Complete disruption of gD binding to HVEM alters virus replication and host receptor homeostasis

Herpes simplex virus 1 (HSV‑1) establishes infection through coordinated interactions of multiple viral glycoproteins with host cell receptors. Interactions between the viral glycoprotein D (gD) and the immunomodulatory cellular receptor HVEM (herpesvirus entry mediator) critically influence HSV‑1 latency and reactivation by regulating both receptor engagement and immunomodulatory signaling in the corneal epithelium, immune cells, and neurons. The underlying mechanisms of these phenotypes remain poorly understood because no tools selectively block their interaction. To dissect the contributions of individual amino acids (aa) to HVEM binding, we co‑immunoprecipitated wild‑type (WT) and mutant (Q27P and Q27A‑L28A‑T29A) forms of gD that disrupt gD binding to HVEM. We found that gD aa Q27-T29 were critical for HVEM binding, with the triple mutant showing greater disruption of HVEM binding than the Q27P single mutation, as assessed by co-immunoprecipitation and immunostaining. To map gD binding to HVEM and assess functional relevance, we constructed two recombinant viruses: a single-amino-acid (aa) gD mutation (v27 virus) and a triple-aa mutation in gD (v27-29 virus). Replication of both mutant viruses was comparable to that of the WT virus in four mammalian cell lines, except in Neuro2a cells. However, using confocal microscopy, HVEM protein expression was reduced in v27-29-infected Neuro2a cells, with no gD colocalization observed. Although Neuro2a cells infected with the single gD v27 mutant also showed less HVEM-gD colocalization than Neuro2a cells infected with WT virus, gD protein levels were not affected. RT‑PCR analysis of Neuro2a cells infected with single and triple gD mutants showed significantly less expression of viral glycoproteins (gB, gC, gD), host receptors (HVEM, nectin‑1, 3‑O‑sulfated heparan sulfate), and HVEM ligands (BTLA, LIGHT, Ltα, CD160), with the most pronounced effects observed for the triple v27‑29 mutant virus. Together, these results show that aa Q27-T29, rather than aa Q27 alone, are essential to completely block gD binding to HVEM and, consequently, affect overall receptor homeostasis.