Insertion Mutations in Herpes Simplex Virus 1 Glycoprotein H Reduce Cell Surface Expression,Slow the Rate of Cell Fusion,or Abrogate Functions in Cell Fusion and Viral Entry

Of the four required herpes simplex virus (HSV) entry glycoproteins, the precise role of gH-gL in fusion remains the most elusive. The heterodimer gH-gL has been proposed to mediate hemifusion after the interaction of another required glycoprotein, gD, with a receptor. To identify functional domains of HSV-1 gH, we generated 22 randomized linker-insertion mutants. Analyses of 22 gH mutants revealed that gH is relatively tolerant of insertion mutations, as 15 of 22 mutants permitted normal processing and transport of gH-gL to the cell surface. gH mutants that were not expressed well at the cell surface did not function in fusion or viral entry. The screening of gH mutants for function revealed the following: (i) for wild-type gH and some gH mutants, fusion with nectin-1-expressing target cells occurred more rapidly than with herpesvirus entry mediator (HVEM)-expressing target cells; (ii) some gH mutants reduced the rate of cell fusion without abrogating fusion completely, indicating that gH may play a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion; and (iii) only one gH mutant, located within the short cytoplasmic tail, completely abrogated function, indicating that the gH cytoplasmic tail is crucial for cell fusion and viral infectivity.Herpes simplex virus (HSV), an enveloped neurotropic virus, infects target cells via membrane fusion, a process executed by viral fusion proteins capable of inserting into target membranes. Unlike many enveloped viruses that induce fusion through the activity of a single viral fusion protein, HSV requires four glycoproteins, glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL), to execute fusion (6, 40, 42). The focus of this study, gH, is expressed as a heterodimer with gL (gH-gL). HSV gH and gL rely on one another for proper folding, posttranslational processing, and transport to the cell and virion surface (5, 23, 35).A sequential model of entry is the prevailing working hypothesis of HSV entry (1-3, 28, 32, 41). Viral attachment is mediated by the binding of glycoprotein C (gC) or gB to cell surface glycosaminoglycans such as heparan sulfate (38). The subsequent fusion between the virion envelope and host cell membrane is thought to result from a series of concerted events. First, gD binds to one of its host cell receptors. These receptors include herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and heparan sulfate modified by specific 3-O-sulfotransferases (39).It was previously proposed that gD binding a receptor induces a conformational change that allows for interactions between gD, gB, and/or gH-gL (1, 2, 8, 10, 16, 25, 32). It is thought that while gD functions primarily in receptor binding, gB and gH-gL function as the core fusion machinery of HSV.Based on its crystal structure, gB has structural features typical of viral fusion proteins in general and is structurally similar to vesicular stomatitis virus (VSV) glycoprotein G, the fusion protein of VSV (22, 34). In addition to its resemblance to other viral fusogens, gB also binds its own receptor, paired immunoglobulin-like receptor (PILRalpha) (36, 37). Importantly, HSV gB does not successfully execute fusion in the absence of gD or gH-gL (41). Compared to the other required HSV entry glycoproteins, relatively little is known about the specific roles of gH-gL during fusion. The structure of gH-gL is unknown, although in silico analyses and studies of synthetic gH peptides suggested that gH also has fusogenic properties (12, 13, 17-20).gD, a gD receptor, and gH-gL have been shown to be sufficient for inducing hemifusion, the mixing of the proximal leaflets of the viral and host cell bilayers (41). Several lines of research suggest that the subsequent step in fusion is an interaction between gH-gL and gB, with the latter glycoprotein being required for a committed and expanding fusion pore (1-3, 16, 28, 41). However, it is still unclear whether the gB and gH-gL interaction requires that gD first bind a receptor (1, 3), indicating that another viable model of HSV entry may be nonsequential gD-gB-gH-gL complex formation.Several domains important for fusion within HSV gH have been discerned. The only function associated with the N-terminal domain of HSV gH, to date, is gL binding. Residues 377 to 397 within a predicted alpha-helix in the gH ectodomain are required for cell-cell fusion and complementation of a gH-null virus (18). The mutation of a predicted heptad repeat region spanning residues 443 to 471 abrogated cell-cell fusion (17). Insertion mutations within what has been termed the pretransmembrane region of gH have also been shown to abrogate fusion and viral entry (11). The glycine residue at position 812 within the predicted gH transmembrane domain was shown previously to be important for fusion (21). Finally, although the deletion of the final six residues of gH (residues 832 to 838), which are within its short cytoplasmic tail, has no effect on fusion, further deletions were shown to decrease polykaryocyte formation by a syncytial HSV strain (4, 43).We used a transposon-based comprehensive random linker-insertion mutagenesis strategy to generate a library of mutants spanning the entire length of HSV-1 gH, an 838-amino-acid type I membrane protein. A panel of 22 insertion mutants was generated, 15 of which were expressed at near-normal levels on the cell surface. Interestingly, some insertions reduced the rate of cell fusion rather than abrogating cell fusion activity altogether, suggesting that gH may have a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion. Additionally, one insertion mutation that completely abrogated cell fusion and viral infectivity is located within the gH cytoplasmic tail, indicating that the short C-terminal tail of gH is critical for cell fusion and entry mediated by HSV-1.     

