单泛素化ORF45蛋白介导卡波氏肉瘤相关疱疹病毒（KSHV）定位自噬体进行组装的机制研究

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of seven currently known human cancer virus. This virus causes Kaposi's sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma （PEL） and some types of multicentric Castleman's disease (MCD). .KSHV exhibits two alternative phases in its life cycle, the lytic and latent. Primary infection of a host cell establishes viral latency by expression of only a limited number of viral (latent) genes and no infectious virions are produced. The latent viral genome undergoes periodic lytic reactivation followed by a temporally regulated cascade of viral gene expression. The newly expressed viral proteins assemble to form infectious mature virions which egress out of the cell and initiate primary infection of uninfected cell. In KSHV, the lytic cycle is also responsible to tumorigenesis by spreading viruses to target cells and providing paracrine regulation for KS development. Given the importance of the KSHV lytic cycle in both viral propagation and tumorigenesis, a thorough understanding of viral lytic replication processes including virion assembly and egress becomes vital.. KSHV is a large double-stranded DNA virus with a protein covering that packages its nucleic acids, called capsid, which is then surrounded by an amorphous protein layer called tegument, and finally enclosed in a lipid envelope derived in part from the cell membrane. The tegument layer, containing the most of viral structure proteins which orderedly built through specific protein–protein interactions, is critical for viral assembly. .Our recent study found the tegument protein ORF45, which is believed as an outer tegument protein, located in lipid raft (LR) and trafficked from trans-Golgi network (TGN) to early endosome and autophagosome with LR during KSHV assembly process. Others also found the formation of autophagosome was augmented by KSHV lytic replication, while the fusion of autophagosome and lysosome was suppressed. It is still understudied how autophagy will benefit KSHV assembly..To address how ORF45 is involved in autophagosome for KSHV assembly, we will examine the SIAH induced monoubiquitination in autophagosome localization of ORF45. It has been reported SIAH could catalyze ubiquitination of ORF45. We also found monoubiquitination on Lys297 of ORF45 is vital for its LR-localization. And the K297R mutant of ORF45 could not locate in autophagosome. In the following study we will reveal the mechanism of ubiquitination mediating autophagosome localization of ORF45..In additionally, we have found the wild type KSHV (BAC-16) nucleocapsid could locate in autophagosome, while the ORF45 mutant virus (BAC-K297R) could not locate in autophagosome, but colocalize with lysosome. And much less progeny virus was released than wild type. It indicated ORF45 might mediate KSHV assembly in autophagosome. In the going-on study, we will disclose the mechanism of ORF45 regulating KSHV assembly autophagosome..Overall, our study will provide a reasonable alternative for KSHV regulating autophagy. The virus may hijack autophagy as their assembly relay station, and avoid degradation by lysosome. ORF45 may serve as a shuttle for viral structure protein transportation and localization.

卡波氏肉瘤相关疱疹病毒（KSHV）是人类七种致瘤病毒之一，可致淋巴和皮肤粘膜等系统肿瘤。KSHV在裂解复制期组装释放子代病毒，感染更多组织细胞，是病毒的持续感染的重要原因。有研究发现KSHV在裂解复制期促进自噬体的形成，同时又抑制自噬体与溶酶体融合，但KSHV是否利用自噬进行组装尚未见报道。我们前期研究发现，单泛素化的ORF45可以定位于脂筏，参与病毒组装，在此过程中ORF45和衣壳蛋白都能与自噬体部分共定位，ORF45还能促进自噬。我们推测ORF45可能以脂筏为平台，通过与自噬体相关蛋白相互作用定位于自噬体，并组织KSHV的一些结构蛋白进入自噬体进行组装。本课题将通过激光共聚焦显微镜和免疫电镜等技术可视化地探究ORF45定位于自噬体的机制，及ORF45介导KSHV在自噬体组装的机制，阐明宿主细胞的自噬对KSHV组装的影响，为寻找抑制KSHV裂解复制的靶点提供更多思路。

卡波氏肉瘤相关疱疹病毒（Kaposi's sarcoma associated herpesvirus, KSHV），又称HHV8，是一类γ型疱疹病毒，与皮肤、口腔、生殖系统等多种肿瘤的发生密切相关。病毒组装是裂解性复制中的重要环节，阻断KSHV组装将有效的抑制子代病毒的产生，抑制KSHV的持续感染，但目前这方面的研究报道较少。我们重点研究KSHV在宿主细胞浆中的组装机制，为寻找阻断KSHV持续感染的靶点提供更多思路。我们的前期研究发现，KSHV的被膜蛋白ORF45的单泛素化对于病毒在胞浆的定位和组装有着重要的作用，但其作用机制尚不明确。因此，通过IP-MS（免疫共沉淀联合质谱）进一步分析与ORF45相互作用的蛋白谱，筛选到了一个泛素连接酶RNF14与ORF45相互作用，并通过免疫共沉淀，验证了二者的相互作用，并发现RNF14介导ORF45的泛素化，起到了稳定ORF45的作用，并且介导ORF45定位于脂筏和自噬体，参与病毒的组装释放。初步阐明了ORF45介导KSHV借助自噬体组装释放的机制。