Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. While most other enveloped viruses use a single viral catalyst, called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH–gL, plus other non-conserved components. gB is a class III viral fusogen but, unlike other members of its class, does not function alone. We determined the crystal structure of the gH ectodomain bound to gL, from herpes simplex virus 2. gH–gL is an unusually tight complex of a novel architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH–gL activates gB for fusion, possibly through direct binding. Formation of a gB–gH–gL complex is critical for fusion and is inhibited by a neutralizing antibody, making gB–gH–gL interface a promising antiviral target.

Herpesviridae comprise a large family of enveloped DNA viruses all of whom employ orthologs of the same three glycoproteins, gB, gH and gL. Additionally, herpesviruses often employ accessory proteins to bind receptors and/or bind the heterodimer gH/gL or even to determine cell tropism. Sorting out how these proteins function has been resolved to a large extent by structural biology coupled with supporting biochemical and biologic evidence. Together with the G protein of vesicular stomatitis virus, gB is a charter member of the Class III fusion proteins. Unlike VSV G, gB only functions when partnered with gH/gL. However, gH/gL does not resemble any known viral fusion protein and there is evidence that its function is to upregulate the fusogenic activity of gB. In the case of herpes simplex virus, gH/gL itself is upregulated into an active state by the conformational change that occurs when gD, the receptor binding protein, binds one of its receptors. In this review we focus primarily on prototypes of the three subfamilies of herpesviruses. We will present our model for how herpes simplex virus (HSV) regulates fusion in series of highly regulated steps. Our model highlights what is known and also provides a framework to address mechanistic questions about fusion by HSV and herpesviruses in general.

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...

Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N-or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.BiMC ͉ fusion ͉ HSV ͉ interaction ͉ EYFP

Herpes simplex virus type 1 (HSV-1) is a large (150-kb) double-stranded DNA virus that forms latent infections in neuronal cells of the human peripheral nervous system. Previous work determined that the HSV-1 genome is found in an ordered nucleosomal structure during latent infection. However, during lytic infection, it was unclear whether viral DNA was in a chromatin state. We examined HSV-1 during lytic infection using micrococcal nuclease digestion and chromatin immunoprecipitation. The HSV-1 genome is at least partially nucleosomal, although apparently not in a regular repeating structure. Analysis of histones associated with HSV-1, within both the promoter and the transcribed regions, revealed covalent amino tail modifications similar to those associated with active host mammalian genes. Certain of the modifications were detected in the temporal order expected of the immediate-early, early, and late gene classes. These data suggest that productive infection may be accompanied by acquisition of a permissive chromatin state.