新型冠状病毒（SARS-CoV-2）感染发展到晚期，患者多处器官损伤，尤其是心脏、肾脏会被累及，其细胞的线粒体正常网络结构受损，氧化磷酸化基因的表达水平显著降低。线粒体自噬调节失调和线粒体介导的凋亡通路激活，是造成感染患者多器官衰竭的重要原因之一。探究SARS-CoV-2对细胞线粒体自噬和细胞凋亡的影响及途径，有利于找到缓解SARS-CoV-2感染并发的多器官损伤的治疗靶点。

SARS-CoV-2的非结构蛋白NSP7，它能与线粒体相关蛋白相互作用，抑制线粒体功能。NSP7也与一种溶酶体膜蛋白RAB7A相互作用。RAB7A通过其磷酸化介导溶酶体膜与线粒体膜接触，从而促进线粒体的正常分裂和自噬降解。线粒体自噬的过度发生会引起Caspase9依赖性的细胞凋亡。本论文探究了SARS-CoV-2的NSP7蛋白对细胞内线粒体自噬和细胞凋亡的影响以及相关机制。

本论文主要的研究结果如下：

1. GEO数据分析表明COVID19患者线粒体功能和细胞凋亡基因显著富集

KEGG富集分析结果显示COVID-19患者的线粒体氧化磷酸化基因以及程序性细胞死亡通路基因被显著富集。凋亡基因的Caspase9 mRNA，自噬基因RAB7A mRNA显著升高。

2. 构建SARS-CoV-2非结构蛋白NSP7真核表达质粒pLV3/NSP7/GFP

通过CLUSTALW网站对SARS-CoV及SARS-CoV-2中不同变异株的NSP7氨基酸进行序列比对，发现二者的NSP7同源性达到98.8%。选择SARS-CoV-2的 NSP7，用pLV3-Puro质粒载体成功构建pLV3/NSP7/GFP真核表达质粒。该质粒可在HEK-293T细胞系中表达NSP7/GFP融合蛋白，其转染率约为60%-80%。

3. NSP7/GFP蛋白干扰HEK-293T细胞中线粒体形态结构

通过Expasy_ProtParam数据库分析NSP7氨基酸序列发现其含有潜在跨膜区。线粒体染料MitoTracker示踪和激光共聚焦显微镜技术分析显示在HEK-293T细胞中，NSP7/GFP蛋白与线粒体呈现共定位关系。表达NSP7/GFP蛋白的细胞中线粒体网络形态分支长度显著低于对照组，流式细胞术分析显示表达NSP7/GFP的HEK-293T细胞线粒体的数量显著降低。蛋白免疫印迹（Western Blot, WB）分析显示线粒体标志性蛋白TOM20表达量降低。以上结果说明NSP7/GFP定位于线粒体膜，抑制了细胞中线粒体正常分裂状态，也干扰了线粒体结构。

4. NSP7/GFP蛋白与自噬相关溶酶体蛋白RAB7A相互作用

为探究NSP7/GFP降低线粒体数量，引起其形态异常的机制，Co-IP（Co-immunoprecipitation）技术证明NSP7/GFP与RAB7A蛋白能够直接相互作用。

5. NSP7/GFP蛋白促进HEK 293 T细胞自噬的发生

由于线粒体数量和形态改变与细胞自噬发生相关，我们探测了NSP7/GFP对细胞自噬产生的影响。WB结果显示NSP7/GFP上调HEK-293T细胞中RAB7A的表达。并且促进细胞自噬标志性蛋白LC3 Ⅱ和p62的表达。通过细胞线粒体和溶酶体共定位分析发现，相较于对照组，表达NSP7/GFP细胞的线粒体和溶酶体共定位增加。这些数据提升NSP7/GFP促进了线粒体自噬的发生。

6. NSP7/GFP蛋白促进细胞凋亡

将pLV3/NSP7/GFP转染到HEK-293T细胞中48 h和72 h后，发现细胞碎片变多，细胞收缩，细胞与细胞之间出现空洞。流式细胞术评估NSP7/GFP细胞凋亡状态显示NSP7/GFP诱导了细胞凋亡。48 h时与对照相比表达，NSP7/GFP的细胞早期凋亡增加5.47%。转染72 h，与48 h相比，NSP7/GFP的细胞早期凋亡占比增加12.19%。WB结果证明表达NSP7/GFP的HEK-293T中凋亡蛋白Caspase9和BAX表达量增加。

综上所述，本论文结合前期研究发现，SARS-CoV-2的NSP7/GFP蛋白与细胞内的RAB7A互作，导致线粒体自噬增加，细胞凋亡途径激活。该研究揭示了SARS-CoV-2非结构蛋白NSP7对宿主细胞损伤的一个潜在机制。

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection to the advanced stage, patients with multiple organ damage, especially the heart, kidney will be involved, the normal network structure of the mitochondria of their cells is damaged, and the expression level of oxidative phosphorylation genes is significantly reduced. Dysregulation of mitophagy and activation of mitochondria-mediated apoptosis pathway are one of the important causes of multiple organ failure in infected patients. To explore the effects and pathways of SARS-CoV-2 on mitophagy and apoptosis is beneficial to the treatment of multiple organ damage caused by SARS-CoV-2 infection.

