Sugimoto Atsuko, Abe Yuichi, Watanabe Tadashi, Hosokawa Kohei, Adachi Jun, Tomonaga Takeshi, Iwatani Yasumasa, Murata Takayuki, Fujimuro Masahiro
Department of Cell Biology, Kyoto Pharmaceutical University, Yamashina, Kyoto, Japan.
Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Japan.
J Virol. 2021 Apr 26;95(10). doi: 10.1128/JVI.02194-20. Epub 2021 Feb 24.
During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication, host cell functions including protein expression and post-translational modification pathways are dysregulated by KSHV to promote virus production. Here, we attempted to identify key proteins for KSHV lytic replication by profiling protein expression in the latent and lytic phases using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis, immunoblotting, and quantitative PCR demonstrated that antigen-F (HLA-F) adjacent transcript 10 (FAT10) and UBE1L2 (also known as ubiquitin-like modifier-activating enzyme 6, UBA6) were upregulated during lytic replication. FAT10 is a ubiquitin-like protein (UBL). UBE1L2 is the FAT10-activating enzyme (E1), which is essential for FAT10 modification (FAT10ylation). FAT10ylated proteins were immediately expressed after lytic induction and increased over time during lytic replication. Knockout of UBE1L2 suppressed KSHV production but not KSHV DNA synthesis. In order to isolate FAT10ylated proteins during KSHV lytic replication, we conducted immunoprecipitations using anti-FAT10 antibody and Ni-NTA chromatography of exogenously expressed His-tagged FAT10 from cells undergoing latent or lytic replication. LC-MS/MS was performed to identify FAT10ylated proteins. We identified KSHV ORF59 and ORF61 as FAT10ylation substrates. Our study revealed that the UBE1L2-FAT10 system is upregulated during KSHV lytic replication, and it contributes to viral propagation.Ubiquitin and UBL post-translational modifications, including FAT10, are utilized and dysregulated by viruses for achievement of effective infection and virion production. The UBE1L2-FAT10 system catalyzes FAT10ylation, where one or more FAT10 molecules are covalently linked to a substrate. FAT10ylation is catalyzed by the sequential actions of E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase) enzymes. The E1 enzyme for FAT10ylation is UBE1L2, which activates FAT10 and transfers it to E2/USE1. FAT10ylation regulates the cell cycle, IFN signaling, and protein degradation; however, its primary biological function remains unknown. Here, we revealed that KSHV lytic replication induces UBE1L2 expression and production of FAT10ylated proteins including KSHV lytic proteins. Moreover, UBE1L2 knockout suppressed virus production during the lytic cycle. This is the first report demonstrating the contribution of the UBE1L2-FAT10 system to KSHV lytic replication. Our findings provide insight into the physiological function(s) of novel post-translational modifications in KSHV lytic replication.
在卡波西肉瘤相关疱疹病毒（KSHV）的裂解复制过程中，包括蛋白质表达和翻译后修饰途径在内的宿主细胞功能会被KSHV失调，以促进病毒产生。在此，我们试图通过使用液相色谱-串联质谱（LC-MS/MS）分析潜伏和裂解阶段的蛋白质表达，来鉴定KSHV裂解复制的关键蛋白。蛋白质组学分析、免疫印迹和定量PCR表明，抗原-F（HLA-F）相邻转录本10（FAT10）和UBE1L2（也称为泛素样修饰激活酶6，UBA6）在裂解复制过程中上调。FAT10是一种泛素样蛋白（UBL）。UBE1L2是FAT10激活酶（E1），对FAT10修饰（FAT10ylation）至关重要。FAT10化蛋白在裂解诱导后立即表达，并在裂解复制过程中随时间增加。敲除UBE1L2可抑制KSHV产生，但不影响KSHV DNA合成。为了在KSHV裂解复制过程中分离FAT10化蛋白，我们使用抗FAT10抗体进行免疫沉淀，并对来自经历潜伏或裂解复制的细胞中外源表达的His标签FAT10进行镍-氮三乙酸（Ni-NTA）色谱分析。进行LC-MS/MS以鉴定FAT10化蛋白。我们鉴定出KSHV ORF59和ORF61为FAT10化底物。我们的研究表明，UBE1L2-FAT10系统在KSHV裂解复制过程中上调，并有助于病毒传播。包括FAT10在内的泛素和UBL翻译后修饰被病毒利用并失调，以实现有效的感染和病毒粒子产生。UBE1L2-FAT10系统催化FAT10ylation，其中一个或多个FAT10分子与底物共价连接。FAT10ylation由E1（激活酶）、E2（缀合酶）和E3（连接酶）酶的顺序作用催化。FAT10ylation的E1酶是UBE1L2，它激活FAT10并将其转移到E2/USE1。FAT10ylation调节细胞周期、干扰素信号传导和蛋白质降解；然而，其主要生物学功能仍然未知。在此，我们揭示KSHV裂解复制诱导UBE1L2表达和包括KSHV裂解蛋白在内的FAT10化蛋白的产生。此外，UBE1L2敲除抑制了裂解周期中的病毒产生。这是第一份证明UBE1L2-FAT10系统对KSHV裂解复制有贡献的报告。我们的发现为KSHV裂解复制中新型翻译后修饰的生理功能提供了见解。
