【作者】 张震；

【导师】 贾大；

【作者基本信息】 四川大学 ， 药学， 2021， 硕士

【摘要】 新型冠状病毒(SARS-CoV2)是一种于2019年下半年爆发并引起全球大流行的烈性传染性病毒。临床研究发现,感染该病毒后轻症患者出现类似流感的症状,如肌肉乏力、咳嗽、发热等;而在有基础疾病的老龄患者中则易出现呼吸困难、低血氧症、多器官衰竭等严重症状。截至目前,WHO数据显示全球新型冠状病毒肺炎确诊病例累计超过1亿例,其中死亡病例累计突破280万例。遗憾的是,从病毒爆发至今全球的医疗和科研攻坚力量仍未发现能应用于临床的有效药物来遏止新型冠状病毒对患者的侵害。但是随着一些疫苗的成功开发和应用,SARS-CoV2在人群中的感染效率明显下降,新冠大流行也得到了良好的控制。然而,SARS-CoV2具有易突变的狡猾特点,突变毒株会弱化抗体的免疫效果且传染性更强,再次危害全人类的生命安全。因此,了解SARS-CoV2的生物学特点和病毒关键蛋白功能显得尤为重要。SARS-CoV2与重症急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸系统综合症冠状病毒(MERS-CoV)同属冠状病毒科冠状病毒属,冠状病毒基因组全长有近三万个碱基对(30 kb),其在5’端有两个开放阅读框(open-reading frame,ORF1a/ORF1ab),编码16种非结构蛋白(non-structural proteins,NSP1-NSP16)。NSPs不仅在病毒复制过程中形成转录/复制复合物(Replication and Transcription Complex,RTC),还会通过与宿主细胞中的蛋白相互作用,来利用或劫持宿主细胞的部分细胞通路。NSPs在冠状病毒当中高度保守,NSP2作为十六种非结构蛋白保守度较低的一种,其序列相似性只有82.9%。早期研究发现,在SARS-CoV中,NSP2作为NSP3的调节因子存在,NSP2的缺失会影响病毒的生长和病毒RNA的合成,但NSP2其他的生物学功能尚不清楚,在SARSCoV2中,NSP2是否有相同的功能也尚不清楚。亲和纯化质谱检测发现,SARS-CoV2-NSP2与小G蛋白二磷酸鸟苷解离促进剂(small G-protein GDP dissociation stimulator,Smg GDS)可能有相互作用。根据文献报道,Smg GDS是一种鸟嘌呤核苷酸交换因子(GEF),能特异性促进Rho A与Rho C(Rho家族鸟苷三磷酸酶A/C)的非活性形式(Rho A-GDP)向活性形式(Rho A-GTP)的转变。Rho A作为在细胞正常生命活动中关键的开关蛋白,在细胞肌动蛋白骨架(actin stress fiber)的重组,细胞运动和附着能力中起着重要作用。根据以上报道,我们提出以下几个科学问题:SARS-CoV2的NSP2蛋白与Smg GDS是否存在相互作用?这种相互作用在SARS-CoV2和SARS-CoV是否具有保守性?NSP2结合Smg GDS后对其GEF酶活性会有什么影响?在细胞中又会有什么样的表型?首先我们通过pulldown实验发现,SARS-CoV2-NSP2可以特异性结合Smg GDS,而SARS-CoV-NSP2不能与Smg GDS特异性结合。SARS-CoV2-NSP2与SARS-CoV-NSP2序列对比发现两者氨基酸差异主要集中在蛋白碳端,其次我们通过设计大量SARS-CoV2-NSP2的截短体,发现SARS-CoV2-NSP2与Smg GDS特异性结合位置确实位于SARS-CoV2-NSP2碳端区域,即第506位氨基酸至第638位氨基酸(506-C);我们通过鸟嘌呤核苷酸交换实验,发现SARSCoV2-NSP2与Smg GDS的互作降低Smg GDS的GEF活性,干扰Rho A上核苷酸的解离;同时我们通过Rho A活化检测实验,发现在HEK293T细胞中SARSCoV-NSP2会影响Rho A的活化,即Rho A-GTP的生成。以上研究结果,为我们进一步理解SARS-CoV2-NSP2在病毒复制和繁殖过程中的作用和被其感染引发的新型冠状病毒肺炎的发病机制提供了关键的分子基础。更多还原

