Analysis of the mechanism of HCMV cytoplasmic envelopment

虽然在很大程度上无症状，但人类巨细胞病毒（HCMV）可在一部分易感个体中引起严重甚至致命的疾病。虽然在了解HCMV复制的基本阶段方面取得了很大进展，但对其中许多过程缺乏详细描述。本研究特别关注的是HCMV病毒粒子的成熟，即被膜获取和细胞质包膜。为了提供这些事件的分子描述，重要的是要确定涉及的因素，包括病毒和细胞。本课题将重点研究两种病毒蛋白UL88和UL71。我们之前已经发表了UL88在将被膜蛋白子集包装到病毒粒子被膜层中的作用，并且UL88的缺失会降低病毒的适应性。先前的工作已经确定UL71是一种包膜因子，可能介导膜分裂，因为缺乏UL71的病毒在出芽的各个阶段被捕获。本提案中的实验将试图阐明UL88和UL71分别如何驱动被膜获取和包膜的分子细节。这包括对每种蛋白质功能区域的详细分析，以及对每种过程潜在贡献的其他因素的调查。我们将研究EEA1+内体在被膜获取中的作用以及膜分裂因子DNM1在细胞质包膜中的作用。该建议将利用一种新的基于荧光的包膜来识别参与包膜的其他细胞蛋白质。综上所述，这些研究将进一步加深我们对驱动HCMV成熟后期的分子事件的理解，并确定HCMV组装可以作为潜在干预手段的新方法。

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.