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.     

A Double Mutation in Glycoprotein gB Compensates for Ineffective gD-Dependent Initiation of Herpes Simplex Virus Type 1 Infection

Herpes simplex virus (HSV) entry into cells is triggered by the binding of envelope glycoprotein D (gD) to a specific receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), resulting in activation of the fusion effectors gB and gH and virus penetration. Here we report the identification of a hyperactive gB allele, D285N/A549T, selected by repeat passage of a gD mutant virus defective for nectin-1 binding through cells that express a gD-binding-impaired mutant nectin-1. The gB allele in a wild-type virus background enabled the use of other nectins as virus entry receptors. In addition, combination of the mutant allele with an epidermal growth factor receptor (EGFR)-retargeted gD gene yielded dramatically increased EGFR-specific virus entry compared to retargeted virus carrying wild-type gB. Entry of the gB mutant virus into nectin-1-bearing cells was markedly accelerated compared to that of wild-type virus, suggesting that the gB mutations affect a rate-limiting step in entry. Our observations indicate that ineffective gD activation can be complemented by hypersensitization of a downstream component of the entry cascade to gD signaling.Entry of herpes simplex virus type 1 (HSV-1) into susceptible cells involves the coordinated activities of at least five viral envelope glycoproteins (9, 18, 33, 40). Virions initially bind to glycosaminoglycan (GAG) moieties of cell surface proteoglycans through glycoproteins B and C (gB and gC, respectively) (32, 51), facilitating the interaction of gD with one of its specific receptors, herpesvirus entry mediator (HVEM, or HveA), nectin-1 (HveC), or 3-O-sulfated heparan sulfate (24, 45, 50). Receptor binding is believed to result in a conformational change in gD, which in turn activates the fusion mechanism mediated by gB and the gH/gL heterodimer; fusion merges the virus envelope with the cell surface or endosomal membrane, resulting in capsid release into the cytoplasm (11, 23, 30, 37, 44, 47, 48). Prior to receptor binding, the N-terminal region of the gD ectodomain is folded back over the immunoglobulin (Ig)-like core domain in a position to engage the C-terminal effector region (pro-fusion domain), thereby keeping the effector domain in an inactive state (23, 37). Receptor binding disrupts this engagement and liberates the effector domain to activate gB and/or gH/gL. The crystal structure of the gB ectodomain shows unexpected homology to the postfusion form of glycoprotein G from vesicular stomatitis virus (VSV G), a well-characterized fusion protein (30), providing strong evidence that gB plays a major role in membrane fusion. In addition, gH displays structural hallmarks of fusion proteins (26, 27), and gB and gH each have fusogenic activity, as indicated by the finding that either alone is sufficient for membrane fusion during nuclear egress (20). However, gB and gH/gL are both required for complete fusion during virus entry, although gB is dispensable for hemifusion, an intermediate state (53).Results from biochemical and bimolecular-complementation assays have shown that gD binds individually to both gB and gH/gL, regardless of the presence of gD receptors (4, 5, 25), while complexes of gB and gH/gL assemble only in the presence of receptor-bound gD (4, 5). These observations suggested that receptor-dependent gD activation brings gB and gH/gL together for execution of the fusion event. However, based on new evidence that gB and gH/gL can also interact in the absence of gD, an alternative model has been proposed in which activated gD signals to preformed gB-gH/gL complexes (6). While these models are not mutually exclusive, the functional significance of the detected complexes remains to be firmly established (15). However, there is broad consensus that the gD-receptor interaction triggers the initiation of fusion by direct interaction with either or both gB and gH/gL, indicating that the quality of the gD-receptor interaction is key to the efficiency of HSV infection.Viruses have an intrinsic ability to evolve and adapt to changes in the environment, including the acquisition of an extended host range which can lead to epidemic infections (56). We previously described gain-of-function derivatives of a gD mutant virus, K26-gD:R222N/F223I, that was impaired in its ability to use nectin-1 as an entry receptor (54). Repeated passage of this virus through cells that express nectin-1 as the sole gD receptor yielded phenotypic revertants that had regained the ability to use nectin-1 for infection. This phenotype resulted from reversion or forward mutations at the parental mutant positions or from substitutions elsewhere in gD that likely affect the integrity of the discontinuous interface with nectin-1. Since these types of experiments can reveal novel factors or interactions that are important for virus entry, we performed a similar study at higher stringency in an attempt to avoid simple reversion mutations. The strategy was to use our previous gD:R222N/F223I mutant virus that is defective for entry via nectin-1 and ask if this virus could adapt to host cells expressing a mutant form of nectin-1 whose binding to wild-type gD is severely impaired. A specific goal of this effort was to find mutations in gD or other envelope glycoproteins that could enhance infection through atypical receptors, including cell-type-specific receptors that can be engaged by retargeted HSV vectors.Here we report the identification of a hyperactive gB double mutation, gB:D285N/A549T, referred to herein as gB:N/T, that allows virus entry in the absence of authentic gD receptors, enhances virus entry through unconventional receptors, including a targeted receptor, and appears to act by sensitizing gB to activation by gD, directly or indirectly via gH/gL, and increasing the rate of virus entry into different host cells. Our observations demonstrate that hyperactive gB can compensate for ineffective gD-receptor interactions in the process of HSV entry into cells.     

