Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the transGolgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.egress ͉ nuclear envelope ͉ deenvelopment ͉ perinuclear space T he nuclear envelope (NE) is composed of inner and outer nuclear membrane (NM) separated by the perinuclear space and connected by nuclear pore complexes. A dense network of lamins forms a rigid girdle underneath the inner NM, organizing nuclear pores and chromatin (reviewed in ref. 1). For viruses that replicate their genomes in the nucleus, the NE often serves to confine and orchestrate nucleic acid synthesis and assembly of virus particles. However, the NE can also present a formidable barrier to virus egress from cells. Nonenveloped viruses such as SV40 and adenoviruses rupture the NE, as well as the plasma membrane, to gain release from cells (2, 3). Herpesviruses assemble large capsids in the nucleus but, because they are enveloped viruses, have evolved the capacity to cross membranes by budding and fusion mechanisms.There has been extensive debate over how herpesviruses exit cells (4-8). Earlier models suggested that perinuclear enveloped virions are ferried from the perinuclear space/endoplasmic reticulum through the Golgi apparatus to the cell surface in exocytic transport vesicles (reviewed in ref. 9). However, a substantial body of genetic, biochemical, and morphologic evidence has substantiated the envelopment3deenvelopment3reenvelopment model for egress, at least for ␣-herpesviruses (reviewed in refs. 4 and 6). In this model, capsids become enveloped at the inner leaflet of the NE (primary envelopment) and are deenveloped at the outer NM releasing unenveloped cytoplasmic capsids, which acquire other tegument proteins and a second envelope by budding into the trans-Golgi network. For herpes simplex virus (HSV), this secondary envelopment requires glycoproteins gD or gE, and removal of both abolishes this process, but removal of either individually has little effect (10). Recently, it was suggested th...

The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.Herpes simplex virus (HSV) is in many respects the best studied of the herpesviruses and the paradigm for ␣-herpesvirus replication and assembly. As with other herpesviruses, viral DNA is packaged into nucleocapsids in the nucleus (43). To escape the nucleus, capsids become enveloped by regions of the inner nuclear envelope and then move into the cytoplasm by fusion with the outer lamellae of the nuclear envelope (45,47). In the cytoplasm, nucleocapsids bind several tegument proteins (reviewed in reference 35). Tegument-coated capsids bind onto cytoplasmic membranes, including the trans-Golgi network (TGN) and endosomes, enriched in viral glycoproteins (reviewed in reference 24). Nascent virions bud into the lumen of these cytoplasmic vesicles as the virion envelope wraps around tegument-coated capsids. Enveloped virions are transported to the cell surface where fusion of cytosolic vesicles with the plasma membrane delivers virus particles into the extracellular environment or onto the cell surface.The molecular details of how ␣-herpesviruses become enveloped at either nuclear or cytoplasmic membranes are poorly characterized. In the case of cytoplasmic envelopment, it is clear that viral membrane glycoproteins accumulate extensively in the TGN or endosomes and this likely promotes incorporation into the virion envelope (reviewed in references 24 and 35). The cytosolic domains of these membrane glycoproteins probably provide a surface on which the last stage of assembly, i.e

