State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan Universitygrid.13291.38 and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan, People's Republic of China.
Institute of Life Sciences, Chongqing Medical Universitygrid.203458.8, Chongqing, People's Republic of China.
J Virol. 2022 May 25;96(10):e0190521. doi: 10.1128/jvi.01905-21. Epub 2022 Apr 28.
Removal of 5' cap on cellular mRNAs by the African swine fever virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate-linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and the hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer is composed of a unique N-terminal helical domain and a C-terminal classic Nudix domain. As g5Rp is an mRNA-decapping enzyme, we identified key residues, including K, K, K, K, K, R, and K located on the substrate RNA binding interfaces of g5Rp which are important to RNA binding and decapping enzyme activity. Furthermore, the g5Rp-mediated mRNA decapping was inhibited by InsP. The g5Rp-InsP complex structure showed that the InsP molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP on mRNA decapping by g5Rp. ASF is a highly contagious hemorrhagic viral disease in domestic pigs which causes high mortality. Currently, there are still no effective vaccines or specific drugs available against this particular virus. The protein g5Rp is the only viral mRNA-decapping enzyme, playing an essential role in the machinery assembly of mRNA regulation and translation initiation. In this study, we solved the crystal structures of g5Rp dimer and complex with InsP. Structure-based mutagenesis studies revealed critical residues involved in a candidate RNA binding region, which also play pivotal roles in complex with InsP. Notably, InsP can inhibit g5Rp activity by competitively blocking the binding of substrate mRNA to the enzyme. Our structure-function studies provide the basis for potential anti-ASFV inhibitor designs targeting the critical enzyme.
非洲猪瘟病毒（ASFV）的 5' 帽状结构去除酶 g5R 蛋白（g5Rp）有利于病毒在感染早期的基因表达。作为 ASFV 基因组中唯一编码的核苷二磷酸连接部分 X（Nudix）脱帽酶，g5Rp 既参与细胞 mRNA 的降解，也参与二磷酸肌醇多磷酸的水解。在这里，我们报告了二聚体 g5Rp 及其与肌醇六磷酸（InsP）复合物的结构。两个 g5Rp 蛋白亚基以头对头的方式相互作用形成二聚体，二聚体界面由广泛的极性和非极性相互作用形成。每个亚基由独特的 N 端螺旋结构域和 C 端经典 Nudix 结构域组成。由于 g5Rp 是一种 mRNA 脱帽酶，我们鉴定了关键残基，包括 g5Rp 上位于底物 RNA 结合界面的 K、K、K、K、K、R 和 K，这些残基对于 RNA 结合和脱帽酶活性很重要。此外，InsP 抑制 g5Rp 介导的 mRNA 脱帽。g5Rp-InsP 复合物结构表明，InsP 分子占据主要介导 g5Rp-RNA 相互作用的相同区域，阐明了 InsP 在调节 g5Rp 在 mRNA 降解中病毒脱帽活性的作用。总之，这些结果提供了 RNA 与 g5Rp 相互作用的结构基础，并强调了 InsP 对 g5Rp 介导的 mRNA 脱帽的抑制机制。ASF 是一种高度传染性的猪出血性病毒病，可导致高死亡率。目前，针对这种特定病毒，仍然没有有效的疫苗或特定药物。蛋白 g5Rp 是唯一的病毒 mRNA 脱帽酶，在 mRNA 调节和翻译起始的机制组装中发挥重要作用。在本研究中，我们解析了 g5Rp 二聚体及其与 InsP 复合物的晶体结构。基于结构的突变研究揭示了参与候选 RNA 结合区域的关键残基，这些残基在与 InsP 复合物中也起着关键作用。值得注意的是，InsP 可以通过竞争性地阻止底物 mRNA 与酶结合来抑制 g5Rp 的活性。我们的结构功能研究为针对关键酶的潜在抗 ASFV 抑制剂设计提供了基础。
