Development of the human cytomegalovirus assembly complex

描述（由申请方提供）：人巨细胞病毒（HCMV）是一种重要的人类病原体，可在免疫功能低下的个体和先天性感染儿童中引起严重疾病。需要改进的方法来预防和控制HCMV感染。我们最近进行了新的观察，中心的人巨细胞病毒（HCMV）的细胞质病毒粒子装配区室（AC）窝藏组装的囊泡，标志着早期内体抗原1（EEA 1）。这项工作和进一步的工作导致了AC结构的新模型的开发。AC是在感染后的前2至4天发生的剧烈细胞质重塑过程中产生的。重构导致细胞外排途径的重新定向，使得早期内吞隔室最终位于同心排列的高尔基体和反高尔基体囊泡的网络的中心。虽然最初是违反直觉的，但三维AC结构提供了病毒体成熟和排出的合理途径。拟开展的工作的目的是鉴定HCMV感染细胞中负责细胞分泌器重定向的HCMV基因产物。AC的发展依赖于先前的病毒DNA合成，表明一个或多个病毒早-晚或晚基因对该过程至关重要。因此，在目标1中，我们将系统地消除已知对有效病毒复制重要的病毒早-晚和晚基因的表达。将使用共聚焦显微镜检测作为AC发展指标的独特细胞质重排。在目的2中，这些蛋白质的作用将通过候选基因在未感染细胞中的异位表达、通过异位基因表达和通过混合感染（例如，如果涉及两个基因，则与所述基因单独缺失的病毒共感染），以及鉴定与负责AC发展的病毒蛋白相互作用的细胞蛋白。这项工作将提供（i）更深入地了解HCMV成熟和出口，（ii）扩展HCMV基因的已识别功能阵列，（iii）识别此过程中的细胞参与者，以及（iv）控制细胞器官发生的机制的照明。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.