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.     

Differential Effects on Cell Fusion Activity of Mutations in Herpes Simplex Virus 1 Glycoprotein B (gB) Dependent on Whether a gD Receptor or a gB Receptor Is Overexpressed

Glycoprotein B (gB) of herpes simplex virus (HSV) is one of four glycoproteins essential for viral entry and cell fusion. Recently, paired immunoglobulin-like type 2 receptor (PILRα) was identified as a receptor for HSV type 1 (HSV-1) gB. Both PILRα and a gD receptor were shown to participate in HSV-1 entry into certain cell types. The purpose of this study was to determine whether insertional mutations in gB had differential effects on its function with PILRα and the gD receptor, nectin-1. Previously described gB mutants and additional newly characterized mutants were used in this study. We found that insertional mutations near the N terminus and C terminus of gB and especially in the central region of the ectodomain reduced cell fusion activity when PILRα was overexpressed much more than when nectin-1 was overexpressed. Most of the insertions reduced the binding of gB to PILRα, for at least some forms of gB, but this reduction did not necessarily correlate with the selective reduction in cell fusion activity with PILRα. These results suggest that the regions targeted by the relevant mutations are critical for functional activity with PILRα. They also suggest that, although both the binding of gB to a gB receptor and the binding of gD to a gD receptor may be required for HSV-induced cell fusion, the two receptor-binding activities may have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.Manifestations of disease caused by herpes simplex virus (HSV) include recurrent mucocutaneous lesions in the mouth or on the face or genitalia and, more rarely, meningitis or encephalitis. The infection of host cells occurs by the fusion of the virion envelope with a cell membrane to deliver the nucleocapsid containing the viral genome into the host cell. This entry process and virus-induced cell fusion require glycoprotein B (gB), along with gD, gH, and gL. The membrane-fusing activity of HSV depends in part on the binding of gD to one of its receptors, herpesvirus entry mediator (HVEM), nectin-1, nectin-2, or 3-O-sulfated heparan sulfate (18). HVEM is a member of the tumor necrosis factor receptor family and is expressed by cells of the immune system, as well as many other cell types, such as epithelial, stromal, and endothelial cells (23). Nectin-1 and nectin-2 are cell adhesion molecules belonging to the immunoglobulin superfamily and are widely expressed by a variety of cell types, including epithelial cells and neurons (20). Specific sites in heparan sulfate generated by particular 3-O-sulfotransferases can serve as gD-binding entry receptors (17). This binding of gD to a receptor is associated with conformational changes in gD that are thought to enable gD to interact with gB and/or the heterodimer gH-gL to trigger fusogenic activity (8, 12). Both gB and gH have properties of fusogenic viral proteins (1, 7). Although evidence has been presented that gD and gH-gL are sufficient for hemifusion and that gB, in addition, is required for fusion pore formation (19), the specific roles each plays in HSV-induced membrane fusion have not been fully defined.gB was recently discovered to bind to paired immunoglobulin-like type 2 receptor (PILRα) in an interaction that can mediate viral entry and cell fusion, provided that gD also binds to one of its receptors (14). For cells such as CD14⁺ monocytes, antibodies specific for either HVEM or PILRα were shown to block HSV entry. Also, entry requires the presence of both gD and gB in the virion. Although the overexpression of either a gD receptor or a gB receptor can enhance the susceptibility of cells to HSV entry and HSV-induced cell fusion, there are very few, if any, cell types that do not express at least low levels of endogenous receptors. Thus, the possibility exists that these endogenous receptors are cooperating with the introduced receptors to render the cells susceptible to HSV-induced membrane fusion.PILRα belongs to the paired-receptor families, which consist of activating and inhibitory receptors (4, 11, 19). They are conserved among mammals (24). The inhibitory form PILRα has an immunoreceptor tyrosine-based inhibition motif in its cytoplasmic domain and transduces inhibitory signals (4). On the other hand, the activating form PILRβ associates with the immunoreceptor tyrosine-based activation motif-bearing DAP12 adaptor molecule and delivers activating signals (16). Both PILRα and PILRβ are expressed on cells of the immune system, especially monocytes, dendritic cells, and NK cells (4, 11, 19), and also in neurons (14). CD99 has been identified as a natural ligand for both PILRα and PILRβ (16). The binding of either PILRα or PILRβ to CD99 depends on the presence of sialyated O-linked glycans on CD99 (22).In addition to binding to PILRα, gB can bind to heparin and heparan sulfate and may contribute, along with gC, to the binding of HSV to cell surface heparan sulfate (17). Also, gB and gC can bind to DC-SIGN, which serves as a binding receptor for the infection of dendritic cells (2). An X-ray structure of the HSV-1 gB ectodomain reveals a homotrimeric conformation with structural homology to vesicular stomatitis virus (VSV) G glycoprotein, the single glycoprotein responsible for the entry of VSV. Both HSV-1 gB and VSV G glycoprotein have features of class 1 and class 2 viral fusion proteins and have been designated class 3 fusion proteins (7, 14, 15). The heparan sulfate-binding determinant of gB has been localized to a lysine-rich domain in the N terminus and shown to be dispensable for viral entry (9). It lies within a region that is probably disordered and was not included in the defined coordinates of the X-ray structure. The binding of DC-SIGN to gB probably depends on high-mannose N-glycans of gB (6).In a previous study (10), 81 insertion mutants of HSV-1 gB were characterized to assess the effects of the insertions on protein processing and function in cell fusion with gD receptors, in relation to structural domains of gB identified in an X-ray structure (7). Only 27 mutants were found to be processed into mature glycosylated forms and transported to the cell surface. Only 11 of these retained fusion activity toward target cells expressing nectin-1 or HVEM. For the present study, we used 25 previously described gB insertion mutants shown to be expressed on cell surfaces and also identified an additional 10 such mutants.The present study was designed to determine whether the effects of insertions in gB on cell fusion activity would be dependent on whether a gD receptor (nectin-1) or a gB receptor (PILRα) was overexpressed in target cells that also expressed unidentified weak endogenous receptors. In addition, we assessed the abilities of the gB mutants to bind to PILRα. Our results showed that some insertions inhibited cell fusion activity when PILRα was overexpressed significantly more than when nectin-1 was overexpressed, but without necessarily preventing the binding of PILRα to gB, at least to some stable oligomeric forms of gB. The results indicate that, although both a gB receptor and a gD receptor may be required for cell fusion activity, the two receptor-binding activities have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.     

