped viruses, most of which induce fusion through the activity of a single fusion protein. Although the specific mode of HSV entry is cell type dependent—fusion with neurons and Vero cells occurs at the plasma membrane at neutral pH; fusion with HeLa and CHO cells involves pH-dependent endocytosis, and fusion with C10 cells involves pH-independent endocytosis (42, 45)—all routes of entry require gD, gB, and gH/gL. Furthermore, although some discrepancies between virus-cell and cell-cell fusion have been observed (8, 44, 55, 58), both generally require the actions of gD, gB, and gH/gL.Much work has gone toward the understanding of how the required HSV entry glycoproteins work together to accomplish fusion, and many questions remain. After viral attachment, mediated by glycoprotein C and/or gB (54), the first step in HSV fusion is thought to be gD binding a host cell receptor (either herpesvirus entry mediator [HVEM], nectin-1, nectin-2, or heparan sulfate modified by specific 3-O-sulfotransferases) (56). The gD-receptor interaction induces a conformational change in gD (39) that is thought to trigger gD-gB and/or gD-gH/gL interactions that are required for the progression of fusion (1-4, 13, 18, 23, 49).gB and gH/gL are considered the core fusion machinery of most herpesviruses. The HSV-1 gB structure revealed surprising structural homology to the postfusion structures of two known viral fusion proteins (31, 35, 51). This structural homology indicates that despite not being sufficient for HSV fusion, gB is likely a fusion protein. Although the gB cytoplasmic tail (CT) is not included in the solved structure, it acts as a regulator of fusion, as CT truncations can cause either hyperfusion or fusion-null phenotypes (5, 17). The gB CT has been proposed to bind stably to lipid membranes and negatively regulate membrane fusion (12). Another proposed regulator of gB function is gH/gL. Despite conflicting accounts of whether gD and a gD receptor are required for the interaction of gH/gL and gB (1, 3, 4), a recent study indicates that gH/gL and gB interact prior to fusion and that gB may interact with target membranes prior to an interaction with gH/gL (2). The gB-gH/gL interaction seems to be required for the progression of fusion.Compared to the other required HSV entry glycoproteins, the role of gH/gL during fusion remains enigmatic. Mutational studies have revealed several regions of the gH ectodomain, transmembrane domain (TM), and CT that are required for its function (19, 25, 26, 30, 33). gH/gL of another herpesvirus, Epstein-Barr virus (EBV), have been shown to bind integrins during epithelial cell fusion, and soluble forms of HSV gH/gL have been shown to bind cells and inhibit viral entry in vitro (24, 46). However, the role of gH/gL binding to target cells in regard to the fusion process remains to be determined.There are some lines of evidence that suggest that gH/gL is a fusion protein. The gH/gL complexes of VZV and CMV have been reported to independently execute some level of cell-cell fusion (14, 37). HSV-1 gH/gL has been reported to independently mediate membrane fusion during nuclear egress (15). In silico analyses and studies of synthetic HSV gH peptides have proposed that gH has fusogenic properties (20, 21, 25-28). Finally, of most importance to the work we report here, gH/gL has been shown to be sufficient for induction of hemifusion in the presence of gD and a gD receptor, further promoting the premise that gH/gL is a fusion protein (59). However, the recently solved crystal structure of HSV-2 gH/gL revealed a tight complex of gH/gL in a “boot-like” structure, which bears no structural homology to any known fusion proteins (11). The HSV-2 gH/gL structure and research demonstrating that gH/gL and gB interactions are critical to fusion (2) have together prompted a new model of HSV fusion in which gH/gL is required to either negatively or positively regulate the activity of gB through direct binding.We wanted to investigate the ability of a previously reported gH CT mutant, 824L, to execute hemifusion. 824L gH contains a five-residue insertion at gH residue 824, just C-terminal of the TM domain. 824L is expressed on cell surfaces and incorporated into virions at levels indistinguishable from those of wild-type gH by either cell-based ELISA or immunoblotting, yet it is nonfunctional (33). We relied on a fusion assay capable of detecting hemifusion, developed by Subramanian et al. (59), which we modified to include an additional control for hemifusion or nonenlarging pore formation, glycosylphosphatidylinositol (GPI)-linked hemagglutinin (GPI-HA). GPI-HA is a variant of the influenza virus fusion protein, HA, that is known to stall the fusion process before enlarging fusion pores are formed.We were surprised to find that in our hands, gD, a gD receptor, and gH/gL were insufficient for the induction of hemifusion or lipid mixing in both cell-based and virus-based fusion assays. We found that gD, gB, and gH/gL are all required to observe lipid mixing. Further, we found that gB, gD, gL, and 824L gH are insufficient for lipid mixing. Our findings support the emerging view, based on gH/gL structure, that the gH/gL complex does not function as a fusion protein and does not insert into target membranes to initiate the process of fusion through a hemifusion intermediate. Our findings also further demonstrate that mutations in the CT of gH can have a dramatic effect on the ability of gH/gL to function in fusion.     

