| Summary: | Herpes simplex virus type-1 (HSV-1) morphogenesis occurs in multiple stages within infected cells. Initially, the virion capsid assembles within the nucleus and buds through the nuclear membrane into the cytoplasm. Within the cytoplasm, additional tegument proteins attach to the capsid and the fully tegumented capsids bud into trans-Golgi network (TGN) derived vesicles. Enveloped virions are ultimately secreted to extracellular spaces. The process by which the cytoplasmic capsids bud into TGN-derived vesicles is not well understood. The prevalent model calls for specific interactions among viral tegument proteins and membrane proteins and glycoproteins embedded within TGN membranes. To further investigate the roles of tegument proteins in cytoplasmic virion envelopment, we constructed deletion mutants of UL11, UL20, both UL11 and UL20, and UL16. UL11 is involved in cytoplasmic virion envelopment. The ΔUL11 virus exhibits large amounts of unenveloped capsids in the cytoplasm of infected cells. The phenotype of the double null virus most closely resembled that of the UL20 single null virus (ΔUL20) in all areas: plaque phenotype, growth kinetics, and ultrastructural characteristics. To asses whether UL11 has any affect on UL20/gK localization, confocal experiments to determine the localization of UL11, UL20 and gK were undertaken, revealing that UL11 transport was completely independent of UL20/gK. Taken together these results indicate that UL11 acts at a step in cytoplasmic envelopment downstream of UL20, and UL20 is required for proper UL11 function. However, UL11 is not dependent upon the UL20/gK heterodimer for its transport. To assess the role of UL16 in virion morphogenesis and egress, the YEbac102ΔUL16 virus was constructed using a recently described RED markerless recombination system. ΔUL16 showed a large accumulation of intranuclear capsids not seen in the ΔUL11 virus. This result indicates a two-fold role for UL16 in virion morphogenesis and egress: 1) The nuclear accumulation of capsids seems to suggest that the first and most important role of UL16 is in intranuclear capsid assembly/egress. 2) The cytoplasmic accumulation of capsids suggests that UL16 also plays a role in cytoplasmic envelopment. These results indicate a possible pathway for the juxtaposition of cytoplasmic capsids with TGN-derived vesicles for final cytoplasmic envelopment.
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