The final assembly of herpes simplex virus (HSV) involves binding of tegument-coated capsids to viral glycoprotein-enriched regions of the trans-Golgi network (TGN) as enveloped virions bud into TGN membranes. We previously demonstrated that HSV glycoproteins gE/gI and gD, acting in a redundant fashion, are essential for this secondary envelopment. To define regions of the cytoplasmic (CT) domain of gE required for secondary envelopment, HSVs lacking gD and expressing truncated gE molecules were constructed. A central region (amino acids 470 to 495) of the gE CT domain was important for secondary envelopment, although more C-terminal residues also contributed. Tandem affinity purification (TAP) proteins including fragments of the gE CT domain were used to identify tegument proteins VP22 and UL11 as binding partners, and gE CT residues 470 to 495 were important in this binding. VP22 and UL11 were precipitated from HSV-infected cells in conjunction with full-length gE and gE molecules with more-C-terminal residues of the CT domain. gD also bound VP22 and UL11. Expression of VP22 and gD or gE/gI in cells by use of adenovirus (Ad) vectors provided evidence that other viral proteins were not necessary for tegument/glycoprotein interactions. Substantial quantities of VP22 and UL11 bound nonspecifically onto or were precipitated with gE and gD molecules lacking all CT sequences, something that is very unlikely in vivo. VP16 was precipitated equally whether gE/gI or gD was present in extracts or not. These observations illustrated important properties of tegument proteins. VP22, UL11, and VP16 are highly prone to binding nonspecifically to other proteins, and this did not represent insolubility during our assays. Rather, it likely reflects an inherent "stickiness" related to the formation of tegument. Nevertheless, assays involving TAP proteins and viral proteins expressed by HSV and Ad vectors supported the conclusion that VP22 and UL11 interact specifically with the CT domains of gD and gE.Herpesvirus capsids cross nuclear membranes by envelopment at the inner nuclear membrane followed by fusion or deenvelopment at the outer nuclear membrane, delivering nucleocapsids into the cytoplasm (reviewed in references 35 and 46 to 48). Secondary envelopment occurs as herpesvirus tegument-coated capsids bind onto viral glycoprotein-enriched regions of the Golgi apparatus, trans-Golgi network (TGN), or endosomes. This delivers enveloped virions into cytoplasmic vesicles, which are subsequently trafficked to cell surfaces.How tegument-coated nucleocapsids interact with membranes to promote herpesvirus budding is not well understood. Recent studies have suggested that redundant interactions between tegument proteins and the cytoplasmic (CT) domains of specific viral glycoproteins are required. Herpes simplex virus (HSV) produces as many as 12 membrane glycoproteins as well as other nonglycosylated membrane proteins (54). Any one of these membrane proteins can be deleted without substantially reducing the numbers of enveloped virio...

Herpes simplex virus (HSV) glycoprotein heterodimer gE/gI is necessary for virus spread in epithelial and neuronal tissues. Deletion of the relatively large gE cytoplasmic (CT) domain abrogates the ability of gE/gI to mediate HSV spread. The gE CT domain is required for the sorting of gE/gI to the trans-Golgi network (TGN) in early stages of virus infection, and there are several recognizable TGN sorting motifs grouped near the center of this domain. Late in HSV infection, gE/gI, other viral glycoproteins, and enveloped virions redistribute from the TGN to epithelial cell junctions, and the gE CT domain is also required for this process. Without the gE CT domain, newly enveloped virions are directed to apical surfaces instead of to cell junctions. We hypothesized that the gE CT domain promotes virus envelopment into TGN subdomains from which nascent enveloped virions are sorted to cell junctions, a process that enhances cell-to-cell spread. To characterize elements of the gE CT domain involved in intracellular trafficking and cell-to-cell spread, we constructed a panel of truncation mutants. Specifically, these mutants were used to address whether sorting to the TGN and redistribution to cell junctions are necessary, and sufficient, for gE/gI to promote cell-to-cell spread. gE-519, lacking 32 C-terminal residues, localized normally to the TGN early in infection and then trafficked to cell junctions at late times and mediated virus spread. By contrast, mutants gE-495 (lacking 56 C-terminal residues) and gE-470 (lacking 81 residues) accumulated in the TGN but did not traffic to cell junctions and did not mediate cell-to-cell spread. A fourth mutant, gE-448 (lacking most of the CT domain), did not localize to cell junctions and did not mediate virus spread. Therefore, the capacity of gE/gI to promote cell-cell spread requires early localization to the TGN, but this is not sufficient for virus spread. Additionally, gE CT sequences between residues 495 and 519, which contain no obvious cell sorting motifs, are required to promote gE/gI traffic to cell junctions and cell-to-cell spread.Herpes simplex virus (HSV) commonly infects mucosal and ocular epithelial tissues, causing oral and genital lesions. During primary infection in epithelial tissues, HSV enters sensory and autonomic neurons where the virus replicates and can establish latency. Periodic reactivation in neurons leads to transient replication and spread along neuronal axons, leading to the reinfection of epithelial tissues. This cycle of HSV spread in epithelial tissues, entry into neurons, spread to sensory ganglia, and return to epithelial tissues involves directed intracellular transport to specific cell surfaces and extremely rapid spread between cells (reviewed in reference 23). As evidence of the speed of this process, HSV can spread from a single infected cell to over 250 cells in the cornea within 48 h (34). HSV is largely cell associated and spreads across cell junctions and resists the effects of high concentrations of virus-neutralizing antibodie...

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