Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane

Abstract

ABSTRACT Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection. IMPORTANCE Many viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.