Membrane requirement for folding of the herpes simplex virus 1 gB cytodomain suggests a unique mechanism of fusion regulation

Herpes simplex virus type 1 (HSV-1) enters cells by fusion of its envelope with a host cell membrane, which requires four viral glycoproteins and a cellular receptor. Viral fusion glycoprotein B (gB) mediates membrane fusion through the action of its ectodomain, while its cytoplasmic domain (cytodomain) regulates fusion from the opposite face of the membrane by an unknown mechanism. The gB cytodomain appears to restrict fusion, because point or truncation mutations within it increase the extent of fusion (syn mutations). Previously, we showed that the hyperfusion phenotype correlated with reduced membrane binding in gB syn truncation mutants and proposed that membrane binding was important in regulating fusion. Here, we extended our analysis to three syn point mutants: A855V, R858H, and A874P. These mutations produce local conformational changes, with some affecting membrane interaction, which suggests that while syn mutants may deregulate fusion by somewhat different mechanisms, maintaining the wild-type (WT) conformation is critical for fusion regulation. We further show that the presence of a membrane is necessary for the cytodomain to achieve its fully folded conformation and propose that the membrane-bound form of the cytodomain represents its native conformation. Taken together, our data suggest that the cytodomain of gB regulates fusion by a novel mechanism in which membrane interaction plays a key role.     

Pseudorabies virus glycoprotein M inhibits membrane fusion

A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873-875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.     

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.     

A heptad repeat in herpes simplex virus 1 gH, located downstream of the alpha-helix with attributes of a fusion peptide, is critical for virus entry and fusion

Entry of herpes simplex virus 1 (HSV-1) into cells occurs by fusion with cell membranes; it requires gD as the receptor binding glycoprotein and the trigger of fusion, and the trio of the conserved glycoproteins gB, gH, and gL to execute fusion. Recently, we reported that the ectodomain of HSV-1 gH carries a hydrophobic alpha-helix (residues 377 to 397) with attributes of an internal fusion peptide (T. Gianni, P. L. Martelli, R. Casadio, and G. Campadelli-Fiume, J. Virol. 79:2931-2940, 2005). Downstream of this alpha-helix, a heptad repeat (HR) with a high propensity to form a coiled coil was predicted between residues 443 and 471 and was designated HR-1. The simultaneous substitution of two amino acids in HR-1 (E450G and L453A), predicted to abolish the coiled coil, abolished the ability of gH to complement the infectivity of a gH-null HSV mutant. When coexpressed with gB, gD, and gL, the mutant gH was unable to promote cell-cell fusion. These defects were not attributed to a defect in heterodimer formation with gL, the gH chaperone, or in trafficking to the plasma membrane. A 25-amino-acid synthetic peptide with the sequence of HR-1 (pep-gH(wt25)) inhibited HSV replication if present at the time of virus entry into the cell. A scrambled peptide had no effect. The effect was specific, as pep-gH(wt25) did not reduce HSV-2 and pseudorabies virus infection. The presence of a functional HR in the HSV-1 gH ectodomain strengthens the view that gH has attributes typical of a viral fusion glycoprotein.     

Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.Entry of herpes simplex virus (HSV) into the cell requires a multipartite apparatus made of a quartet of viral glycoproteins, gD, gB, and the heterodimer gH/gL, and a multistep process that culminates in the fusion of the virion envelope with cell membranes (5, 6, 10, 25, 36, 41). gD serves as the receptor-binding glycoprotein, able to interact with alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and, in some cells, modified heparan sulfate (9, 13, 30, 39). It can also be engineered to accept heterologous ligands able to interact with selected receptors present on tumor cells and thus represents a tool to redirect HSV tropism (21, 28, 29, 42). The heterodimer gH/gL and gB execute fusion and constitute the conserved fusion apparatus across the Herpesviridae family. gB structure in the postfusion conformation shows a trimer with a central coiled coil (19). gH shows elements typical of type 1 fusion glycoproteins, in particular, helices able to interact with membranes, and two heptad repeats potentially able to form a coiled coil (12, 15-18). The discovery that a soluble form of gD enables entry of gD-null virions revealed that gD serves the additional function of triggering fusion and led to the view that the major roles of gD are to sense that virus has reached a receptor-positive cell and to signal to gB and gH/gL that fusion is to be executed (8). Biochemical and structural analyses showed that the C-terminal region of the gD ectodomain, containing the profusion domain required for fusion but not for receptor binding, can undergo major conformational changes (11, 24). Specifically, it binds the gD core and masks or hinders the receptor binding sites, conferring upon the molecule a closed, auto-inhibited conformation (24). Alternatively, it may unfold, conferring upon gD an open conformation. It was proposed that the C terminus of gD unfolds from gD core at receptor binding and recruits gH/gL and gB to a quaternary complex. A key feature of the model was that complexes among the glycoprotein quartet were not preformed, but, rather, they would assemble at the onset of or at fusion execution.Central to understanding HSV entry/fusion has become the dissection of the interactions that occur among the members of the glycoprotein quartet and their significance to the process. A first evidence of a gD-gH/gL interaction was provided in coimmunoprecipitation studies (35). Interactions between gD and gH/gL and between gD and gB were subsequently detected by split green fluorescence protein (GFP) complementation assays, implying that gD can recruit gB and gH/gL independently of one another, a result that argues against a stepwise recruitment of the glycoproteins to gD. In agreement with the proposed model, the interaction between gH/gL and gB was detected in the presence of transfected or soluble gD (1, 2). However, further studies highlighted levels of complexity not foreseen in the initial model. Thus, pull-down analyses showed that the interaction sites in gD with gB and with gH/gL lie in part outside the C-terminal portion of the gD ectodomain, that resting virions contain small amounts of gD in complex with gB and with gH/gL prior to encountering cells, and that de novo gD-gB complexes were not detected at virus entry into the cell (14).A major objective of current studies was to analyze the interaction of gB with gH/gL. We documented the interaction by two independent assays, i.e., by a complementation assay of split yellow fluorescent protein Venus (herein indicated as YFP) (31) in whole cells and, biochemically, by a pull-down assay. The latter was applied recently in our laboratory and is based on the ability of One-Strep-tagged proteins (e.g., gH) to specifically absorb to Strep-Tactin resin and thus retain any protein in complex (14). To preliminarily search for gB regions critical for the interaction with gH/gL, we engineered chimeric forms of HSV-1 and human herpesvirus 8 (HHV-8) gB in which the cysteines were preserved. While none of the chimeras was completely defective in the interaction, the interactions in the chimeras carrying substitutions in the pleckstrin-like domain 1—the domain that carries the bipartite fusion loops—were hampered. Altogether, the results underscore the ability of gB to interact with gH/gL in the absence of gD and favor the view that sites in gB for interaction with gH/gL involve multiple contacts, one of which is located in the domain that carries the fusion loops.     

Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH+gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.     

Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.     

Fusion between Perinuclear Virions and the Outer Nuclear Membrane Requires the Fusogenic Activity of Herpes Simplex Virus gB

