目的：在SARS-CoV-2的感染过程中，S蛋白在病毒识别并结合宿主细胞受体进入细胞的过程中发挥重要的作用。在病毒传播过程中，SARS-CoV-2进化出多种变异毒株，变异毒株的S蛋白都存在不同程度的氨基酸突变。本研究通过对SARS-CoV-2变异毒株的S蛋白氨基酸序列进行生物信息学分析来预测不同突变株对S蛋白修饰后修饰是否产生影响，以期预测氨基酸突变引起的翻译后修饰改变对病毒受体识别和细胞入侵的影响。另一方面，低剂量放射治疗被认为可以改善和治疗新冠肺炎患者的症状，但是电离辐射是否对SARS-CoV-2的致病机制发挥作用还缺乏证明。本论文探究了电离辐射能否造成细胞中参与病毒入侵细胞条件的改变从而改变病毒传染性。

方法：通过将SARS-CoV-2标准株和变异株的氨基酸序列构建系统进化树，筛选具有代表性的变异株，并通过蛋白修饰位点预测网站预测SARS-CoV-2标准株和代表性变异株S蛋白的糖基化修饰和磷酸化修饰位点，分析变异毒株是否会通过蛋白修饰的改变来改变S蛋白与宿主细胞的相互作用；在实验研究中，我们以HIV-1病毒骨架和S蛋白构建HIV-S假病毒，通过检测荧光素酶活性来评价电离辐射对假病毒感染细胞的影响；Western Blot、PCR检测电离辐射对细胞中关键受体ACE2的基因和蛋白表达水平影响；PCR检测电离辐射对细胞中NRP1和CD147等可替代性受体的基因表达水平影响；免疫荧光检测电离辐射对细胞表面硫酸乙酰肝素表达的影响；FITC荧光预测细胞表面能够促进S蛋白与细胞受体结合的糖基化修饰；溶酶体酸性荧光探针检测电离辐射对细胞内溶酶体酸性环境的影响；Western Blot检测电离辐射对细胞内组织蛋白酶D表达的影响。

结果：预测结果S蛋白上主要有17个潜在的N-糖基化位点，受变异株序列改变影响的修饰位点是第17和654位点。S蛋白上潜在的O-糖基化位点有15个，变异株中发生了第12、71、255和686位点的修饰改变。潜在的43个磷酸化修饰位点中受氨基酸突变影响的位点主要位于S1亚基上，只有一个位点位于S2亚基；在0.1Gy、0.5Gy、1Gy和2Gy的不同剂量照射后，HIV-S假病毒感染细胞的荧光素酶活性是提高的；照射后，293T-ACE2和CHO-ACE2细胞的ACE2蛋白和基因表达水平提高；在293T-ACE2和Vero细胞中的TMPRSS2、NRP1、CD4、CD147、ASGR1受照射影响后基因表达水平提高，Vero细胞中的KREMEN1受照射影响后基因表达提高；293T-ACE2表面的硫酸乙酰肝素在照射后表达增加；FITC荧光观察PNA-FITC、LCA-FITC和ECL-FITC三种凝集素在不同剂量的照射后与293T-ACE2细胞的结合增加；照射后，溶酶体酸性荧光探针显示293T-ACE2和CHO-ACE2细胞的黄色荧光占比增加；照射后，293T-ACE2和CHO-ACE2细胞内组织蛋白酶D表达增加。

结论：通过生物信息学的方法预测了SARS-CoV-2不同变异毒株上S蛋白的氨基酸位点突变改变了S蛋白的糖基化修饰和磷酸化修饰，提出S蛋白修饰对于SARS-CoV-2病毒与宿主细胞的结合可能存在作用。电离辐射会使细胞表面关键受体、可替代功能受体和辅助受体的增加，也会使细胞表面的糖蛋白HS表达增加促进S蛋白与ACE2受体结合。凝集素与电离辐射后的细胞结合增加预测了细胞表面三种潜在可以与SARS-CoV-2 S蛋白亲和的糖基化修饰。通过细胞溶酶体酸性程度和组织蛋白酶D表达结果表明电离辐射会使细胞组织蛋白酶D表达增加，从而可能会促进SARS-CoV-2通过内吞作用进入细胞，所以SARS-CoV-2与细胞受体结合进入细胞和内吞作用释放到细胞内两种入侵细胞的方式都会受电离辐射的影响。

