Biogenesis and operation of the human cytomegalovirus assembly complex

描述（由申请人提供）：人巨细胞病毒（HCMV）是美国最具公共卫生和医学意义的疱疹病毒。除了作为人类病原体的社会重要性之外，病毒在与人类共同进化期间“学习”的人类生物学方面使HCMV能够发展成为非常有价值的模式微生物，有助于揭示免疫学，分子生物学和细胞生物学领域中病毒-宿主相互作用的许多方面。HCMV细胞质病毒体组装复合物（cVAC）是一种约细胞核大小的结构，其中发育中的病毒体获得其大部分被膜，被包膜，然后被转运到细胞表面释放。cVAC生物发生涉及在感染后的前2至4天期间发生的显著的细胞质重塑。我们发现，cVAC被安排为一组嵌套的圆柱体，外圆柱体由来自高尔基体和trans-Golgi网络的管状囊泡的网络组成;内圆柱体由来自回收内体的囊泡组成，其中病毒粒子被包裹，然后运输到细胞表面。从定量分析的分泌和内体机械的各种组件的标记物的分布，我们确定了引人注目的例子，HCMV诱导的转变中的身份参与蛋白质运输的细胞器，我们已经确定了三个HCMV基因作为候选人调节cVAC生物合成。我们的中心假设是HCMV蛋白通过病毒蛋白与细胞蛋白和细胞器的相互作用协调cVAC的产生，以优化感染性病毒体的产生和排出。为了验证这一假设，在目标1中，我们将定义我们已确定为cVAC生物发生的候选调节因子的病毒基因的作用，并将其用作探针来描绘导致cVAC组装的分子途径。在目标2中，我们将确定重组的分泌细胞器和机制如何有助于病毒体组装和成熟的过程。我们将采用重组病毒来表达候选病毒和细胞调节蛋白，这些蛋白已被工程改造为含有蛋白质去稳定化结构域，从而能够在cVAC生物发生和病毒复制期间的所需时间破坏或重新稳定目标蛋白。然后，我们将使用一组试验来测量对病毒复制的影响，并比较受影响的细胞器和机器的功能，以及是否灭活感兴趣的调节剂。我们的研究将导致（i）病毒粒子成熟过程的照明和HCMV如何管理细胞系统的好处，（ii）定义新的目标，开发新的抗病毒化合物，和（iii）新的见解调节细胞蛋白质运输和处理细胞器的生物发生的机制。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is the herpesvirus of greatest public health and medical significance in the US. In addition to its societal importance as a human pathogen, the aspects of human biology "learned" by the virus during its co-evolution with humans have enabled development of HCMV into an extraordinarily valuable model microbe that has helped to reveal many aspects of virus-host interactions in the areas of immunology, molecular biology, and cell biology. The HCMV cytoplasmic virion assembly complex (cVAC) is a structure about the size of a nucleus where developing virions acquire most of their tegument, are enveloped, and are then transported to the cell surface for release. cVAC biogenesis involves dramatic cytoplasmic remodeling that takes place during the first 2 to 4 days after infection. We found that the cVAC is arranged as a set of nested cylinders, with the outer cylinder consisting of networks of tubular vesicles derived from the Golgi apparatus and the trans-Golgi network; the inner cylinder consists of vesicles derived from recycling endosomes where virions are enveloped and then transported to the cell surface. From quantitative analyses of the distribution of markers for various components of the secretory and endosomal machinery, we identified striking examples of HCMV-induced shifts in the identities of organelles involved in protein transport, and we have identified three HCMV genes as candidate regulators of cVAC biogenesis. Our central hypothesis is that HCMV proteins orchestrate creation of the cVAC via interactions of viral proteins with cellular proteins and organelles to optimize infectious virion production and egress. To test this hypothesis, in Aim 1, we will define the roles of the virus genes we have identified as candidate regulators of cVAC biogenesis and use them as probes to delineate the molecular pathway that results in assembly of the cVAC. In Aim 2, we will determine how the reorganized secretory organelles and machinery contribute to the process of virion assembly and maturation. We will employ recombinant viruses to express candidate viral and cellular regulatory proteins that have been engineered to contain protein destabilization domains, enabling either destruction or restabilization of the protein of interest at desired times during cVAC biogenesis and virus replication. We will then use a battery of assays to measure the effects on virus replication, and to compare the functions of the affected organelles and machinery with and without inactivation of the regulator of interest. Our studies will result in (i) illumination of the process of virion maturation and how HCMV manages cellular systems for its benefit, (ii) definition of new targets for development of novel antiviral compounds, and (iii) new insights into the mechanisms that regulate the biogenesis of cellular protein transport and processing organelles.