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic “fusion loops.” These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.Herpesvirus glycoproteins gB and gH/gL participate in two separate membrane fusion events that occur during different stages of virus replication. First, during virus entry into cells, gB and gH/gL promote fusion between the virion envelope and either the plasma membrane or endosomes (reviewed in references 6, 21, 27, and 39). Second, herpes simplex virus (HSV) gB and gH (likely complexed to form a heterodimer with gL), and likely homologues in other herpesviruses, promote nuclear egress (12). Herpesvirus capsids are produced in the nucleus and cross the nuclear envelope (NE) by envelopment at the inner nuclear membrane (NM), producing perinuclear virions that then fuse with the outer NM (reviewed in references 35 and 36). There is evidence that HSV gB and gH/gL function in a redundant fashion in fusion between enveloped, perinuclear virus particles and the outer NM (12), whereas both gB and gH/gL are essential for entry fusion (8, 13, 38). Much more is known about the mechanisms involved in entry fusion than those involved in egress fusion, and many important questions remain in terms of how these two membrane fusion processes relate to each other.Entry of HSV into cells involves interactions between the viral receptor-binding protein gD and the gD receptors (16, 28, 30, 37). When gD binds to its receptors, there are conformational changes in gD which apparently activate gB and gH/gL, so that these glycoproteins promote fusion involving the virion envelope and cellular membranes (21, 32). By using split green fluorescent protein fusion proteins, also denoted bimolecular complementation, two groups showed that gD binding to gD ligands triggers interactions between gB and gH/gL and that this is accompanied by cell-cell fusion (1, 2). There is also evidence that gB and gH/gL contribute to different stages of membrane fusion. When gH/gL is expressed with gD, there is hemifusion (mixing of the outer leaflets of membranes) of adjacent cells, and this partial fusion is apparently mediated by gH/gL (41). However, full fusion (mixing of both inner and outer leaflets) occurs only when gB is coexpressed with gD and gH/gL (41). Also supporting a role for gH in membrane fusion, peptides based on heptad repeats in gH can disrupt model membranes (14, 15, 17). HSV gB is a class III fusion protein, structurally similar to vesicular stomatitis virus G protein, with a three-stranded coil-coil barrel in the central region of the molecule reminiscent of class I fusion proteins, e.g., influenza virus hemagglutinin (22). Therefore, herpesvirus gB and gH/gL differ substantially from the fusion proteins expressed by all other well-studied viruses because both gB and gH/gL participate directly in membrane fusion, apparently functioning in different aspects of entry fusion.HSV gB and other viral class III fusion proteins differ from class I fusion proteins that have N-terminal, hydrophobic fusion peptides because class III fusion proteins possess internal bipartite “fusion loops” composed of both hydrophobic and hydrophilic residues (3, 22). In the solved structure of the HSV gB ectodomain, which might represent a postfusion form of the protein, the fusion loops are located near the base of the molecule, adjacent to the virion envelope (22). Mutant forms of gB with single amino acid substitutions in these fusion loops displayed diminished cell-cell fusion activity when transfected into cells with gD and gH/gL (20). Cell-cell fusion approximates the fusion that occurs during entry, defining the minimal fusion machinery, although there are differences between entry and cell-cell fusion (10). Moreover, full-length gB molecules with fusion loop mutations failed to complement gB null HSV (19). Recently, it was demonstrated that the HSV gB extracellular domain can interact with liposomes in vitro and that this binding depends upon gB''s fusion loops (19).Herpesvirus capsids are assembled in the nucleus and acquire an envelope by budding through the inner NM. For a short time, enveloped virus particles are found in the space between the inner and outer NMs (perinuclear space), but then the envelopes of these particles fuse with the outer NM, releasing capsids into the cytoplasm (reviewed in references 35 and 36). Cytoplasmic capsids acquire a second envelope by budding into the trans-Golgi network, and this secondary envelopment involves redundant or additive functions of gE/gI and