SARS-CoV-2（严重急性呼吸窘迫综合征冠状病毒2型），与SARS-CoVs严重急性呼吸窘迫综合征冠状病毒）同源，均属于冠状病毒家族成员，且在结构及功能上具有一定的相似性。直径约为50-200，全长约30kb。5端包括四种结构蛋白和几种辅助蛋白。与其他冠状病毒一样，SARS-CoV-2在宿主细胞内部，ORF1a和ORF1ab转化为多蛋白pp1a和pp1b。这些多蛋白通过NSP3（即木瓜样蛋白酶）和NSP5（即主要蛋白酶）进行的蛋白水解切割产生16个非结构蛋白 （NSP）。SARS-CoV-2 3端具有四种结构蛋白，分别为S（刺突），E（包膜），M（膜）和N（核衣壳）蛋白。SARS-CoV-2自2019年12月爆发以来，截至2021年4月10日，全球已有192个国家和地区累计报告逾1.34亿名确诊病例，其中逾600万人死亡、7653.7万人被治愈，目前感染及死亡人数仍在迅速攀升中。然而由于病毒的高突变性，使得病毒的流行毒株正在不断地发生变化。影响病毒感染及传播特性的突变主要集中在S蛋白上，这些突变影响了病毒S蛋白与宿主ACE2受体的结合能力，同时S蛋白上的众多突变也促进了病毒的免疫逃逸能力，而不同毒株的感染及复制能力也存在一定的差异。目前有研究表明SARS-CoV-2 NSP1可以通过与40S核糖体亚基结合从而抑制宿主翻译，而病毒自身RNA 5端特殊的茎环结构可以逃避抑制作用，但另一些转录组的数据却表明NSP1在宿主内可能还发挥着其他一些重要的作用。

首先我们通过在线程序表征了SARS-CoV-2不同毒株（Prototype、Alpha、Beta、Delta、Omicron）的S蛋白在抗原性及三级结构上的差异，各个毒株S蛋白的突变对抗原性均存在一定的影响，除Omicron外其他毒株差异似乎并不特别明显，而突变株中D614G突变使得S蛋白RBD区域更加暴露，这可能是引起突变株传播更加迅速的原因。随后我们为了验证不同突变株的S蛋白与ACE2结合能力，采取了假病毒感染实验及不同突变株S蛋白与ACE2蛋白体外结合能力实验，我们发现了Delta株S蛋白与ACE2的高结合能力及Omicron S蛋白与ACE2的低结合能力。为了进一步验证这些结果，我们又采用了不同毒株感染Vero细胞实验，并在不同时间点检测了病毒的载量，发现在感染后2h Omicron 病毒基因组载量最低Prototype最高，而在第24小时则是Delta最高，Omicron第二。这说明Omicron的ACE2结合能力及细胞感染能力并不够强，但是其免疫逃逸能力却很强，同时其细胞毒性较其他毒株弱。

之后为了研究SARS-CoV-2 NSP1在宿主内的其他功能及分子机制，寻找NSP1互作蛋白及其调控的信号通路，我们分别在原核及真核细胞中进行了NSP1全长表达。使用H1299细胞总蛋白进行GST-pull down实验，银染分析，并送质谱，寻找互作蛋白。将真核表达载体转染至H1299细胞，送组学分析，结合质谱及组学数据初步分析NSP1所调控的信号通路及可能作用机制。我们通过质谱发现了大量与NSP1结合的宿主蛋白，包括大量的核糖体蛋白，并最终验证到了与NSP1相互结合的hnRNPK及FXR1蛋白。而在蛋白组中发现并验证到了高表达的干扰素信号抑制因子GPATCH3蛋白。最终通过干扰素报告基因检测系统证明了NSP1及GPATCH3对干扰素的抑制作用，而敲低或过表达FXR1未检测到GPATCH3表达差异。病毒感染实验发现了NSP1促进了病毒的感染而GPATCH3似乎表现出相反的作用。总之我们通过一系列实验发现了NSP1对干扰素的抑制作用可能是由于NSP1与宿主某个蛋白相互作用进而促进了GPATCH3的表达而引起的，同时NSP1为病毒感染创造了有利的条件，但是具体机制仍然需要进一步研究。

我们针对不同SARS-CoV-2突变株的研究为进一步分析及评估病毒感染传播以及预防方面提供了一定的参考。此外我们对于非结构蛋白的研究揭示了非结构蛋白对病毒感染的促进作用，为病毒的预防及靶点的开发提供了一定的参考。

