HSV-1 induces stable associations between the Golgi and endosomes within the cytoplasmic viral assembly compartment of neuronal cells

-Golgi network (TGN) and endosomal compartments have been implicated as membrane sources for cytoplasmic envelopment, how secretory and endosomal pathways are reorganized and associated within the cVAC remains unclear. Here, we treated HSV-1-infected cells with nocodazole at a post-entry stage to disperse membrane components of the cVAC and identify microtubule-independent membrane organizations and associations using immunofluorescence imaging. We found that the Golgi apparatus was fragmented during infection but maintained cisternal stacking, as the Golgi markers GM130, GalT, and GRASP65 remained colocalized in both the absence and presence of nocodazole. We further demonstrated that Golgi mini-stacks retained their association with Rab6 during HSV-1 infection and formed microtubule-independent associations with early and recycling endosomes, resulting in the formation of Golgi-endosome super-mini-stacks. Viral capsid, glycoprotein, and tegument proteins were colocalized with Golgi-endosome super-mini-stacks in both the presence and absence of microtubules, suggesting that this membrane complex serves as a site of cytoplasmic envelopment regardless of clustering at the MTOC. Formation of these stable Golgi-endosome associations did not require viral capsids. Neither actin filaments nor acetylated microtubules colocalized with Golgi-endosome super-mini-stacks, suggesting that cytoskeletal elements are not required for this association.IMPORTANCEMany viruses remodel host cytoplasmic membranes to facilitate their assembly. Elucidating the molecular mechanisms underlying this remodeling may reveal novel antiviral targets while also providing insight into fundamental principles of intracellular membrane organization. Infection with an important human pathogen, herpes simplex virus type 1 (HSV-1), induces extensive membrane remodeling that leads to the formation of the cytoplasmic viral assembly compartment (cVAC) in neuronal cells, yet the underlying mechanisms remain poorly understood. Here, we demonstrate that HSV-1 induces stable associations between membranes within the cVAC. Our findings support a model in which HSV-1 generates a dynamic hybrid membrane compartment during infection, potentially reconciling conflicting views on the roles of distinct membrane sources in cytoplasmic assembly.