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Unlabelled: Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (∼400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wild-type HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes., Importance: An essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important for identifying novel targets to reduce alphaherpesvirus disease., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The cellular requirements for activation of herpesvirus fusion and entry remain poorly understood. Low pH triggers change in the antigenic reactivity of the prefusion form of the herpes simplex virus (HSV) fusion protein gB in virions, both in vitro and during viral entry via endocytosis (S. Dollery et al., J. Virol. 84:3759-3766, 2010). However, the mechanism and magnitude of gB conformational change are not clear. Here we show that the conformation and oligomeric state of gB with mutations in the bipartite fusion loops were similarly altered despite the fusion-inactivating mutations. Together with previous studies, this suggests that fusion loop mutants undergo conformational changes but are defective for fusion because they fail to make productive contact with the outer leaflet of the host target membrane. A direct, reversible effect of low pH on the structure of gB was detected by fluorescence spectroscopy. A soluble form of gB containing cytoplasmic tail sequences (s-gB) was triggered by mildly acidic pH to undergo changes in tryptophan fluorescence emission, hydrophobicity, antigenic conformation, and oligomeric structure and thus resembled the prefusion form of gB in the virion. In contrast, soluble gB730, for which the postfusion crystal structure is known, was only marginally affected by pH using these measures. The results underscore the importance of using a prefusion form of gB to assess the activation and extent of conformation change. Further, acidic pH had little to no effect on the conformation or hydrophobicity of gD or on gD's ability to bind nectin-1 or HVEM receptors. Our results support a model in which endosomal low pH serves as a cellular trigger of fusion by activating conformational changes in the fusion protein gB.

Egress of herpes simplex virus (HSV) and other herpesviruses from cells involves extensive modification of cellular membranes and sequential envelopment and deenvelopment steps. HSV glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Capsids in the nucleus undergo primary envelopment at the inner nuclear membrane (INM), and then enveloped virus particles undergo deenvelopment by fusing with the outer nuclear membrane (ONM). Capsids delivered into the cytoplasm then undergo secondary envelopment, involving trans-Golgi network (TGN) membranes. The deenvelopment step involves HSV glycoproteins gB and gH/gL acting in a redundant fashion. This fusion has features common to the fusion that occurs between the virion envelope and cellular membranes when HSV enters cells, a process requiring gB, gD, and gH/gL. Whether HSV gD also participates (in a redundant fashion with gB or gH/gL) in deenvelopment has not been characterized. Secondary envelopment in the cytoplasm is known to involve HSV gD and gE/gI, also acting in a redundant fashion. Whether gB might also contribute to secondary envelopment, collaborating with gD and gE/gI, is also not clear. To address these questions, we constructed an HSV double mutant lacking gB and gD. The HSV gB(-)/gD(-) mutant exhibited no substantial defects in nuclear egress. In contrast, secondary envelopment was markedly reduced, and there were numerous unenveloped capsids that accumulated in the cytoplasm, as well as increased numbers of partially enveloped capsids and morphologically aberrant enveloped particles with thicker, oblong tegument layers. These defects were different from those observed with HSV gD(-)/gE(-)/gI(-) mutants, which accumulated capsids in large, aggregated masses in the cytoplasm. Our results suggest that HSV gB functions in secondary envelopment, apparently acting downstream of gE/gI.

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 capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.