Analysis of the mechanism of HCMV cytoplasmic envelopment

Although largely asymptomatic, human cytomegalovirus (HCMV) can cause severe and even fatal disease in a subset of susceptible individuals. While great progress has been made in understanding essential stages of HCMV replication, a detailed description of many of these processes is lacking. Of particular interest in this proposal is the maturation of HCMV virions, namely tegument acquisition and cytoplasmic envelopment. To provide a molecular description of these events, it is important to identify the factors involved, both viral and cellular. This proposal will focus on two viral proteins, UL88 and UL71. We have previously published a role for UL88 in packaging a subset of tegument proteins into the virion tegument layer and the absence of UL88 decreases viral fitness. Previous work has identified UL71 as an envelopment factor that potentially mediates membrane scission, as viruses lacking UL71 are trapped at various stages of budding. The experiments in this proposal will seek to elucidate the molecular details of how UL88 and UL71 drive tegument acquisition and envelopment, respectively. This includes a detailed analysis of functional regions on each protein as well as an investigation into additional factors that potentially contribute to each process. We will investigate the role of EEA1+ endosomes in tegument acquisition and for the membrane scission factor DNM1 in cytoplasmic envelopment. This proposal will utilize a novel fluorescence-based envelopment to identify additional cellular proteins that participate in envelopment. Taken together, these studies will further our understanding of the molecular events that drive the late stages of HCMV maturation and identify novel ways in which HCMV assembly can be targeted as a potential intervention.