Herpes simplex virus gE/gI must accumulate in the trans-Golgi network at early times and then redistribute to cell junctions to promote cell-cell spread.

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.