gD, i.e., either of these glycoproteins will suffice (11). The second step of the nuclear egress pathway involving membrane fusion between the envelope of perinuclear particles and the outer NM requires HSV glycoproteins gB and gH/gL (12). HSV double mutants lacking both gB and gH accumulate enveloped virus particles in the perinuclear space and in herniations, i.e., membrane vesicles that bulge into the nucleoplasm and derive from the inner NM (12). These observations, coupled with the evidence that gB and gH/gL are fusion proteins, suggested that gB and gH/gL promote the fusion between virus particles and the outer NM. However, there is one important difference between nuclear egress fusion and entry fusion. Virus mutants lacking either gB or gH are unable to enter cells, but such mutants have fewer defects in nuclear egress than double mutants lacking both gB and gH (12). Thus, as with secondary envelopment that involves gD and gE/gI, glycoproteins gB and gH/gL act in a redundant or additive fashion to mediate the fusion between the envelope of perinuclear virus particles and the outer NM. It is also important to note that there appear to be other mechanisms by which HSV particles can exit the perinuclear space. For example, although a substantial number of gB gH null double mutants accumulated in herniations (increased by ∼10-fold), some virions were seen on cell surfaces, although their numbers were reduced by ∼2.5- to 5-fold compared with those of wild-type HSV (12, 46).HSV entry fusion is triggered by gD binding to one of its ligands. However, it is not clear what triggers fusion of the envelope of perinuclear particles with the outer NM. gD, gB, gH, gM, gK, and other viral membrane proteins are all present in NMs and in perinuclear virus particles (4, 12, 25, 40, 42, 44). It seems unlikely that there are substantial quantities of known gD receptors in NMs, although this has not been carefully examined and there may well be unidentified gD receptors present in NMs. However, if fusion at NMs is not activated by gD binding to gD receptors, there must be other mechanisms to trigger this fusion. There is evidence that HSV gK negatively regulates fusion at the NE because (i) overexpression of gK causes enveloped virus particles to accumulate in the perinuclear space (25) and (ii) gK is primarily localized to the endoplasmic reticulum and NM and is not substantially found in extracellular virions (26, 34). Another potential regulatory mechanism for fusion at the outer NM involves phosphorylation of the cytoplasmic domain of gB by the HSV kinase US3 (46). An HSV recombinant lacking gH and expressing a mutant gB with a substitution, T887A, affecting an amino acid in the gB cytoplasmic domain displayed reduced US3-dependent phosphorylation and accumulated enveloped virus particles in herniations (46). This mutation in gB did not alter HSV entry into cells (31, 46). Together, these results suggest that HSV fusion with the outer NM differs from entry fusion in some, but likely not all, important mechanistic details.Given that both gB and gH/gL are well established as fusion proteins for virus entry, we hypothesized that these glycoproteins directly mediate the membrane fusion that occurs between the envelope of perinuclear virus particles and the outer NM (12, 46). However, there are other possibilities. For example, it is conceivable that loss of both gB and gH alters the structure of the envelope of perinuclear HSV virions so that other HSV glycoproteins (that directly promote fusion) are affected. To address this issue and extend our understanding of how gB functions in nuclear egress fusion, we constructed HSV recombinants that express mutant forms of gB with substitutions in the fusion loops. These viruses also lacked gH, making nuclear egress totally dependent on a functional form of gB. By propagating these recombinants using gH-expressing cells, we could produce virus particles including gH and the mutant gB molecules. These HSV recombinants expressing gH as well as gB fusion loops, W174R, W174Y, Y179K, and A261D, were all unable to enter cells. However, two recombinants, expressing W174Y and Y179K, exhibited some cell-to-cell spread while the other two, expressing W174R and A261D, did not spread beyond single infected cells. All four recombinants infected into cells lacking gH exhibited defects in nuclear egress. These results provide strong support for the hypothesis that gB acts directly to mediate the fusion of the virion envelope with the outer NM during HSV egress.     

