Development of the human cytomegalovirus assembly complex

描述（由申请人提供）：人巨细胞病毒（HCMV）是一种重要的人类病原体，在免疫功能低下的个体和先天性感染的儿童中引起严重疾病。需要改进预防和控制HCMV感染的方法。我们最近观察到人类巨细胞病毒（HCMV）细胞质病毒粒子组装室（AC）的中心含有一组由早期内体抗原1 （EEA1）标记的囊泡。这项工作和进一步的工作导致了一种新的交流结构模型的发展。AC是在感染后的最初2至4天内发生的戏剧性的细胞质重塑过程中产生的。重构导致细胞胞外通路的重新定位，使得早期的内噬室最终位于集中排列的高尔基囊泡和反高尔基囊泡网络的中心。虽然最初违反直觉，但三维AC结构提供了病毒粒子成熟和退出的合理途径。本研究的目的是确定HCMV感染细胞中负责细胞分泌装置重新定向的HCMV基因产物。AC的发育依赖于先前的病毒DNA合成，这表明一个或多个病毒的Early-Late或Late基因对这一过程至关重要。因此，在Aim 1中，我们将系统地去除病毒早期晚期和晚期基因的表达，这些基因已知对病毒的有效复制很重要。共聚焦显微镜将用于分析作为AC发展指标的独特细胞质重排。在目标2中，这些蛋白的作用将通过在未感染细胞中候选基因的异位表达，通过异位基因表达和混合感染（例如，如果涉及两个基因，则与所讨论的基因单独删除的病毒共同感染）来补充缺乏这些基因的病毒，以及鉴定与负责AC发展的病毒蛋白相互作用的细胞蛋白来确定。这项工作将提供(i)更深入地了解HCMV的成熟和输出，（ii）扩展HCMV基因的已识别功能阵列，（iii）识别这一过程中的细胞参与者，以及（iv）阐明控制细胞细胞器发生的机制。由于AC对HCMV病毒粒子成熟的重要性，这项工作将导致抗病毒药物开发新的分子靶点和机制。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is an important human pathogen, causing serious disease in immune compromised individuals and in congenitally infected children. Improved methods are needed for prevention and control of HCMV infections. We recently made the novel observation that the center of the human cytomegalovirus (HCMV) cytoplasmic virion assembly compartment (AC) harbors an assemblage of vesicles that are marked by early endosome antigen 1 (EEA1). This and further work led to development of a new model of AC structure. The AC is created during a dramatic cytoplasmic remodeling process that takes place during the first 2 to 4 days after infection. The remodeling results in reorientation of the cellular exocytic pathway such that the early endocytic compartment ultimately resides at the center of a network of concentrically-arranged Golgi and trans-Golgi vesicles. While initially counterintuitive, the three dimensional AC structure offers a rational pathway of virion maturation and egress. The objective of the proposed work is to identify the HCMV gene product(s) responsible for reorientation of the cellular secretory apparatus in HCMV infected cells. AC development is dependent on prior viral DNA synthesis, indicating that one or more viral Early-Late or Late genes are essential for the process. Thus, in Aim 1 we will systematically ablate expression of viral Early-Late and Late genes that are known to be important for efficient viral replication. Confocal microscopy will be used to assay for the distinctive cytoplasmic rearrangements that are indicators of AC development. In Aim 2, the roles of these proteins will be identified by ectopic expression of candidate genes in uninfected cells, complementation of viruses lacking these genes by ectopic gene expression and by mixed infections (e.g., if two genes are involved, co-infection with viruses singly deleted for the genes in question), and identification of cellular proteins that interact with the viral proteins responsible for AC development. This work will provide (i) deeper understanding of HCMV maturation and egress, (ii) expansion of the array of identified functions for HCMV genes, (iii) identification of cellular participants in this process, and (iv) illumination of mechanisms that control cellular organellogenesis. Because of the importance of the AC to HCMV virion maturation, this work will result in new molecular targets and mechanisms to be exploited by antivirals.