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

1 因 COVID-19 罹患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 种综合征，以线粒体功能受损而闻名的人群。此外，最常见的 住院新冠患者的3个症状是呼吸短促和疲劳，表明缺氧和 4.能量代谢，也提示线粒体有缺陷。 COVID-19 患者也有显着 5 血清乳酸脱氢酶升高并增加氧化应激，表明可能降低 6 线粒体氧化磷酸化（OXPHOS）。这些信息共同引导我们考虑是否 7 线粒体功能障碍可能导致 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 种位于线粒体的 SARS-CoV-2 病毒蛋白， 9 种被证明与参与关键 OXPHOS 途径汇聚的宿主细胞线粒体蛋白相互作用 10 关于呼吸复合物 I 生物发生。我们的实验室已经建立了线粒体研究方面的专业知识 11 生物学和线粒体医学，特别是与复合物 I 相关的 OXPHOS 生物合成。这些年来我们 12 开发了一系列具有不同类型复杂 I 缺陷的独特细胞模型，包括细胞组 13个具有不同含量的功能复合物I亚基，一组具有不同复合物I组装能力的细胞， 14 和一组携带复杂 I 亚基基因致病性突变的细胞，以及一个工程系统 15 通过引入酵母复合物 I 对应的 NDI1 基因来挽救复合物 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能力和总体能力 17 呼吸机械组装，以及不同的复合物 I 和总体线粒体 OXPHOS 活性。 18 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.诱导表达，可以打开和关闭 我们的细胞模型中的 21 种 SARS-CoV-2 蛋白处于适当水平； 2.可表达的多基因表达 在我们的细胞模型中，22 种多种 SARS-CoV-2 蛋白同时针对一种或多种 OXPHOS 途径； 23 3.A549-hACE2细胞的建立，其中转导人肺泡上皮细胞系A549 24 种表达人 ACE2 的慢病毒。 A549-hACE2 细胞很容易支持 SARS-CoV2 感染和复制； 25 4.生成可作为对照的突变 SARS-CoV2 系。这些为 26 我们利用我们独特的系统和专业知识来实现两个独立且综合的目标，以研究 27 SARS-CoV2 和线粒体之间的相互作用及其对氧化应激和细胞死亡的影响， 28 在具有调节线粒体功能的细胞模型和感染 29 SARS-CoV2。我们预计这些研究将有助于识别宿主体内 SARS-CoV2 蛋白的分子靶点 30 细胞，还将提供防止 COVID-19 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