SARS-CoV-2 (severe acute respiratory distress syndrome coronavirus 2), homologous to SARS-CoVs severe acute respiratory distress syndrome coronavirus), both belong to the coronavirus family, and have certain structural and functional characteristics. similarity. The diameter is about 50-200, and the total length is about 30kb. The 5-terminal includes four structural proteins and several accessory proteins. Like other coronaviruses, SARS-CoV-2 converts ORF1a and ORF1ab into the polyproteins pp1a and pp1b inside the host cell. Proteolytic cleavage of these polyproteins by NSP3 (i.e. papain-like protease) and NSP5 (i.e. major protease) yields 16 nonstructural proteins (NSPs). The 3-terminal of SARS-CoV-2 has four structural proteins, namely S (spike), E (envelope), M (membrane) and N (nucleocapsid) proteins. Since the outbreak of SARS-CoV-2 in December 2019, as of April 10, 2021, more than 134 million confirmed cases have been reported in 192 countries and regions around the world, of which more than 6 million have died and 76.537 million have been cured , the number of infections and deaths is still rising rapidly. However, due to the high mutagenicity of the virus, the circulating strains of the virus are constantly changing. The mutations that affect the characteristics of virus infection and transmission are mainly concentrated in the S protein. These mutations affect the binding ability of the virus S protein to the host ACE2 receptor. At the same time, many mutations on the S protein also promote the immune escape ability of the virus. There are also some differences in the infection and replication ability of the strains. Current studies have shown that SARS-CoV-2 NSP1 can inhibit host translation by binding to the 40S ribosomal subunit, while the special stem-loop structure at the 5 end of the virus's own RNA can escape the inhibition, but other transcriptome data have shown that NSP1 It may also play some other important roles in the host.

First, we characterized the differences in antigenicity and tertiary structure of the S protein of different strains of SARS-CoV-2 through an online program. The mutation of the S protein of each strain has a certain impact on the antigenicity. Except for Omicron, other viruses The strain difference does not seem to be particularly pronounced, and the D614G mutation in the mutant strain makes the RBD region of the S protein more exposed, which may be the reason for the more rapid spread of the mutant strain. Then, to verify the binding ability of S protein and ACE2 of different mutant strains, we carried out pseudovirus infection experiments and in vitro binding ability experiments of S protein and ACE2 protein of different mutant strains. Low binding capacity of S protein to ACE2. To further verify these results, we used different strains to infect Vero cells, and detected the viral load at different time points. It was found that the Omicron virus genome load was the lowest at 2h after infection, and the Prototype was the highest at 24 hours after infection. Delta is the highest, Omicron second. This shows that Omicron's ACE2 binding ability and cell infection ability are not strong enough, but its immune evasion ability is strong, and its cytotoxicity is weaker than other strains.

Then, to study other functions and molecular mechanisms of SARS-CoV-2 NSP1 in the host, and to search for NSP1-interacting proteins and their regulated signaling pathways, we expressed the full-length NSP1 in prokaryotic and eukaryotic cells, respectively. The total protein of H1299 cells was used for GST-pull down experiment, silver staining analysis, and mass spectrometry was used to search for interacting proteins. The eukaryotic expression vector was transfected into H1299 cells and sent to omics analysis. Combined with mass spectrometry and omics data, the signaling pathway regulated by NSP1 and its possible mechanism were preliminarily analyzed. We found many host proteins that bind to NSP1 by mass spectrometry, including many ribosomal proteins, and finally verified the hnRNPK and FXR1 proteins that bind to NSP1. In the proteome, a highly expressed interferon signal inhibitor GPATCH3 protein was found and verified. Finally, the inhibitory effect of NSP1 and GPATCH3 on interferon was proved by the interferon reporter gene detection system, but no difference in GPATCH3 expression was detected by knockdown or overexpression of FXR1. Viral infection experiments found that NSP1 promotes viral infection while GPATCH3 appears to have the opposite effect. In short, we found through a series of experiments that the inhibitory effect of NSP1 on interferon may be caused by the interaction between NSP1 and a certain protein in the host to promote the expression of GPATCH3. At the same time, NSP1 creates favorable conditions for virus infection, but the mechanism still needs further study. Our research on different SARS-CoV-2 mutants provides a certain reference for further analysis and evaluation of viral infection transmission and prevention. In addition, our research on non-structural proteins revealed the promoting effect of non-structural proteins on virus infection, which provided a certain reference for virus prevention and target development.