Roles of KSHV Tegument Proteins in Virion Assembly

KSHV 外皮蛋白在病毒粒子组装中的作用

基本信息

批准号：
10480736
负责人：
Chi-Der Chen
金额：
$ 40.73万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-02 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10480736
关键词：
3-Dimensional Amino Acid Sequence Amino Acids Antibodies Architecture Binding Biochemistry C-terminal Capsid Cell membrane Cells Cellular Membrane Chemicals Complex Comprehension Cryo-electron tomography Cryoelectron Microscopy Cytoplasmic Vesicles DNA Data Disease Electron Microscopy Episome Etiology Fluorescence Microscopy Foundations Glycoproteins Golgi Apparatus Herpesviridae Herpesviridae Infections Human Herpesvirus 8 In Situ Joints Kaposi Sarcoma Knowledge Label Laboratories Lesion Life Cycle Stages Light Lipid Bilayers Lytic Mediating Membrane Membrane Microdomains Microscopy Microtubules Modeling Molecular Molecular Biology Motor Movement Multicentric Angiofollicular Lymphoid Hyperplasia Mutagenesis Nature Nuclear Organelles Photons Population Process Property Protein-Protein Interaction Map Proteins Proteomics Research Research Proposals Resolution Role Side Site Sorting - Cell Movement Structure Systems Biology Techniques Testing Ubiquitin Vaccines Vesicle Viral Viral Pathogenesis Virion Virus Virus Latency design gammaherpesvirus lytic replication novel strategies nucleocytoplasmic transport particle primary effusion lymphoma protein protein interaction reconstruction segregation therapy design three dimensional structure trans-Golgi Network

项目摘要

Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Lytic KSHV replication is crucial to tumorogenicity because in KS lesions it sustains the population of latently infected cells that would otherwise be lost by segregation of latent viral episomes as spindle cells divide. Despite the importance of lytic replication in viral pathogenesis, many processes of KSHV lytic life cycle, especially the roles of tegument proteins in virion assembly, remain poorly understood, partly due to the technical challenges inherent in studying such complex, host-related and membrane-associated processes. Through over a decade of efforts, the two collaborating groups of this application have overcome many technical hurdles and obtained a large body of preliminary data on KSHV structure and assembly. Our persistent efforts, though characterized by slow progress, has paved the road finally for rapid progress towards a thorough understanding of KHSV virion assembly. For example, the Yuan group used state-of-the-art proteomics and systems biology approaches to analyze KSHV virion protein composition and to draw a virion-wide protein interaction map. The Zhou lab has advanced cryo electron microscopy (cryoEM) and cryo electron tomography (cryoET) to visualize both ordered and pleomorphic (disordered) components of large viruses at near-atomic resolution and molecular resolution, respectively. These efforts have led to our hypotheses concerning the roles of the largest tegument protein ORF64 and a KSHV-specific tegument protein ORF45: (i) ORF64 interacts with the capsid through its C- terminal end helix bundle and connects ORF45 at its other end; (ii) tegumented KSHV capsids are transported from nuclear periphery to the trans-Golgi network (TGN) through ORF45-mediated movement along microtubules; (iiI) ORF64 guides the particles to TGN membrane where viral glycoproteins are present; and (iv) ORF45 promotes internalization of viral particles into TGN vesicles for final envelopment through a mono- ubiquitin-mediated recognition by cellular membrane sorting machinery. The proposed studies harnesses our complementary expertise to test these hypotheses. Our three specific aims are: (1) We will establish the structural basis of ORF45 and ORF64 to target lipid rafts and to bind viral glycoproteins by determining in situ tegument organization in the virion by cryoET and near-atomic resolution structure of the ordered domains ORF64 in tegumented capsids by cryoEM; (2) Guided by structure data, molecular biology approaches will be utilized to define the roles of ORF64 in virion envelopment and egress at the Golgi-derived vesicles; (3) By an integrative approach of molecular biology and 3D correlative photon/electron microscopy, we will elucidate the mechanism of ORF45-mediated targeting and internalization of viral particles into the lumen of TGN vesicles. Overall, the proposed study will yield long sought-after data on the final envelopment step and regulatory mechanisms of KSHV assembly and should inform new strategies for designing KS therapies and vaccines.

概括卡波西肉瘤相关疱疹病毒（KSHV）是卡波西肉瘤（KS）的病原体，原发性渗出性淋巴瘤（PEL）和多中心卡斯尔曼病（MCD）。裂解性 KSHV 复制对于致瘤性，因为在 KS 病变中，它维持了潜伏感染细胞的群体，否则这些细胞将当纺锤体细胞分裂时，潜伏病毒附加体的分离会丢失。尽管裂解复制很重要在病毒发病机制中，KSHV 裂解生命周期的许多过程，特别是被膜蛋白在病毒粒子组装仍然知之甚少，部分原因是研究此类病毒粒子组装所固有的技术挑战复杂的、与宿主相关的和与膜相关的过程。经过十多年的努力，两人该应用的合作小组克服了许多技术障碍并获得了大量成果 KSHV 结构和组装的初步数据。我们坚持不懈的努力，尽管速度缓慢进展，最终为全面了解 KHSV 病毒体的快速进展铺平了道路集会。例如，袁团队使用最先进的蛋白质组学和系统生物学方法分析 KSHV 病毒粒子蛋白组成并绘制病毒粒子范围内的蛋白质相互作用图。周实验室有先进的冷冻电子显微镜 (cryoEM) 和冷冻电子断层扫描 (cryoET) 可可视化有序以及近原子分辨率和分子分辨率的大型病毒的多形（无序）成分，分别。这些努力导致了我们关于最大的皮层蛋白的作用的假设 ORF64 和 KSHV 特异性被膜蛋白 ORF45：(i) ORF64 通过其 C-与衣壳相互作用末端螺旋束并在其另一端连接ORF45； (ii) 运输有皮的 KSHV 衣壳通过 ORF45 介导的运动从核外围到跨高尔基体网络 (TGN) 微管； (iii) ORF64引导颗粒到达存在病毒糖蛋白的TGN膜； (四) ORF45 促进病毒颗粒内化到 TGN 囊泡中，通过单- 通过细胞膜分选机制进行泛素介导的识别。拟议的研究利用了我们的互补的专业知识来检验这些假设。我们的三个具体目标是： (1) 我们将建立 ORF45和ORF64的结构基础通过原位测定靶向脂筏并结合病毒糖蛋白通过冷冻电子显微镜观察病毒颗粒的外被组织和有序域的近原子分辨率结构通过冷冻电镜观察有皮衣壳中的 ORF64； (2)以结构数据为指导，分子生物学方法将用于定义 ORF64 在病毒粒子包封和高尔基体衍生囊泡排出中的作用； (3) 通过分子生物学和 3D 相关光子/电子显微镜的综合方法，我们将阐明 ORF45介导的病毒颗粒靶向和内化到TGN囊泡腔内的机制。总体而言，拟议的研究将产生有关最终包封步骤和监管的长期追捧的数据 KSHV 组装机制，并为设计 KS 疗法和疫苗的新策略提供信息。