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.     

Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.Herpes simplex virus (HSV) entry into cells requires four viral envelope glycoproteins (gB, gD, and the heterodimer gH/gL) as well as a cell surface gD receptor (reviewed in references 31, 42, 43, and 49). When gD binds its receptor, it undergoes conformational changes that are essential to activate the fusion machinery, gB and gH/gL. In addition to being essential for virus entry, both gH/gL and gB play important roles in primary fusion events that occur during egress of the capsid from the nuclei of infected cells (22). gB and gH/gL constitute the core fusion machinery of all members of the Herpesviridae.The mechanisms by which gB and gH/gL function individually and in concert during fusion are topics of intense investigations. Peptides based on predicted heptad repeats in gH block virus entry and have the ability to bind and disrupt model membranes (24, 26, 27). In addition, gH/gL can achieve hemifusion of adjacent cells in the absence of other herpesvirus proteins (50). These studies imply that gH/gL has fusogenic properties. Previously, we showed that both virion gB and soluble wild-type (WT) gB (gB730t), but not gD or gH/gL, bind to cells and associate with lipid rafts (10). Like gH/gL, several synthetic gB peptides induced the fusion of large unilamellar vesicles and inhibited herpesvirus infection (23, 24). Thus, it appears that both gB and gH/gL may be fusion proteins, a theory strengthened by data showing that either gB or gH/gL is sufficient for membrane fusion during nuclear egress (22). Additionally, gB730t blocks virus entry into cells deficient in heparan sulfate proteoglycans (HSPGs), suggesting that it competes with virion gB for an obligate cell surface receptor (9). A recent study suggested that paired immunoglobulin-like type 2 receptor alpha (PILRα) may serve this role for at least some cell types (47).The crystal structure of gB is now known for both HSV type 1 (HSV-1) (32) and Epstein-Barr virus (EBV) (6). Interestingly, gB is structurally related to two other viral fusion proteins, the vesicular stomatitis virus (VSV) G protein (45) and the baculovirus gp64 protein (34). VSV G, gB, and most recently, baculovirus gp64 were placed into a newly formed group of fusion proteins, the class III proteins. Class III fusion proteins have similar individual domain structures and contain a central three-stranded coiled coil reminiscent of the class I proteins. Whereas class I proteins have an N-terminal fusion peptide, class III proteins have internal bipartite fusion loops within domain I (shown in Fig. ​Fig.1A1A for gB) which are similar to the single fusion loop of class II fusion proteins. However, the class II fusion loop is composed entirely of hydrophobic amino acids, whereas the fusion loops of gB have both hydrophobic and charged residues (32, 34, 45). Unlike G or gp64, which are the sole fusion proteins for their respective viruses, gB requires gH/gL to function in fusion and entry.Open in a separate windowFIG. 1.HSV gB hydrophobic ridge is surrounded by charged residues on the surface of the molecule. A ribbon diagram of the HSV protomer (A) and molecular surface representation of the trimer (B) are shown. In each, one protomer is colored by secondary structure succession, using blue (domain I), green (domain II), yellow (domain III), orange (domain IV), and red (domain V). The box in panel A shows the primary amino acid sequences of the fusion loops. The box in panel B shows the base of the gB trimer, rotated 90°. For the boxes in both panels A and B, highlighted hydrophobic residues are colored in blue and charged residues are shown in red. All structural figures were generated, in part, using PyMOL Molecular Graphics System software.In our previous study, we used site-directed mutagenesis to show that three hydrophobic amino acids within the gB loops (W174, Y179, and A261) are essential for gB function (29). Similar studies of VSV G, gp64, and EBV gB support the notion that hydrophobic amino acids of both fusion loops are critical for fusion (34, 44, 51) and together constitute a fusion domain. Recently, bimolecular complementation was used to show that gB and gH/gL interact with each other concomitantly with fusion and that this interaction is triggered by binding of gD to its cellular receptor (3, 4). Thus, gB may function cooperatively with gH/gL, yet each may have some fusogenic potential on its own.The goal of the experiments reported here was twofold. First, we wanted to complete our mutagenic analysis of all of the residues in the two putative fusion loops of HSV gB. Our data show that the two fusion loops constitute a structural “subdomain” wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. We hypothesize that two charged residues on one side of the ridge enhance the ability of the hydrophobic residues to interact with target membranes and to function in fusion.Our second goal was to assess the effects of mutations in the fusion loops on the function of gB in cell binding, blocking of entry, and insertion into lipid membranes. Therefore, we constructed recombinant baculoviruses, with each carrying the gene for a truncated version (residues 31 to 730) of one of four mutant forms of gB (W174R, Y179S, H263A, and R264A). We found that the mutant proteins were able to efficiently block virus entry, suggesting that the fusion loops do not participate in protein-receptor binding. However, all four mutant proteins were impaired in cell binding compared to WT gB730t. Whereas WT gB730t associated with liposomes in a flotation assay, soluble truncated forms of HSV gD and gH/gL did not, consistent with our previous finding that gB730t associates with lipid rafts on cell surfaces (8). In contrast to WT gB730t, the gB mutant proteins were either impaired or unable to bind liposomes. Our data suggest that gB has an intrinsic ability to associate with a target membrane via its fusion domain.     

Human cytomegalovirus glycoprotein gO complexes with gH/gL, promoting interference with viral entry into human fibroblasts but not entry into epithelial cells

A complex of five human cytomegalovirus virus (HCMV) proteins, gH, gL, UL128, UL130, and UL131 (gH/gL/UL128-131), is essential for virus entry into epithelial cells. We previously showed that gH/gL/UL128-131 expressed in epithelial cells interferes with subsequent HCMV entry into cells. There was no interference with only gH/gL or gB. We concluded that the expression of gH/gL/UL128-131 causes a mislocalization or downregulation of epithelial cell proteins that HCMV requires for entry. In contrast, gH/gL/UL128-131 expression in fibroblasts did not produce interference, suggesting a different mechanism for entry. Here, we show that the coexpression of another HCMV glycoprotein, gO, with gH/gL in human fibroblasts interferes with HCMV entry into fibroblasts but not epithelial cells. However, the coexpression of gO with gH/gL did not increase the cell surface expression level of gH/gL and did not enhance cell-cell fusion, a process that depends upon cell surface gH/gL. Instead, gO promoted the export of gH/gL from the endoplasmic reticulum (ER) and the accumulation of gH/gL in the trans-Golgi network. Thus, interference with gH/gL or gH/gL/gO, i.e., the mislocalization or blocking of entry mediators, occurs in cytoplasmic membranes and not in cell surface membranes of fibroblasts. Together, the results provide additional support for our hypotheses that epithelial cells express putative gH/gL/UL128-1331 receptors important for HCMV entry and that fibroblasts express distinct gH/gL receptors.     

Structure-function analysis of herpes simplex virus type 1 gD and gH-gL: clues from gDgH chimeras

In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.