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.     

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.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.Entry of herpes simplex virus (HSV) into cells requires four viral glycoproteins, gB, gD, gH, and gL, plus one of several cell receptors, either herpesvirus entry mediator (HVEM), nectin-1, or 3-OST (45). Crystal structures and other studies have documented that receptor binding triggers conformational changes to gD that trigger the downstream events leading to fusion (10, 11, 18, 26, 28, 52). Moreover, when HSV receptor-bearing cells are transfected with expression plasmids for glycoproteins gB, gD, gH, and gL, the cells fuse to form multinucleated giant cells or syncytia (39, 48). However, the precise series of events that take place after receptor binding have not yet been fully elucidated. What we do know is that both gB and a heterodimer of gH/gL constitute the core fusion machinery that is conserved and required for the fusion step of entry of all herpesviruses (18, 26, 30, 46, 49).Thus far, we know the crystal structure of one form of the gB ectodomain of HSV type 1 (HSV-1) (19). This protein has the characteristics of a fusion protein and is a charter member of the class III group of viral fusion proteins (4). Others in this class include Epstein-Barr virus gB, vesicular stomatitis virus (VSV) G, and baculovirus gp64 (5, 22, 41). Like VSV G and gp64, gB has two putative fusion loops at the base of each protomer of the crystallized trimer. Single-amino-acid mutations in many of the hydrophobic residues of the putative fusion loops of gB ablate its ability to function in cell-cell fusion assays (16, 17). Moreover, these mutants are unable to complement the entry of a gB-null virus (16). Finally, the ectodomains of these mutants, unlike wild-type protein, failed to coassociate with liposomes, indicating that the putative fusion loops do insert into membranes (16, 17). Recently, it was shown that several of these mutants are also defective for fusion events involved in virus egress (51). Together, these studies provide compelling evidence that HSV gB functions as a fusion protein and that the fusion loops are critical for this function. However, unlike VSV G and baculovirus gp64, gB does not function on its own in entry but, rather, requires the participation of gH/gL. In the absence of crystallographic data for gH/gL, it is not yet clear what role it plays in herpesvirus fusion. In a previous study, we used bimolecular complementation (BiMC) to examine protein-protein interactions that occur among the viral glycoproteins during fusion (1). A similar study was carried out by Avitabile et al. (2). The BiMC assay is based on the observation that N- and C-terminal fragments of green fluorescent protein (GFP) (and derivatives such as enhanced yellow fluorescent protein [EYFP]) do not spontaneously reconstitute a functional fluorophore (20, 29, 40). However, the codons for each half can be appended to the genes for two interacting proteins (23, 24). When these are cotransfected, an interaction between the two proteins of interest brings the two halves of the fluorophore in close enough contact to restore fluorescence.When HSV receptor-bearing cells, such as B78H1 cells that are engineered to express nectin-1, are transfected with plasmids that express gB, gD, gH, and gL, they undergo cell-cell fusion (13, 15, 27, 31, 48). When gD is omitted, no fusion occurs. We found that fusion of these transfected cells could be triggered by addition of a soluble form of gD (the gD ectodomain). We then used this approach to examine interactions between gB and gH/gL during cell fusion (1). Therefore, we tagged gB with the C-terminal half of EYFP and gH with the N-terminal half. When plasmids bearing these forms were cotransfected into C10 cells along with a plasmid for untagged gL, no fusion occurred, but importantly, no BiMC occurred. However, when we added gD306, cells began to fuse within 10 min, and all of the syncytia that formed exhibited bright EYFP fluorescence indicative of BiMC. We concluded that gD triggers both fusion and a physical interaction between gB and gH/gL. However, these experiments did not separate these two events, so we were unable to determine if the interaction preceded fusion or merely was a by-product of it.The purpose of this study was to determine if the gB-gH/gL interaction is essential for fusion and if it occurs prior to fusion. We focused on gB because its structure is known and we have a panel of well-characterized monoclonal antibodies (MAbs) to gB. Our approach was to determine which of these MAbs, if any, could block fusion and also block the interaction with gH/gL. We also examined the effect of mutations to the fusion loops of gB on its interaction with gH/gL. We previously mapped these MAbs to four functional regions (FR) of gB, three of which were resolved in the crystal structure (6, 19). Of these, FR1 contains the fusion loops, FR2 is in the center of the gB structure with no known function, and FR3 is at in the crown of the protein and may be involved in binding to cells (7). Our rationale was that if the interaction between gB and gH/gL is important for fusion, then it should not be blocked by nonneutralizing anti-gB MAbs. At the same time, we thought that some neutralizing MAbs might not only block fusion but also block BiMC. We found that neutralizing MAbs to FR1 and FR2 inhibited both BiMC and fusion. In contrast, we found that neutralizing MAbs that map to FR3 blocked fusion but failed to block the interaction between gB and gH/gL, thereby dissociating the two events. Finally, we found that gB mutants with changes in the fusion loops that were fusion negative were also unable to bind to gH/gL. The latter results suggest that insertion of gB into the target membrane precedes its interaction with gH/gL.     

