Roles of KSHV Tegument Proteins in Virion Assembly

摘要 卡波西肉瘤相关疱疹病毒(KSHV)是原发卡波西肉瘤(KS)的病原体 渗出性淋巴瘤(PEL)和多中心性Castleman病(MCD)。裂解KSHV复制对 致瘤性，因为在KS病变中，它维持着潜伏感染细胞的数量，否则就会 当纺锤形细胞分裂时，由于潜伏的病毒表型分离而丢失。尽管裂解复制很重要 在病毒致病过程中，KSHV裂解生命周期的许多过程，尤其是被膜蛋白在病毒致病中的作用。 病毒粒子的组装仍然知之甚少，部分原因是研究这种病毒粒子所固有的技术挑战 复杂的寄主相关和膜相关过程。经过十多年的努力，两人 该应用程序的协作小组克服了许多技术障碍，并获得了大量的 关于KSHV结构和组装的初步数据。我们坚持不懈的努力，尽管特点是缓慢 进步，最终为彻底了解KHSV病毒粒子的快速进展铺平了道路 集合。例如，袁小组使用最先进的蛋白质组学和系统生物学方法来 分析KSHV病毒粒子的蛋白质组成并绘制病毒粒子范围的蛋白质相互作用图。周实验室已经有 先进的冷冻电子显微镜(CryoEM)和冷冻电子断层扫描(CryoET)以可视化这两种有序 以及大病毒在近原子分辨率和分子分辨率下的多态(无序)成分， 分别进行了分析。这些努力导致了我们关于最大的被膜蛋白的作用的假设 ORF64和KSHV特异的被膜蛋白ORF45：(I)ORF64通过其C- 末端螺旋束并在其另一端连接ORF45；(Ii)运输被覆的KSHV衣壳 从核周到跨高尔基网络(TGN)的ORF45介导的运动 微管；(Iii)ORF64将颗粒引导到存在病毒糖蛋白的TGN膜上；以及(Iv) ORF45促进病毒颗粒内化到TGN囊泡中，最终通过单核细胞被膜包裹。 泛素介导的细胞膜分选机识别。拟议的研究利用了我们的 互补的专业知识来检验这些假说。我们的三个具体目标是：(一)我们将建立 原位检测ORF45和ORF64靶向脂筏和结合病毒糖蛋白的结构基础 病毒粒子的冷冻组织和有序结构的近原子分辨结构 (2)在结构数据的指导下，分子生物学的方法将是 用来确定ORF64在高尔基体衍生小泡的病毒粒子包膜和出口中的作用；(3)通过 分子生物学和三维相关光子/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TGN囊泡的机制。 总体而言，拟议的研究将产生有关最终封存步骤和监管的长期数据 KSHV组装的机制，并应为设计KS疗法和疫苗的新战略提供信息。

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.