Abstract：
A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.
摘要：
严重急性呼吸道综合症冠状病毒2（SARS-CoV-2）感染会引起轻度至重度的呼吸道症状，包括急性呼吸窘迫.尽管在研究SARS-CoV-2的病毒学和病理学影响方面做出了巨大努力，但SARS-CoV-2感染的许多特征仍然未知。干扰素诱导的泛素样蛋白ISG15与几种病毒蛋白共价缀合以抑制它们的功能。据报道，SARS-CoV-2利用其木瓜蛋白酶样蛋白酶（PLpro）来阻止ISG15结合，ISGylation。然而,ISGylation在SARS-CoV-2感染中的作用尚不清楚.我们旨在阐明ISGylation在SARS-CoV-2复制中的作用。我们观察到SARS-CoV-2核衣壳蛋白是培养细胞中HERC5E3连接酶介导的ISG化的靶蛋白。定点诱变表明，C端间隔区B-N3（SB/N3）结构域内的残基K374是核衣壳-ISG化必需的，MERS-CoV(K372)和SARS-CoV(K375)中的保守赖氨酸残基。我们还观察到核衣壳-ISG化导致核衣壳寡聚化的破坏，从而抑制病毒复制。ISG15mRNA的敲除增强了SARS-CoV-2报告复制子细胞中SARS-CoV-2的复制，而ISG化成分的外源表达部分阻碍了SARS-CoV-2的复制。一起来看,这些结果表明,SARS-CoV-2PLpro抑制核衣壳蛋白的ISG化,从而通过逃避ISG化介导的核衣壳寡聚化的破坏来促进病毒复制.IMPORTANCEISG15是一种干扰素诱导的泛素样蛋白，通过特定的Lys残基与病毒蛋白共价结合，并在许多病毒中抑制病毒功能和病毒繁殖。然而,ISGylation在SARS-CoV-2感染中的作用尚不清楚。这里,我们证明SARS-CoV-2核衣壳蛋白是HERC5E3连接酶介导的ISG化的靶蛋白。我们还发现，C端间隔区B-N3（SB/N3）结构域内的残基K374是核衣壳-ISG化所必需的。我们获得的证据表明，核衣壳-ISG化导致核衣壳-寡聚化的破坏，从而抑制SARS-CoV-2复制。我们发现SARS-CoV-2木瓜蛋白酶样蛋白酶通过其脱偶联酶活性抑制核衣壳蛋白的ISG15偶联。本研究可能有助于对ISGylation介导的抗病毒功能在SARS-CoV-2感染中的作用获得新的认识，并可能导致开发针对SARS-CoV-2核衣壳蛋白的更有效和选择性抑制剂。