The UL31 and UL34 Gene Products of Herpes Simplex Virus 1 Are Required for Optimal Localization of Viral Glycoproteins D and M to the Inner Nuclear Membranes of Infected Cells

U_L31 and U_L34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU_L31, pU_L34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U_L34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U_L34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U_L34 (P < 0.0001). pU_L34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU_L34/gD interaction in recruiting gD to the NM. The effects of U_L34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU_L31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U_L31 and U_L34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.Herpesvirus virions comprise a nucleocapsid containing genomic viral DNA, a proteinaceous tegument layer surrounding the nucleocapsid, and a virion envelope surrounding the tegument. The envelope of extracellular herpes simplex virus (HSV) virions contains glycoproteins gB, gC, gD, gE, gI, gG, gH, gK, gL, and gM (23, 51).As viewed by electron microscopy, nascent virions form as the nucleocapsid buds through densely staining regions of the nuclear membrane (NM) (21, 41). Electron tomograms of HSV perinuclear virions compared to those of extracellular virions infer that the former contain glycoproteins of considerably less glycosylation and a relatively sparse tegument layer compared to their counterparts in mature extracellular virions (6). The lower levels of glycosylation in HSV perinuclear virions are consistent with the fact that the lumen of the perinuclear space is continuous with that of the endoplasmic reticulum. Thus, the polysaccharide moieties of virion glycoproteins become fully processed as virions access Golgi enzymes during their egress to the extracellular space. Although the full proteome of the nascent perinuclear virion is unknown, immunogold studies have shown that they contain at least pU_L31, pU_L34, pUS3, gB, gC, gD, gH, gM, and the VP16 and pU_L11 tegument proteins in addition to the proteins that comprise the viral capsid (4, 5, 15, 25, 37, 40, 47, 50, 55).The U_L31 and U_L34 gene products of HSV-1 (pU_L31 and pU_L34, respectively) form a complex that localizes at the inner and outer NMs (INM and ONM, respectively) of infected cells (40). Both proteins are essential for nucleocapsid envelopment at the INM and become incorporated into nascent virions when nucleocapsids bud through the INM into the perinuclear space (39, 40, 42). The proteins and their essential role in nucleocapsid envelopment are conserved in all herpesvirus subfamilies (14, 20, 32, 45). pU_L31 of HSV-1 is a mostly hydrophobic phosphoprotein that is held in close approximation to the nucleoplasmic face of the INM by interaction with pU_L34, an integral membrane protein of type II orientation (33, 40, 46, 56). The first 248 amino acids of pU_L34 are predicted to reside in the nucleoplasm or cytoplasm, depending on whether the protein localizes in the INM or ONM, respectively. This is followed by an approximately 22-amino acid transmembrane domain with up to 5 amino acids residing in the perinuclear space or lumen of the endoplasmic reticulum.In the most prominent model of herpesvirion egress, the envelope of the perinuclear virion fuses with the ONM, releasing the deenveloped nucleocapsid into the cytoplasm, where it subsequently buds into cytoplasmic membranous organelles such as the Golgi or trans-Golgi network (34, 49). This model is supported by the observation that pU_L31 and pU_L34 are located in the perinuclear virion but not extracellular virions (18, 40). Thus, these proteins are lost from the virion upon fusion of the virion envelope with the ONM. Also supporting this egress model is the observation that deletion of both gB and gH causes virions to accumulate aberrantly in the perinuclear space (15). The involvement of gH and gB is potentially satisfying because these proteins comprise essential components of the machinery that mediates fusion of the virion envelope with the plasma or endosomal membranes during the initiation of infection (9, 12, 16, 44, 52). Moreover, expression of a combination of gB, gD, gH, and gL is sufficient to mediate fusion of cell membranes, whereas coexpression with gM or gK inhibits this fusion (3, 8, 11). Although the mechanism of fusion is unclear, gD is known to bind viral receptors on cell surfaces, and the structure of gB indicates features reminiscent of other viral fusion proteins (24, 35, 48). gD has been shown to interact with gB and gH at least transiently, suggesting that these interactions may be important for the fusion reaction (1, 2). Thus, fusion between the nascent and mature virion envelopes with target membranes may share mechanistic similarities.On the other hand, it is likely that the two fusion events are mechanistically distinct because (i) single deletion of either gH or gB precludes viral entry and cell/cell fusion but does not cause nascent virions to accumulate in the perinuclear space (9, 16, 31, 43) and (ii) the activity of a viral kinase encoded by U_S3 is dispensable for entry but believed to promote fusion of the perinuclear virion and ONM (28, 40). Moreover, the lack of glycoproteins from the pseudorabies virus perinuclear virion suggests that fusion is mediated by an entirely different mechanism in this system (26).The current study focuses on how glycoproteins are incorporated into the nascent virion. We show that optimal recruitment of gD to both leaflets of the NM and gM to the INM requires pU_L34 and pU_L31. We also show that immature gD interacts with pU_L34, suggesting a mechanism by which pU_L34 might recruit gD to the NM.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

