Characterization the disruption of mitochondrial function and induction of oxidative stress by SARS-CoV2

1患新冠肺炎严重疾病风险较高的人群是老年人和有代谢障碍的人 2个综合征，这些人群以线粒体功能受损而闻名。此外，最常见的 3住院的COVID患者的症状是呼吸急促和疲劳，表明缺氧和 4能量代谢，也提示线粒体有缺陷。新冠肺炎患者也明显 5血清乳酸脱氢酶升高和氧化应激增加，表明 6线粒体氧化磷酸化(OXPHOS)。总而言之，这些信息让我们思考是否 7线粒体功能障碍可能参与了新冠肺炎的发病。全面的蛋白质组学 8调查和其他研究确定了至少6种线粒体定位的SARS-CoV-2病毒蛋白 9被证明与宿主细胞线粒体蛋白相互作用，参与关键的OXPHOS通路聚合 10呼吸复合体I的生物发生。我们的实验室已经在线粒体的研究方面建立了专业知识 11生物学和线粒体医学，特别是关于复杂的I-相关OXPHOS的生物发生。多年来，我们 12个人开发了一系列独特的细胞模型，具有不同类型的复杂I缺陷，包括几组细胞 13个不同含量的功能复合体I亚基，一组具有不同复合体I组装能力的细胞， 14和一组携带复杂I亚单位基因致病突变的细胞，以及一个工程系统 15通过引入酵母复合体I对应的NDI1基因来挽救复合体I的相关功能。这些 16个模型表现出不同水平的复合体I亚基表达，不同的复合体I和总体能力 17呼吸机械组装，以及不同复合体I和整体线粒体OXPHOS活性。 因此，这些细胞模型对氧化应激和细胞死亡也表现出不同的敏感性。我们有 19还发起了一系列关于病毒对线粒体的影响以及由此对人类的影响的研究 20种疾病。此外，我们还实现了1.可以开启和关闭的诱导表达 21个SARS-CoV-2蛋白在我们的细胞模型中的适当水平；2.多基因的表达 22在我们的细胞模型中，多个SARS-CoV-2蛋白同时靶向一个或多个OXPHOS通路； 23.人肺泡上皮细胞系A549-hACE2细胞的建立 24个表达人血管紧张素转换酶2的慢病毒。A549-hACE2细胞容易支持SARS-CoV2的感染和复制； 25.获得了可作为对照的SARS-CoV2突变株。这些都提供了一个独特的机会 26我们将利用我们独特的系统和专业知识，实现两个独立和综合的目标，以研究 27 SARS-CoV2与线粒体的相互作用及其对氧化应激和细胞死亡的影响， 28在线粒体功能受调控的细胞模型和感染人肺泡上皮细胞系中都是如此 29例SARS-CoV2。我们希望这些研究将有助于确定SARS-CoV2蛋白在宿主中的分子靶点 30个细胞，还将提供防止新冠肺炎有害影响的新方法。 31 32

1 Populations at higher risk of severe disease from COVID-19 are the elderly and those with metabolic 2 syndromes, the populations known for compromised mitochondrial function. In addition, the most common 3 symptoms in hospitalized COVID patients are shortness of breath and fatigue, indicating deficient oxygen and 4 energy metabolism, also suggesting defective mitochondria. COVID-19 patients also have significantly 5 elevated serum lactate dehydrogenase and increases oxidative stress, pointing to a possibility of reduced 6 mitochondrial oxidative phosphorylation (OXPHOS). Together, these information leads us to consider whether 7 mitochondrial dysfunction might contribute to the pathogenesis of COVID-19. A comprehensive proteomics 8 investigation and other studies identified at least 6 mitochondrially-localized SARS-CoV-2 viral proteins which 9 were shown to interact with host cell mitochondrial proteins involved in critical OXPHOS pathways converging 10 on respiratory Complex I biogenesis. Our lab has established expertise on the investigation of mitochondrial 11 biology and mitochondrial medicine, especially on Complex I-related OXPHOS biogenesis. Over the years we 12 have developed a series of unique cell models with different types of complex I defects, including sets of cells 13 with different contents of functional complex I subunits, a set cells with different complex I assembly capacity, 14 and a set of cells carrying pathogenic mutations in complex I subunit genes, as well as an engineered system 15 to rescue complex I-related function with the introduction of a yeast Complex I counterpart NDI1 gene. These 16 models exhibit different levels of complex I subunit expression, different capacities of complex I and overall 17 respiratory machinery assembly, and different complex I and overall mitochondrial OXPHOS activities. 18 Accordingly, these cell models also exhibit different sensitivities to oxidative stress and cell death. We have 19 also initiated a line of study on the effect of viruses on mitochondria and consequent implications on human 20 diseases. In addition, we have achieved to obtain 1.Inducible expression which could turn on and off the 21 SARS-CoV-2 proteins in our cell models at proper levels; 2.Multiple genes expression which can express 22 multiple SARS-CoV-2 proteins targeting one or multiple OXPHOS pathways simultaneously in our cell models; 23 3.Establishment of A549-hACE2 cell, where a human alveolar epithelial cell line, A549 was transduced with 24 lentiviruses expressing human ACE2. A549-hACE2 cells readily support SARS-CoV2 infection and replication; 25 4.Generated mutant SARS-CoV2 lines which could serve as controls. These provide a unique opportunity for 26 us to utilize our unique systems and expertise to fulfill with two independent and integrated aims to study the 27 interactions between SARS-CoV2 and mitochondria, and their implications on oxidative stress and cell death, 28 both in cell models with regulated mitochondrial function and in human alveolar epithelial cell line infected with 29 SARS-CoV2. We expect these research will help identify molecular targets of SARS-CoV2 proteins in host 30 cells and will also provide novel approaches for protecting against the harmful effects of COVID-19. 31 32