【作者】 黄国锋；

【导师】 张文艳；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2024， 硕士

【摘要】 新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)是新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)感染所导致的,SARS-CoV-2和SARS-CoV均属于Sarbecovirus亚属。这类亚属病毒会编码独特的辅助蛋白,其与病毒在细胞中的生存、适应以及天然免疫逃逸密切相关。近期有研究表明SARS-CoV-2辅助蛋白ORF6是天然免疫应答的抑制因子,是干扰素(Interferon,IFN)介导的抗病毒信号传导拮抗剂,对稳定SARS-CoV-2的复制起到重要作用,因此阐明SARS-CoV-2辅助蛋白与宿主蛋白相互作用机制,对于COVID-19的防治意义重大。泛素化在许多生物学过程中起着至关重要的作用,包括蛋白质降解、运输、信号转导、固有免疫应答和病毒复制等。我们最近的研究发现,宿主利用不同E3泛素连接酶,通过泛素-蛋白酶体系统诱导SARS-CoV-2包膜(Envelope,E)和ORF9b泛素化降解,从而影响病毒感染和复制。在病毒-宿主相互作用的微妙动态中,二者都利用泛素化和去泛素化的复杂过程参与竞争、防御和共同进化。然而,泛素化和去泛素化对于Sarbecovirus编码的ORF6蛋白功能调节机制的影响尚不清楚。针对泛素化机制在Sarbecovirus致病性中的作用,本文进行了如下实验:我们首先使用不同亚型Sarbecovirus ORF6通过利用荧光素酶报告基因的方法检测该蛋白对IFN-β启动子活性的影响;接下来我们使用蛋白酶体抑制剂、溶酶体抑制剂和自噬体抑制剂来探究ORF6蛋白在宿主中的具体降解方式,IP实验明确ORF6的降解与泛素-蛋白酶体途径之间的关系;接下来我们筛选了能够抑制ORF6降解的USP家族成员,分别将USPs与ORF6共转染HEK293T细胞,筛选去泛素化酶USPs对ORF6表达的影响,结果发现USP1可以显著稳定ORF6蛋白的表达,IP实验结果也验证了USP1和ORF6之间的相互作用;通过使用USP1不同功能缺失截短体,探究USP1和ORF6相互作用区域;Luciferase报告基因实验和RT-q PCR实验检测USP1对ORF6介导的I型干扰素信号通路的影响;流式细胞术检测USP1对SARS-CoV-2重组缺陷型病毒感染性的影响;最后利用RT-q PCR检测USP1对新冠病毒毒力的影响。在本研究中,我们发现Sarbecovirus ORF6蛋白能够被泛素化修饰,并通过泛素-蛋白酶体途径降解。我们还发现去泛素化酶家族蛋白USP1可以通过去泛素化和阻断ORF6的降解来稳定其能力。进一步发现,USP1通过增强ORF6介导的IFN和NF-κB抑制促进SARS-CoV-2毒力。综上所述,SARS-CoV-2辅助蛋白ORF6通过泛素-蛋白酶体途径降解,USP1有效降低了SARS-CoV-2 ORF6的K48连接泛素化,并稳定其蛋白水平。我们的研究揭示了宿主去泛素化酶USP1和SARS-CoV-2 ORF6之间未被识别的作用机制,为SARS-CoV-2的预防和治疗提供了新的靶点。更多还原

【Abstract】 Corona Virus Disease 2019(COVID-19)is caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection.Both SARS-CoV-2 and SARS-CoV belong to the Sarbecovirus subgenus.Viruses of this subgenus encode unique accessory proteins,which are closely related to viral survival,escape from adaptation and natural immune in cells.Recent studies have shown that SARS-CoV-2 accessory protein ORF6 is an inhibitor of innate antiviral responses and an antagonist of antiviral signaling mediated by interferon,which plays an important role in stabilizing the replication of SARS-CoV-2.Therefore,elucidating the interaction mechanism between SARS-CoV-2 accessory protein and host protein is of great significance for the prevention and treatment of COVID-19.Ubiquitination plays a critical role in many biological processes,including protein degradation,transportation,participation in signaling pathways,innate immune responses,and viral replication.Our recent study shows that the host utilizes different E3 ubiquitin ligases to induce ubiquitination degradation of SARS-CoV-2 envelope(E)protein and ORF9 b via the ubiquitin proteasome system,thereby affecting viral infection and replication.In the subtle dynamics of virus-host interactions,both engage in competition,defense,and co-evolution using complex processes of ubiquitination and deubiquitination.However,the regulatory mechanisms by which ubiquitination and deubiquitination affect the function of Sarbecovirus ORF6 protein are still unclear.Aiming at the role of ubiquitin in the pathogenicity of Sarbecovirus ORF6,we conducted the following experiments: First,we used different subtypes of Sarbecovirus ORF6 to detect the effects of this protein on IFN-β promoter activity by using luciferin reporter gene.Next,we used proteasome inhibitors,lysosome inhibitors and autophagosome inhibitors to explore the specific degradation mode of ORF6 protein in the host.IP experiment confirmed the relationship between ORF6 degradation and ubiquitin-proteasome pathway.Next,we screened USP family members that could inhibit ORF6 degradation.We co-transfected USPs and ORF6 into HEK293 T cells,respectively,and screened the effects of deubiquitination enzyme USPs on ORF6 expression.The results showed that USP1 could significantly stabilize the expression of ORF6 protein.The interaction between USP1 and ORF6 is also verified by IP experiment results.By truncating USP1,the interaction region between USP1 and ORF6 is defined.Luciferase reporter gene assay and RT-q PCR assay were used to detect the effects of USP1 on ORF6-mediated type I interferon signaling pathway,including IFN-β and downstream response genes.The effect of USP1 on SARS-CoV-2trvlps infection was detected by flow cytometry.The relationship between USP1 and COVID-19 was detected by RT-q PCRIn this study,we found that Sarbecovirus ORF6 protein could be ubiquidized and degraded through proteasome pathway.We also found that the Deubiquitinating enzymes(DUBs)USP1 can stabilize their ability by deubiquitinating and blocking the degradation of ORF6.Further,we found that USP1 promotes SARS-CoV-2 virulence by enhancing ORF6-mediated IFN induction and NF-κB inhibition.In summary,we suggest that SARS-CoV-2 accessory protein ORF6 is degraded through the ubiquitin-proteasome pathway,and USP1 effectively reduces the K48 link ubiquitination of SARS-CoV-2 ORF6 and stabilizes its protein level.Our study reveals a previously unrecognized interaction between host protein DUBs USP1 and SARSCoV-2 ORF6,providing new targets for the prevention and treatment of SARS-CoV-2.更多还原