Glycoprotein D of Bovine Herpesvirus 5 (BoHV-5) Confers an Extended Host Range to BoHV-1 but Does Not Contribute to Invasion of the Brain

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.Bovine herpesvirus 1 (BoHV-1) and BoHV-5 belong to the subfamily Alphaherpesvirinae and are closely related pathogens of cattle (22). The protein repertoire of the two viruses averages 82% amino acid identity (20). Both viruses are neurotropic, but only BoHV-5 can significantly replicate in the central nervous system (CNS) to cause encephalitis of either naturally infected cattle or experimentally inoculated laboratory animals (2, 5, 6, 12, 40, 41, 44). Glycoprotein D (gD) is accepted as the critical and essential receptor-binding protein of many alphaherpesviruses (reviewed in references 8 and 48). The main gD receptors identified to date include members of the tumor necrosis factor (TNF) receptor family (HveA) and the poliovirus receptor family (HveB or nectin 2 and HveC or nectin 1) (28, 42, 51). Furthermore, a modified form of heparan sulfate, 3-O-sulfated heparan sulfate, can mediate herpesvirus entry (46). J1.1-2 cells (J cells) represent a subpopulation of thymidine kinase-negative baby hamster kidney (BHK) cells selected for their property of being resistant to infection with herpes simplex virus type 1 (HSV-1), HSV-2, and BoHV-1. The expression of nectin 1 in those cells rendered them susceptible to BoHV-1 infection and replication, which suggests that nectin 1 can serve as a receptor for BoHV-1 gD (gD1) (16, 18, 28). Interestingly, we observed that BoHV-5 was able to productively replicate in J cells without the nectin 1 receptor.According to a previously reported sequence comparison of BoHV-1 and BoHV-5 (20), the highest divergence between the two viruses mapped to the latency-related region and the immediate-early proteins (less than 75% amino acid identity) BICP0, BICP4, and BICP22. Glycoprotein E (gE) was also listed in this category, with 74% amino acid identity between gE of BoHV-1 (gE1) and gE5. This fact also gave ample reason for attempts to map the neurovirulent phenotype of BoHV-5 to the gE5 molecule (3, 4, 13). In contrast, the highest sequence similarities between the two viruses were described for proteins involved in viral DNA replication and processing as well as certain virion proteins. Among others, the predicted amino acid sequences of gD1 and gD5 were listed as being 98% identical (20). However, our own analysis using the European Molecular Biology software suite (43) revealed only 79.9% amino acid identity. Obviously, the most extensive difference between gD1 and gD5 maps to a glycine-rich stretch located in the molecule''s ectodomain, between amino acids (aa) 280 and 330 of gD5, in close vicinity to the transmembrane region.Based on these considerations, we hypothesized that BoHV-5 was able to make use of a cellular receptor that is unavailable to BoHV-1. To test this hypothesis, the gD genes were removed from bacterial artificial chromosomes (BACs) harboring the genome of either BoHV-5 or BoHV-1 (27). In a second step, gD exchange viruses were created by the cotransfection of the gD-less BACs with appropriate plasmids carrying either the gD1 or gD5 gene and appropriate flanking sequences. The newly generated viruses included an intertypic BoHV-5 mutant carrying gD1 in the place of gD5 and a corresponding BoHV-1 carrying gD5. These mutants, together with appropriate revertant mutants, were then used to explore their ability to infect J cells in vitro and their ability to cause neurological disease and invade the brain in vivo, in a previously established mouse model (2). Our results indicate that gD5 confers an extended host range to BoHV-1 but is nonessential for the invasion of the brain.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

