Development of the human cytomegalovirus assembly complex

描述(申请人提供)：人类巨细胞病毒(HCMV)是一种重要的人类病原体，在免疫功能受损的人和先天性感染的儿童中会引起严重疾病。需要改进方法来预防和控制人巨细胞病毒感染。我们最近做了一项新的观察，发现人巨细胞病毒(HCMV)细胞质病毒粒子组装室(AC)的中心含有以早期内吞体抗原1(EEA1)为标志的囊泡。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开发。AC是在感染后的头2到4天内发生的戏剧性的细胞质重塑过程中产生的。这种重塑导致细胞外分泌途径的重新定位，使得早期的内吞隔室最终位于同心排列的高尔基体和跨高尔基体小泡网络的中心。虽然最初是违反直觉的，但三维AC结构提供了病毒粒子成熟和外泄的合理途径。本研究的目的是确定在巨细胞病毒感染细胞中负责细胞分泌器官重定位的巨细胞病毒基因产物S。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早-晚或晚基因在这一过程中是必不可少的。因此，在目标1中，我们将系统地去除病毒早-晚和晚基因的表达，这些基因对于有效的病毒复制是重要的。共聚焦显微镜将被用来分析作为AC发展指标的独特的细胞质重排。在目标2中，这些蛋白的作用将通过以下方式确定：候选基因在未感染细胞中的异位表达，缺乏这些基因的病毒通过异位基因表达和混合感染(例如，如果涉及两个基因，与单个缺失的病毒共感染)，以及与负责AC发展的病毒蛋白相互作用的细胞蛋白的鉴定。这项工作将提供(I)更深入的了解人巨细胞病毒成熟和出口，(Ii)扩大已发现的巨细胞病毒基因的功能，(Iii)鉴定细胞在这一过程中的参与者，(Iv)阐明控制细胞器官发生的机制。由于AC对HCMV病毒粒子成熟的重要性，这项工作将导致新的分子靶点和机制被抗病毒药物所利用。

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.