Assembly compartment formation and nuclear alterations mediated by HCMV

HCMV 介导的装配区室形成和核改变

• 批准号: 8665535
• 负责人: JAMES C ALWINE
• 金额: $ 8.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8665535
• 关键词: ATP phosphohydrolase; Accounting; Affect; Area; Atherosclerosis; Cell Nucleus; Cells; Characteristics; Complex; Cytomegalovirus; Cytomegalovirus Infections; Cytoplasm; Cytoplasmic Structures; Cytoskeleton; DNA Sequence Rearrangement; Data; Disease; Dynein ATPase; Electron Microscopy; Endoplasmic Reticulum; Fatty Acids; Glioblastoma; Infection; Lamins; Life; Link; Malignant neoplasm of prostate; Mediating; Membrane; Microscopy; Molecular; Molecular Biology; Molecular Chaperones; Morphology; Nuclear; Nuclear Envelope; Nuclear Lamin; Nuclear Lamina; Nuclear Outer Membrane; Nuclear Structure; Nucleocapsid; Pathogenesis; Phosphorylation; Process; Production; Protein Binding; Proteins; Role; Shapes; Signal Transduction; Tertiary Protein Structure; Therapeutic Intervention; Viral; Viral Proteins; Virion; Virus; cofactor; design; glucose-regulated proteins; insight; small hairpin RNA; virology

DESCRIPTION (provided by applicant): Virion assembly and egress are among the least understood areas of virology. This is particularly true for human cytomegalovirus (HCMV), whose nucleocapsids must exit from the nucleus, then proceed through complex steps in the cytoplasm acquiring the tegument layer and envelope before they exit from the cell. The lack of understanding of HCMV assembly and egress presents a significant barrier to understanding HCMV pathogenesis and developing anti-viral treatments that might target these processes. The significance of this proposal is that it brings together data showing that HCMV utilizes both viral and cellular proteins to remodel the nucleus and the cytoplasm, forming a highly interconnected and interdependent "assembly-egress continuum" between the nucleus, the cytoplasmic viral assembly compartment (AC), and the cytoskeleton. Our previous studies of the assembly-egress continuum (5) have shown that the nuclear periphery is dramatically altered in HCMV infected cells, especially near the AC. We have demonstrated that the endoplasmic reticulum chaperone glucose regulated protein 78 (GRP78) functions as a component of the AC (6). Depletion of GRP78 results in the loss of AC integrity, virus-induced nuclear lamin phosphorylation, lamina rearrangement and the nuclear morphology characteristic of HCMV infection. Our data also confirmed the role of dynein in assembly compartment formation, and showed that the nuclear morphology characteristic of an HCMV infection is, in part, due to virus-mediated alterations in dynein function (5). The specific aims will build on these observations and further define the assembly-egress continuum. In Aim 1 we will define HCMV- mediated reorganization of the shape and size of the nucleus and AC formation. We will focus on dynein's function in these processes, as well as the potential role of fatty acid synthesis with respect to the production of nuclear membranes to maintain the enlarged nuclei in infected cells. In Aim 2 we will define the role of GRP78, SUN domain and KASH domain proteins in the assembly-egress continuum. We previously established the importance of GRP78 in assembly compartment formation/integrity and have established a link between assembly compartment-localized GRP78, lamin phosphorylation and lamina reorganization characteristic of infected cells (5, 6). We propose mutational analysis of GRP78 to further understand the role(s) of its functional domains (DNAJ, protein binding, ATPase and the KDEL ER retention signal)(1, 64) in AC formation/localization, lamina phosphorylation/rearrangement and nuclear morphology. SUN and KASH domain proteins span the nuclear envelope and connect with the cytoskeleton. Our data (5) show that SUN domain protein depletion during HCMV infection facilitates alteration in nuclear membrane structure. The role of SUN and KASH domain proteins in the assembly-egress continuum will be further examined.

描述（由申请人提供）：病毒装配和出口是病毒学领域最少的领域之一。对于人类巨细胞病毒（HCMV）尤其如此，其核皮质必须从细胞核中退出，然后通过细胞质中的复杂步骤进行，在从细胞退出之前获得Tegument层和包膜。缺乏对HCMV组装和出口的了解为理解HCMV发病机理和开发可能针对这些过程的抗病毒治疗的重大障碍。该提议的重要性是，它将数据汇总在一起，表明HCMV利用病毒和细胞蛋白来重塑细胞核和细胞质，形成了核之间高度相互联系和相互依存的“组装式连续”，胞质病毒组装组装（AC）和cytoskeleton。我们先前对装配式连续体（5）的研究表明，在HCMV感染的细胞中，尤其是在AC附近，核周围发生了巨大改变。我们已经证明，内质网状伴侣葡萄糖调节的蛋白78（GRP78）是AC的组成部分（6）。 GRP78的耗竭导致AC完整性丧失，病毒诱导的核层粘连蛋白磷酸化，层层重排和HCMV感染的核形态特征。我们的数据还证实了动力蛋白在装配室形成中的作用，并表明HCMV感染的核形态特征部分是由于病毒介导的动力蛋白功能的改变（5）。具体的目标将基于这些观察结果，并进一步定义了装配式连续性。在AIM 1中，我们将定义HCMV介导的细胞核形状和大小的重组和AC形成。我们将专注于动力蛋白在这些过程中的功能，以及脂肪酸合成在核膜产生中的潜在作用，以维持感染细胞中的核。在AIM 2中，我们将定义GRP78，Sun域和Kash域蛋白在装配式连续体中的作用。我们先前确定了GRP78在装配室形成/完整性中的重要性，并已经在受感染细胞的装配室 - 定位GRP78，lamin磷酸化和层层重组特征之间建立了联系（5，6）。我们建议对GRP78的突变分析，以进一步了解其功能结构域（DNAJ，蛋白质结合，ATPase和KDEL ER保留信号）（1，64）在交流/定位，层磷酸化/重排/重排和核形态中的作用。太阳和喀什结构域蛋白跨越了核包膜，并与细胞骨架连接。我们的数据（5）表明，HCMV感染期间的太阳域蛋白质耗竭促进了核膜结构的改变。将进一步研究太阳和Kash域蛋白在装配式连续体中的作用。