Objective：During the infection of SARS-CoV-2, the S protein plays an important role in the recognition and binding of the host cell receptor into the cell by the virus. During virus transmission, multiple mutant strains of SARS-CoV-2 have evolved, all of which have varying degrees of amino acid mutations in the S protein. In this study, bioinformatics analysis of the S protein amino acid sequences of SARS-CoV-2 mutant strains was performed to predict the effect of different mutant strains on post-translational modifications of S proteins, with a view to predicting the effect of post-translational modifications caused by amino acid mutations on virus receptor recognition and cellular invasion. On the other hand, low-dose radiation therapy is thought to improve and treat symptoms in patients with neocrown pneumonia, but there is a lack of proof whether ionising radiation plays a role in the pathogenesis of SARS-CoV-2. This thesis investigates whether ionising radiation can cause alterations in cellular conditions involved in viral invasion of cells and thus alter viral infectivity.

Methods：The amino acid sequences of SARS-CoV-2 standard and variant strains were used to construct phylogenetic trees to screen representative variant strains, and the glycosylation modification and phosphorylation modification sites of S proteins of SARS-CoV-2 standard and representative variant strains were predicted by protein modification site to analyze whether the variant strains would change the S proteins with host cells through the alteration of protein modification; In our experimental study, we constructed HIV-S pseudovirus with HIV-1 viral backbone and S protein, and evaluated the effect of ionizing radiation on pseudovirus-infected cells by detecting luciferase activity; Western Blot and PCR were used to detect the effect of ionizing radiation on the gene and protein expression levels of key receptors ACE2 in cells; PCR was used to detect the effect of ionizing radiation on the gene expression levels of alternative receptors such as NRP1 and CD147 in cells. Immunofluorescence detects the effect of ionizing radiation on the expression of acetyl heparan sulfate on the cell surface; FITC fluorescence predicts glycosylation modifications on the cell surface that promote the binding of S proteins to cellular receptors; lysosomal acidic fluorescent probe detects the effect of ionizing radiation on the acidic environment of lysosomes in cells; Western Blot detects the effect of ionizing radiation on the expression of cathepsin D expression by Western Blot.

Results：The predicted results showed that there were mainly 17 potential N-glycosylation sites on the S protein, and the modification sites affected by sequence alterations in the mutant strain were sites 17 and 654. 15 potential O-glycosylation sites on the S protein, and modification changes at sites 12, 71, 255 and 686 occurred in the mutant strain. The 43 potential phosphorylation modification sites affected by amino acid mutations were mainly located on the S1 subunit, and only one site was located on the S2 subunit; luciferase activity was increased in HIV-S pseudovirus-infected cells after irradiation at different doses of 0.1Gy, 0.5Gy, 1Gy and 2Gy; after irradiation, 293T-ACE2 and CHO-ACE2 cells had increased ACE2 protein and gene expression levels were increased; TMPRSS2, NRP1, CD4, CD147, ASGR1 in 293T-ACE2 and Vero cells were increased after irradiation and KREMEN1 in Vero cells were increased after irradiation; acetyl heparan sulfate expression on the surface of 293T-ACE2 was increased after irradiation; Immunofluorescence observation of three lectins, PNA-FITC, LCA-FITC and ECL-FITC, showed increased binding to 293T-ACE2 cells after different doses of irradiation; after irradiation, lysosomal acidic fluorescence probe showed an increased percentage of yellow fluorescence in 293T-ACE2 and CHO-ACE2 cells; after irradiation , the expression of cathepsin D was increased in 293T-ACE2 and CHO-ACE2 cells.

Conclusion：The amino acid site mutations of S proteins on different mutant strains of SARS-CoV-2 were predicted to alter the glycosylation modifications and phosphorylation modifications of S proteins by bioinformatics methods, and it was proposed that S protein modifications may have a role in the binding of SARS-CoV-2 virus to host cells. Ionizing radiation causes an increase in cell surface critical receptors, alternative functional receptors and co-receptors, and also increases the expression of glycoprotein HS on the cell surface to promote S protein binding to ACE2 receptors. Increased cellular binding of lectins following ionizing radiation predicts three glycosylation modifications on the cell surface that can potentially affinity with SARS-CoV-2 S proteins. The results by cytosolic lysosomal acidity and histone D expression suggest that ionizing radiation increases cellular histone D expression, which may promote SARS-CoV-2 entry into cells via endocytosis, so that both modes of SARS-CoV-2 invasion into cells by binding to cellular receptors and release into cells by endocytosis are affected by ionizing radiation.