Herpes simplex virus (HSV) is a ubiquitous human pathogen whose efficient transmission depends on successful viral envelopment and cell-to-cell spread. Alphaherpesviruses acquire their final envelope from cytoplasmic membranes through interactions between viral glycoproteins and the tegument-coated nucleocapsid, with the gE/gi complex playing a central role. This study further characterizes the function of gE/gi in HSV-1 secondary envelopment and spread. We demonstrate that glycoproteins gD and gE act redundantly to mediate secondary envelopment, as deletion of both proteins blocks envelopment and leads to cytoplasmic nucleocapsid accumulation. Specific regions of the gE cytoplasmic tail were identified as critical for envelopment, tegument interaction, intracellular trafficking, and cell-to-cell spread. These findings highlight distinct molecular requirements for HSV envelopment and reveal additional roles for gE beyond envelope acquisition.
