Zhu Junji, Liu GuanQun, Goins Christopher M, Stauffer Shaun R, Gack Michaela U
Florida Research and Innovation Center, Cleveland Clinic, Port St. Lucie, FL 34987, USA.
Department of Microbiology & Immunology, McGill University, Montreal, QC H3A 2B4, Canada.
bioRxiv. 2024 May 20:2024.05.15.594393. doi: 10.1101/2024.05.15.594393.
Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host (immune) proteins such as MDA5 and IRF3 in a process called ISGylation, thereby limiting the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.
干扰素（IFN）刺激基因15（ISG15）是一种类泛素蛋白，在一个称为ISGylation的过程中与宿主（免疫）蛋白如MDA5和IRF3共价结合，从而限制严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的复制。然而，SARS-CoV-2蛋白是否可直接成为ISGylation的靶标仍不清楚。在本研究中，我们确定SARS-CoV-2的核衣壳（N）蛋白是宿主E3连接酶HERC5催化的ISGylation的主要底物；然而，N蛋白的ISGylation可通过NSP3的木瓜样蛋白酶（PLpro）活性进行去ISGylation而轻易去除。质谱分析确定N蛋白在四个赖氨酸残基（K266、K355、K387和K388）处发生ISGylation，并且在SARS-CoV-2复制子（N-4KR）背景下对这些位点进行突变分析消除了N蛋白的ISGylation，并减轻了ISGylation介导的对病毒RNA合成的抑制。此外，我们的结果表明，HERC5优先靶向磷酸化的N蛋白进行ISGylation，以调节其寡聚组装。这些发现揭示了宿主ISGylation机制直接靶向SARS-CoV-2蛋白以限制病毒复制的新机制，并阐明了宿主（HERC5）和病毒（PLpro）酶之间复杂的相互作用如何协调病毒蛋白的ISGylation，从而调节病毒复制。
