Cytoplasmic Residues of Herpes Simplex Virus Glycoprotein gE Required for Secondary Envelopment and Binding of Tegument Proteins VP22 and UL11 to gE and gD

Abstract

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...

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“…5, it is likely that some components of the capsid-associated inner tegument proteins are assembled inside the host nucleus at or near the nuclear lamina prior to budding through the inner nuclear membrane in the early stage of virion maturation. Our observation of the outer tegument layer conforming to the pleomorphic shape of viral envelope implies that the outer tegument proteins might be packaged into maturing virion particles through interactions with the viral glycoproteins decorated on the trans Golgi membrane (Chi et al 2005;Farnsworth et al 2007;Fuchs et al 2002). As the first to be released into the cellular environment upon infection, outer layer proteins are probably essential viral functional proteins during the early stages of infection.…”

Function of glycoprotein E of herpes simplex virus requires coordinated assembly of three tegument proteins on its cytoplasmic tail

“…The limiting membranes of these vesicles are thought to contain gE, with its tail extended into the cytoplasm. Alterations in the tail of gE reduce the numbers of virions at cell junctions, and they instead accumulate on apical surfaces (2,6,17,35). This misrouting results in smaller plaques and reduces epithelial cell-to-cell spread in vivo and in vitro.…”

“…However, we cannot rule out the possibility that the triplex of tegument proteins works independently of gE by forming complexes on the tails of other viral membrane proteins. For example, there is an unconfirmed report that UL11 binds to the tail of gD (17). If so, these three tegument proteins might directly regulate the core fusion machinery.…”

“…There are three tegument proteins known to bind to gE: UL11, UL16, and VP22 (17)(18)(19)(20). UL11 and UL16 were investigated here because they bind each other as well.…”

Glycoproteins gE and gI Are Required for Efficient KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles in Neurons

“…Importantly, unlike Us9 null mutants, gE and gI single or double null mutants display a small-plaque phenotype in cultured epithelial cells (9), suggesting that this protein complex has multiple functions. Indeed, the gE/gI complex is involved in virion assembly, cell-to-cell spread, species-specific binding of immunoglobulin G as an Fc receptor, and mediating full virulence in animal infections (32,(34)(35)(36)(37)(38).…”