Roles of KSHV Tegument Proteins in Virion Assembly

总结 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Castleman病（MCD）。裂解性KSHV复制对于 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 梭形细胞分裂时，潜伏病毒游离体的分离使病毒游离体丢失。尽管裂解复制的重要性 在病毒致病机制中，KSHV裂解生命周期的许多过程，特别是被膜蛋白在 病毒体组装，仍然知之甚少，部分原因是研究这种病毒体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， 该应用的合作小组克服了许多技术障碍，并获得了大量的 KSHV结构和装配的初步数据。我们坚持不懈的努力，虽然缓慢， 的进展，铺平了道路，最终迅速取得进展，对KHSV病毒粒子的彻底了解 组装件.例如，Yuan团队使用最先进的蛋白质组学和系统生物学方法， 分析KSHV病毒体蛋白组成并绘制病毒体蛋白相互作用图谱。Zhou实验室拥有 先进的冷冻电子显微镜（cryoEM）和冷冻电子断层扫描（cryoET）， 和近原子分辨率和分子分辨率的大型病毒的多形（无序）组分， 分别这些努力导致了我们关于最大的皮层蛋白质的作用的假设 ORF 64和KSHV特异性被膜蛋白ORF 45：（i）ORF 64通过其C-末端与衣壳相互作用。 末端螺旋束并在其另一端连接ORF 45;（ii）被膜化的KSHV衣壳被运输 通过ORF 45介导的沿沿着的移动，从核周边到高尔基体网络（TGN） （iiI）ORF 64将颗粒引导至存在病毒糖蛋白的TGN膜;以及（iv） ORF 45促进病毒颗粒内化到TGN囊泡中，通过单- 泛素介导的细胞膜分选机制的识别。拟议的研究利用了我们的 补充专业知识来测试这些假设。我们的三个具体目标是：（一）建立 通过原位测定ORF 45和ORF 64靶向脂筏和结合病毒糖蛋白的结构基础 病毒粒子被膜组织的cryoET和有序结构域的近原子分辨结构 ORF 64的结构分析;（2）在结构数据的指导下， 用于定义ORF 64在高尔基体衍生囊泡的病毒体增殖和外出中的作用;（3）通过一个 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 ORF 45介导的靶向和内化病毒颗粒进入TGN囊泡内腔的机制。 总的来说，拟议的研究将产生长期追求的数据，最后的实施步骤和监管 KSHV的组装机制，并应告知设计KS疗法和疫苗的新策略。

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.