【Abstract】 Severe acute respiratory syndrome coronavirus 2(SARS-CoV2)is a virulent infectious virus that broke out in the second half of 2019 and caused a global pandemic.Clinical studies have found that mild patients infected with the virus have flu-like symptoms,such as muscle weakness,cough,fever,etc.The elderly patients with basic diseases are prone to dyspnea,hypoxemia,multiple organ failure and other serious symptoms.To date,WHO data shows that there have been more than 100 million confirmed cases of COVID-19 worldwide,including more than 2.8 million deaths.Regrettably,since the outbreak of the virus,the global medical and scientific research forces have not yet found an effective drug that can be applied in clinical practice to contain the invasion of SARS-CoV2 on patients.Moreover,SARS-COV2 has the cunning characteristic of being easily mutated,and the mutant strain will weaken the immune effect of antibodies and become more infectious,again endangering the life safety of all mankind.Despite the recent success of vaccine approaches,numerous challenges remain ahead and diversified,Extensive efforts to repurpose drugs strategies have produced limited clinical results.In order to develop specific drugs for COVID-19,it is important to understand the biological characteristics of SARS-COV2 and the function of key viral proteins.Same to severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV),SARS-CoV2 is an RNA betacoronavirus of the family Coronaviridae,possess the RNA viral genomes,approximately 30 kbp in length.The 5’ region of the genome encodes for two open reading frames(ORF1a/ ORF1ab)that produce 16 non-structural proteins(nsp1-nsp16)needed to form the viral replication complex(RTC).It also interacts with host-proteins to exploit or hijack some cellular pathways in the host cell.These non-structural proteins have high sequence similarity among coronaviruses,while NSP2 is one of the sixteen non-structural proteins with low sequence similarity,only 82.9%.Early studies have found that NSP2 exists as a regulatory factor of Nsp3 in SARS-CoV,and the loss of NSP2 will affect the growth of the virus and the synthesis of viral RNA,but other biological functions of NSP2 are still unclear.In SARS-CoV2,affinity purification mass spectrometry found that SARS-CoV2-NSP2 may interact with small G-protein GDP dissociation stimulator(Smg GDS),Previous studies have shown that Smg GDS can interact with multiple small GTPases,and was characterized as a guanine nucleotide exchange factor(GEF)for Rho A and Rho C,it also promotes the conversion of Rho A and Rho C from inactive forms to active forms.As a key switch protein in normal cell life activities,Rho A plays an important role in cellular actin stress fiber recombination,cell movement and adhesion.In conclusion,as a relatively conserved protein in coronavirus,NSP2 is also a key link in the growth and replication process of the virus,so it is of great pathological significance to study its biological function.Based on the above reports,we raise the following scientific questions: Does the SARS-CoV2-NSP2 interact with Smg GDS? Is this interaction conserved in SARSCOV2 and SARS-CoV? What is the effect of NSP2 binding to Smg GDS on its GEF enzyme activity and what kind of phenotype does it have in the cell?First of all,we found through pulldown experiment that SARS-CoV2-NSP2 could specifically bind to Smg GDS,while SARS-CoV-NSP2 could not specifically bind to Smg GDS.Sequence comparison showed that the difference between SARSCOV2-NSP2 and SARS-CoV-NSP2 was mainly concentrated in the protein carbon terminal.Secondly,we designed a large number of SARS-CoV2-NSP2 truncates,and found that the specific binding site of this interaction was located in the C-terminal region of SARS-CoV2-NSP2,namely amino acid 506-638(506-C).Then,through Guanine nucleotide dissociation assay,we found that the interaction between SARSCoV-NSP2 and Smg GDS could affect the GEF activity of Smg GDS and reduce the nucleotide dissociation rate of Rho A.In addition,we also detected the production of Rho A-GTP by Rho A-activation assay,and proved in cells that SARS-CoV-NSP2 can affect the production of Rho A-GTP.These results provide a key molecular basis for further understanding the role of SARS-COV2-NSP2 in viral replication and reproduction and the pathogenesis of COVID-19 induced by SARS-COV2-NSP2 infection.更多还原