The γ134.5 Protein of Herpes Simplex Virus 1 Is Required To Interfere with Dendritic Cell Maturation during Productive Infection

The γ₁34.5 protein of herpes simplex virus 1 is an essential factor for viral virulence. In infected cells, this viral protein prevents the translation arrest mediated by double-stranded RNA-dependent protein kinase R. Additionally, it associates with and inhibits TANK-binding kinase 1, an essential component of Toll-like receptor-dependent and -independent pathways that activate interferon regulatory factor 3 and cytokine expression. Here, we show that γ₁34.5 is required to block the maturation of conventional dendritic cells (DCs) that initiate adaptive immune responses. Unlike wild-type virus, the γ₁34.5 null mutant stimulates the expression of CD86, major histocompatibility complex class II (MHC-II), and cytokines such as alpha/beta interferon in immature DCs. Viral replication in DCs inversely correlates with interferon production. These phenotypes are also mirrored in a mouse ocular infection model. Further, DCs infected with the γ₁34.5 null mutant effectively activate naïve T cells whereas DCs infected with wild-type virus fail to do so. Type I interferon-neutralizing antibodies partially reverse virus-induced upregulation of CD86 and MHC-II, suggesting that γ₁34.5 acts through interferon-dependent and -independent mechanisms. These data indicate that γ₁34.5 is involved in the impairment of innate immunity by inhibiting both type I interferon production and DC maturation, leading to defective T-cell activation.Herpes simplex virus 1 (HSV-1) is a human pathogen responsible for localized mucocutaneous lesions and encephalitis (51). Following primary infection, HSV-1 establishes a latent or lytic infection in which the virus interacts with host cells, which include dendritic cells (DCs), required to initiate adaptive immune responses (36). Immature DCs, which reside in almost all peripheral tissues, are able to capture and process viral antigens (15). In this process, DCs migrate to lymph nodes, where they mature and present antigens to T cells. Mature DCs display high levels of major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40, CD80, and CD86. Additionally, DCs release proinflammatory cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha, alpha interferon (IFN-α), and IFN-β, which promote DC maturation and activation. An important feature of functional DCs is to activate naïve T cells, and myeloid submucosal and lymph node resident DCs are responsible for HSV-specific T-cell activation (2, 45, 52). Moreover, DCs play a direct role in innate antiviral immunity by secreting type I IFN.HSV-1 is capable of infecting both immature and mature DCs (20, 24, 34, 38, 42). A previous study suggested that HSV entry into DCs requires viral receptors HVEM and nectin-2 (42). Upon HSV infection, plasmacytoid DCs detect viral genome through Toll-like receptor 9 (TLR9) and produce high levels of IFN-α (16, 23, 30, 40). In contrast, myeloid DCs, which are major antigen-presenting cells, recognize viral components through distinct pathways, independently of TLR9 (16, 36, 40). It has been suggested previously that HSV proteins or RNA intermediates produced during viral replication trigger myeloid DCs (16, 40). Indeed, a protein complex that consists of HSV glycoproteins B, D, H, and L stimulates the expression of CD40, CD83, CD86, and cytokines in myeloid DCs (41). Hence, DCs sense HSV through TLR-dependent and -independent mechanisms (16, 40, 41). Nevertheless, HSV replication compromises DC functions and subsequent T-cell activation (3, 20, 24, 42). HSV-1 interaction with immature DCs results in the downregulation of costimulatory molecules and cytokines (20, 34, 38, 42). While HSV-2 induces rapid apoptosis, HSV-1 does so with a delayed kinetics in human DCs (4, 20, 38). HSV-1 is also reported to interfere with functions of mature DCs (24, 39). Upon infection, HSV-1 induces the degradation of CD83 but not CD80 or CD86 in mature DCs (24, 25). Additionally, HSV-1 reduces levels of the chemokine receptors CCR7 and CXCR4 on mature DCs and subsequently impairs DC migration to the respective chemokine ligands CCL19 and CXCL12 (39).Although HSV infection impairs DC functions, viral components responsible for this impairment have not been thoroughly investigated. It has been suggested previously that the virion host shut-off protein (vhs) of HSV-1 contributes partially to the viral block of DC activation (43). This activity is thought to stem from the ability of vhs to destabilize host mRNA. Emerging evidence suggests that ICP0 perturbs the function of mature DCs, where it mediates CD83 degradation via cellular proteasomes (25). Findings from related studies show that ICP0 inhibits the induction of IFN-stimulated genes mediated by IFN regulatory factor 3 (IRF3) or IRF7 in other cell types (11, 27, 32, 33). However, the link of ICP0 activities to DC maturation remains to be established. Recently, we found that γ₁34.5, an HSV virulence factor, associates with and inhibits TANK-binding kinase 1 (TBK1), an essential component of TLR-dependent and -independent pathways that activates IRF3 and cytokine expression (49). Interestingly, an HSV mutant lacking γ₁34.5 stimulates MHC-II surface expression in glioblastoma cells (47). These observations raise the hypothesis that γ₁34.5 may modulate DC maturation during HSV infection.In this study, we report that γ₁34.5 is required to perturb DC maturation during HSV infection, leading to impaired T-cell activation. Wild-type virus, but not the γ₁34.5 null mutant, suppresses the expression of costimulatory molecules as well as cytokines in DCs. We provide evidence that the viral block of DC maturation is associated with reduced IFN-α/β secretion. Furthermore, the expression of γ₁34.5 inhibits DC-mediated allogeneic T-cell activation and IFN-γ production. IFN-neutralizing antibodies partially reverse DC maturation induced by the γ₁34.5 null mutant. These results shed light on the role of γ₁34.5 relevant to DC maturation and T-cell responses in HSV infection.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.     

Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways

Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.Virus recognition is essential for activation of innate antiviral immune defense and the subsequent induction of acquired immunity. Conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), are recognized by pattern recognition receptors (PRRs). Virus-recognizing PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and a number of intracellular DNA receptors. Several TLRs have been attributed roles in the recognition of virus. TLR2 and TLR4 recognize viral surface structures (3, 6, 18, 31), TLR3 recognizes double-stranded RNA (dsRNA) (2), and TLR7/8 and TLR9 function as signaling receptors for viral single-stranded RNA (ssRNA) (8, 11, 21) and CpG DNA (12, 20), respectively.Within the cell, cytoplasmic RLRs RIG-I and MDA5 both recognize accumulation of virus-derived dsRNA; in addition, RIG-I recognizes 5′-triphosphated RNA (14, 27, 39, 40). In addition to the RLRs, a number of receptors recognize foreign DNA. Presently, three DNA receptors have been identified: Z-DNA binding protein 1 (ZBP-1, or DAI) (36) and RNA polymerase III (Pol III) (1, 4) both mediate interferon (IFN) and cytokine production, whereas the AIM2 inflammasome is involved in caspase 1 activation in response to cytoplasmic dsDNA (13).Herpes simplex virus type 1 (HSV-1) and HSV-2 are two closely related human DNA viruses associated with a number of serious diseases, including orofacial infections, encephalitis, and genital infections (34). Macrophages play an important role in the first line of defense against viral infection via production of IFNs, cytokines, and chemokines that regulate the progress of the virus infection and activate and support appropriate defense mechanisms (9, 10, 24).TLR2, TLR3, and TLR9 have been identified as mediators of proinflammatory cytokine production during HSV infections. TLR2 mediates an overzealous inflammatory cytokine response following HSV-1 infection in mice, promoting mononuclear cell infiltration of the brain and development of encephalitis (18). TLR3 mediates type I and III IFN production in human fibroblasts (41). TLR9 recognizes genomic DNA from HSV-1 and HSV-2 in murine plasmacytoid dendritic cells (DCs) (17, 20) and mediates tumor necrosis factor alpha (TNF-α) and CCL5 production in murine macrophages (22). Both TLR2 and TLR9 mediate recognition of HSV and cytokine production in murine conventional DCs (35). HSV is recognized by an RLR/MAVS-dependent mechanism in murine macrophages and mouse embryonic fibroblasts (MEFs) (5, 29, 30). Recent data suggest that RNA Pol III mediates IFN production following HSV-1 infection and transfection with HSV-1 DNA in macrophage-like RAW 264.7 cells (4). Finally, murine L929 fibroblast-like cells are moderately inhibited in their ability to produce IFN after HSV-1 infection when ZBP-1 is knocked down (19, 36). Thus, several PRRs have been reported to recognize HSV-1 in murine cells and different cell lines, but the pathways responsible for sensing this virus in human primary macrophages and their impact on cytokine expression have not previously been described.In this work, we investigate the recognition pathways underlying HSV-induced cytokine and chemokine expression in human primary macrophages. We demonstrate that HSV-1-induced IFN and cytokine expression is independent of TLR2 and TLR9 but highly dependent on virus replication and intracellular nucleotide recognition systems. Specifically, induction of IFNs is dependent on MAVS and MDA5, whereas TNF-α is induced by a novel intracellular nucleotide-sensing system.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.