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

1 COVID-19导致严重疾病的风险较高的人群是老年人和代谢性疾病患者， 2综合征，已知线粒体功能受损的人群。此外，最常见的 住院的COVID患者的3个症状是呼吸急促和疲劳，表明缺氧， 4能量代谢，也提示线粒体缺陷。COVID-19患者也有显著的 5血清乳酸脱氢酶升高，氧化应激增加，指向可能性降低 6线粒体氧化磷酸化（OXPHOS）。综合起来，这些信息使我们考虑， 7线粒体功能障碍可能有助于COVID-19的发病机制。全面的蛋白质组学 8项调查和其他研究确定了至少6种位于肠道的SARS-CoV-2病毒蛋白， 9显示与参与关键OXPHOS途径会聚的宿主细胞线粒体蛋白质相互作用 10对呼吸复合物I生物发生的影响。我们的实验室已经建立了研究线粒体的专业知识 11生物学和线粒体医学，特别是关于复合物I相关的OXPHOS生物发生。多年来我们 12已经开发了一系列具有不同类型复合物I缺陷的独特细胞模型，包括细胞集 13个具有不同复合物I亚单位含量的细胞，一组具有不同复合物I组装能力的细胞， 14和一组携带复合物I亚基基因中的致病性突变的细胞，以及工程化系统 15，以通过引入酵母复合物I对应物NDI 1基因来拯救复合物I相关功能。这些 16个模型表现出不同水平的复合物I亚基表达，不同的复合物I和整体能力， 17呼吸机械组装，以及不同的复合物I和整体线粒体OXPHOS活性。 因此，这些细胞模型也表现出对氧化应激和细胞死亡的不同敏感性。我们有 19还启动了一系列关于病毒对线粒体的影响及其对人类的影响的研究。 20种疾病此外，我们还获得了1.可诱导表达的启动子和关闭子， 21种SARS-CoV-2蛋白在我们的细胞模型中处于适当水平; 2.多个基因表达， 在我们的细胞模型中同时靶向一个或多个OXPHOS通路的22种多SARS-CoV-2蛋白; 3. A549-hACE 2细胞的建立，其中人肺泡上皮细胞系A549用 24慢病毒表达人ACE 2。A549-hACE 2细胞易于支持SARS-CoV 2感染和复制; 4.建立了突变型SARS-CoV 2株系，可作为对照。这为以下方面提供了独特的机会： 26我们利用我们独特的系统和专业知识，以实现两个独立和综合的目标，研究 27 SARS-CoV 2和线粒体之间的相互作用，以及它们对氧化应激和细胞死亡的影响， 在线粒体功能受调节的细胞模型和感染的人肺泡上皮细胞系中， 29例SARS-CoV 2.我们希望这些研究将有助于确定SARS-CoV 2蛋白在宿主中的分子靶点 30个细胞，并将提供新的方法来保护免受COVID-19的有害影响。 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