The non-structural protein NSP7 of SARS-CoV-2, which can interact with mitochondria-related proteins and inhibit mitochondrial function. NSP7 also interacts with RAB7A, a lysosomal membrane protein. Through its phosphorylation, RAB7A mediates the contact between lysosomal membrane and mitochondrial membrane, thus promoting the normal division and autophagy degradation of mitochondria. Overdevelopment of mitophagy can lead to Caspase9-dependent apoptosis. In this paper, we investigated the effects of SARS-CoV-2 NSP7 protein on intracellular mitophagy and apoptosis and the related mechanisms.

The main research results of this paper are as follows:

1. GEO data analysis showed significant enrichment of mitochondrial function and apoptosis genes in COVID-19 patients

KEGG enrichment analysis showed that mitochondrial oxidative phosphorylation genes and programmed cell death pathway genes were significantly enriched in COVID-19 patients. The Caspase9 mRNA of apoptosis gene and RAB7A mRNA of autophagy gene were significantly increased.

2. Construct the SARS-CoV-2 non-structural protein NSP7 eukaryotic expression plasmid pLV3/NSP7/GFP

Through CLUSTALW website, NSP7 amino acid sequences of different variants of SARS-CoV and SARS-CoV-2 were compared, and it was found that the homology of NSP7 between the two strains reached 98.8%. pLV3/NSP7/GFP eukaryotic expression plasmid was constructed using PLV3-PURO plasmid vector by selecting SARS-CoV-2 NSP7. The plasmid can be expressed in HEK-293T cell line, expressing NSP7/GFP fusion protein, and its transfection rate is about 60%-80%.

3. NSP7/GFP protein interferes with mitochondrial morphological structure in HEK-293T cells

Expasy_ProtParam database was used to analyze the amino acid sequence of NSP7, which was found to contain potential transmembrane regions. Mitochondrial dye MitoTracker and confocal laser microscopy showed that NSP7/GFP protein and mitochondria showed a co-localization relationship in HEK-293T cells. The morphological branch length of mitochondrial network in the cells expressing NSP7/GFP protein was significantly lower than that in the control group, and flow cytometry analysis showed that the number of mitochondria in HEK-293T cells expressing NSP7/GFP was significantly reduced. Western Blot (WB) analysis showed that the expression of mitochondrial signature protein TOM20 decreased. These results indicate that NSP7/GFP locates in the mitochondrial membrane, inhibits the normal state of mitochondrial division in cells, and interferes with the mitochondrial structure.

4. NSP7/GFP protein interacts with RAB7A, a lysosomal protein associated with autophagy

In order to explore the mechanism by which NSP7/GFP reduces the number of mitochondria and induces morphological abnormal precipitation, the Co-IP (Co-immunoprecipitation) technique proved that NSP7/GFP can interact directly with RAB7A protein.

5. NSP7/GFP protein promotes autophagy in HEK 293 T cells

Since changes in mitochondrial number and morphology are associated with autophagy, we investigated the effect of NSP7/GFP on autophagy. WB results showed that NSP7/GFP up-regulated RAB7A expression in HEK-293T cells. It also promoted the expression of autophagy signature proteins LC3Ⅱ and p62. Mitochondrial and lysosome colocalization analysis showed that compared with the control group, the mitochondrial and lysosome colocalization of NSP7/GFP expressing cells increased. These data suggest that NSP7/GFP promotes mitophagy.

6. NSP7/GFP protein promotes apoptosis

After transfection of pLV3/NSP7/GFP into HEK-293T cells for 48 h and 72 h, more cell debris, cell contraction, and cell-to-cell cavities were found. Flow cytometry showed that NSP7/GFP induced apoptosis. After 48 h, the early apoptosis of NSP7/GFP cells increased by 5.47% compared with the control group. After 72 h transfection, the early apoptosis rate of NSP7/GFP cells increased by 12.19% compared with 48 h. WB results showed that the expressions of apoptosis proteins Caspase9 and BAX increased in HEK-293T expressing NSP7/GFP.

In summary, this paper, combined with previous studies, found that NSP7/GFP protein of SARS-CoV-2 interacts with intracellular RAB7A, resulting in increased mitophagy and activation of apoptosis pathway. This study reveals a potential mechanism by which SARS-CoV-2 nonstructural protein NSP7 damages host cells.