CAMS Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
J Virol. 2022 May 25;96(10):e0059822. doi: 10.1128/jvi.00598-22. Epub 2022 May 5.
Posttranslational modifications (PTMs) of viral proteins play critical roles in virus infection. The role of neddylation in enterovirus 71 (EV71) replication remains poorly defined. Here, we showed that the structural protein VP2 of EV71 can be modified by neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in an E3 ligase X-linked inhibitor of apoptosis protein (XIAP)-dependent manner. Mutagenesis and biochemical analyses mapped the neddylation site at lysine 69 (K69) of VP2 and demonstrated that neddylation reduced the stability of VP2. In agreement with the essential role of VP2 in viral replication, studies with EV71 reporter viruses with wild-type VP2 (enhanced green fluorescent protein [EGFP]-EV71) and a K69R mutant VP2 (EGFP-EV71-VP2 K69R) showed that abolishment of VP2 neddylation increased EV71 replication. In support of this finding, overexpression of NEDD8 significantly inhibited the replication of wild-type EV71 and EGFP-EV71, but not EGFP-EV71-VP2 K69R, whereas pharmacologic inhibition of neddylation with the NEDD8-activating enzyme inhibitor MLN4924 promoted the replication of EV71 in biologically relevant cell types. Our results thus support the notion that EV71 replication can be negatively regulated by host cellular and pathobiological cues through neddylation of VP2 protein. Neddylation is a ubiquitin-like posttranslational modification by conjugation of neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to specific proteins for regulation of their metabolism and biological activities. In this study, we demonstrated for the first time that EV71 VP2 protein is neddylated at K69 residue to promote viral protein degradation and consequentially suppress multiplication of the virus. Our findings advance knowledge related to the roles of VP2 in EV71 virulence and the neddylation pathway in the host restriction of EV71 infection.
病毒蛋白的翻译后修饰（PTMs）在病毒感染中起着关键作用。神经前体细胞表达的发育下调蛋白 8（NEDD8）在肠道病毒 71（EV71）复制中的作用仍未得到明确界定。在这里，我们表明 EV71 的结构蛋白 VP2 可以通过 E3 连接酶 X 连锁凋亡抑制蛋白（XIAP）依赖性的神经前体细胞表达的发育下调蛋白 8（NEDD8）进行修饰。突变和生化分析将 VP2 的赖氨酸 69（K69）上的 neddylation 位点定位，并表明 neddylation 降低了 VP2 的稳定性。与 VP2 在病毒复制中的重要作用一致，使用具有野生型 VP2（增强型绿色荧光蛋白[EGFP]-EV71）和 K69R 突变 VP2（EGFP-EV71-VP2 K69R）的 EV71 报告病毒进行的研究表明，VP2 的 neddylation 消除增加了 EV71 的复制。支持这一发现，NEDD8 的过表达显著抑制了野生型 EV71 和 EGFP-EV71 的复制，但对 EGFP-EV71-VP2 K69R 没有抑制作用，而用 NEDD8 激活酶抑制剂 MLN4924 抑制 neddylation 可促进生物相关细胞类型中 EV71 的复制。因此，我们的结果支持这样一种观点，即 EV71 复制可以通过 VP2 蛋白的 neddylation 被宿主细胞和病理生物学线索负调控。Neddylation 是一种泛素样翻译后修饰，通过将神经前体细胞表达的发育下调蛋白 8（NEDD8）与特定蛋白缀合来调节其代谢和生物学活性。在这项研究中，我们首次证明 EV71 VP2 蛋白在 K69 残基上发生 neddylation，以促进病毒蛋白降解，并因此抑制病毒的复制。我们的发现推进了与 VP2 在 EV71 毒力中的作用以及 neddylation 途径在宿主限制 EV71 感染中的作用相关的知识。
