TRAFFICKING OF CYTOMEGALOVIRUS FROM THE NUCLEAR MEMBRANE TO THE GOLGI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。人类巨细胞病毒颗粒结构复杂，至少由30种不同的蛋白质组成。病毒基因组包含在一个二十面体的核衣壳内，该核衣壳被一层称为被层的无定形层所包围。被膜被包裹在含有病毒编码的糖蛋白的脂膜中。病毒粒子的组装是复杂的，因为病毒的复制和包膜发生在感染细胞的细胞核中，而一些被膜蛋白和成熟的包膜的获取发生在细胞质中。虽然最近提出了一种病毒粒子通过核膜逃离细胞核的机制，但调节亚病毒粒子运输到最终包膜部位的途径尚未确定。由于细胞质组装隔间与感染细胞的微管组织中心(MTOC)重叠，我们假设颗粒在微管上跟踪到这个位置。货物向MTOC的运输是由负端定向的微管马达完成的，其中细胞质动力蛋白是研究最广泛的。在未感染的细胞中，动力蛋白活性调节许多过程，包括从细胞外围输入物质、逆行运输囊泡、细胞器定位、核膜破裂和细胞分裂。因此，病毒似乎已经进化出利用动力蛋白和微管网络的其他组件来有效组装病毒后代并从感染细胞中释放的机制。同样，众所周知，人巨细胞病毒感染会导致关键细胞周期蛋白的失调和细胞周期停滞。细胞信号通路和细胞周期进程的扰动为病毒基因的表达和复制提供了有利的环境；然而，我们最近使用细胞周期蛋白依赖性激酶(CDK)抑制剂的研究表明，这些蛋白也在病毒组装中发挥作用。因为微管马达的活性是由磷酸化来调节的，我们假设马达蛋白是感染细胞中CDK活性的底物。为了阐明微管马达，特别是动力蛋白在巨细胞病毒颗粒组装中的作用，我们建议完成以下特定目标：1)研究动力蛋白和动力蛋白在巨细胞病毒感染细胞中的表达和定位。2)测定动力蛋白抑制对核外流和包膜的影响。3)阐明CDKs对动力蛋白功能的调节。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The human cytomegalovirus particle is structurally complex and consists of at least 30 different proteins. The viral genome is contained within an icosahedral nucleocapsid that is surrounded by an amorphous layer called the tegument. The tegument is enclosed within a lipid membrane containing virus-encoded glycoproteins. The assembly of the virion is complicated because viral replication and encapsidation occur in the nucleus of the infected cell but acquisition of some tegument proteins and a mature envelope takes place in the cytoplasm. While a mechanism by which the virion escapes the nucleus through the nuclear membrane has recently been proposed, the pathways that regulate transport of the subviral particle to the site of final envelopment have not been definitively identified. Because the cytoplasmic assembly compartment overlaps the microtubule-organizing center (MTOC) of the infected cell, we hypothesize that the particles track to this site on microtubules. The transport of cargo towards the MTOC is accomplished by minus-end directed microtubule motors of which cytoplasmic dynein is the most extensively studied. In uninfected cells, dynein activity mediates many processes including import of material from the cell periphery, retrograde vesicle traffic, positioning of cellular organelles, nuclear envelope breakdown, and cell division. It therefore seems likely that the virus has evolved mechanisms to exploit dynein and other components of the microtubule network for efficient assembly of viral progeny and release from the infected cell. Along the same lines, it is well established that HCMV infection leads to dysregulation of key cell cycle proteins and cell cycle arrest. The perturbation of cellular signaling pathways and cell cycle progression provides an environment conducive to viral gene expression and replication; however, our recent studies using inhibitors of the cyclin-dependent kinases (cdks) suggest that these proteins also play a role in virus assembly. Because the activity of microtubule motors is regulated by phosphorylation, we hypothesize that motor proteins are substrates for cdk activity in infected cells. To elucidate the roles of microtubule motors, specifically dynein, in the assembly of HCMV particles we propose to accomplish the following specific aims: 1) Characterization of dynein and dynactin expression and localization in HCMV-infected cells. 2) Determination of the effects of dynein inhibition on nuclear egress and envelopment. 3) Elucidation of the regulation of dynein function by cdks.