Genetic identification of host factors required for SARS-CoV-2 cell infection

新冠肺炎全球大流行正在全球范围内产生前所未有的社会、医疗和经济影响。截至2020年5月，已有400多万人感染，30多万人死亡，SARS-CoV-2危机是当代最大的挑战之一。大约80%的感染是轻微的，但其余20%的人会出现严重的临床并发症，病毒性肺炎恶化需要住院，并可能需要国际电联的机械通气——这一干预措施的发病率和死亡率非常高。目前既没有针对SARS-CoV-2的疫苗，也没有有效的治疗药物，因此这是一项医疗和科学紧急情况。病毒进入宿主细胞是治疗干预的一个有吸引力的步骤，其中活动性感染可能被预防或阻止。对于其他人类病毒，如HIV和HCV，已经开发出有效的进入抑制剂。为了制定针对SARS-CoV-2的有效治疗策略，了解病毒进入和感染所需的宿主因素至关重要。SARS-CoV-2进入的关键是在病毒外部形成冠状突起的病毒刺突蛋白。刺突与其受体血管紧张素转换酶2 （Ace2）在细胞表面相互作用。刺突蛋白被宿主细胞酶激活，包括TMPRSS2，它允许病毒与细胞膜融合，将病毒RNA释放到细胞中，为病毒复制做好准备。除了Ace2和TMPRSS2外，SARS-CoV-2细胞感染可能还需要其他宿主因子，但尚未对病毒进入和感染所需的因子进行全面分析。病毒的进入也可以通过肺部的微环境条件来调节，例如低氧水平（缺氧）。缺氧是否对SARS-CoV-2感染有调节作用尚待确定。我们在这个项目中的目标是利用我们在遗传筛选方面的专业知识来确定病毒进入和感染所使用的宿主因素。在我们的初步研究中，我们建立了不同的SARS-CoV-2进入模型。这些包括：(i)可溶性刺突fc融合蛋白，它诱导Ace2从细胞表面内化；（ii） SARS-CoV-2假病毒，这是一种在细胞表面表达全长刺突蛋白并容易感染靶细胞的病毒颗粒。我们将在正常条件和低氧水平下，将这些病毒进入模型与活的SARS-CoV-2感染一起使用，进行基于CRISPR/Cas9的基因筛选。我们将确定病毒进入宿主细胞所需的一整套宿主因子，并在SARS-CoV-2感染原代人气道细胞的背景下对其进行验证。我们将描述验证命中的特征，以确定其治疗潜力。我们预计，全面确定SARS-CoV-2感染所需的宿主因子将为开发新型抗病毒药物提供蓝图。

The SARS-CoV-2 global pandemic is having an unprecedented social, medical and economic impact across the globe. As of May 2020, with more than 4 million people infected and over 300 000 deaths, the SARS-CoV-2 crisis represents one of the biggest challenges of the modern era. Around 80% of infections are mild, but the remaining 20% of individuals suffer severe clinical complications, with worsening viral pneumonitis requiring hospital admission and potential mechanical ventilation on ITU - an intervention which carries a very high morbidity and mortality. Neither vaccine nor effective therapeutic agents are at present available for SARS-CoV-2, which therefore represents a medical and scientific emergency. Entry of the virus into the host cell represents an attractive step for therapeutic intervention, where active infection may be either prevented or blocked. Efficient entry inhibitors have been developed for other human viruses, such as HIV and HCV. To develop effective therapeutic strategies against SARS-CoV-2 it is essential to understand the host factors required for virus entry and infection. The key player in SARS-CoV-2 entry is the viral Spike protein which forms the corona-shaped projections on the outside of the virus. Spike interacts with its receptor, Angiotensin-converting enzyme 2 (Ace2) at the cell surface. The Spike protein is activated by host cell enzymes, including TMPRSS2 which allow the virus to fuse with the cellular membrane, releasing its viral RNA into the cell, all ready for viral replication. Apart from Ace2 and TMPRSS2, additional host factors are likely to be required for SARS-CoV-2 cell infection, but no comprehensive analysis of factors required for virus entry and infection has been undertaken. Viral entry can also be modulated by microenvironmental conditions in the lung, such as low oxygen levels (hypoxia). Whether hypoxia regulates SARS-CoV-2 infection needs to be determined. Our aim in this project is to use our expertise in genetic screens to identify host factors used by the virus for entry and infection. In our preliminary studies we have established different models of SARS-CoV-2 entry. These include: (i) Soluble Spike Fc-fusion protein, which induces internalisation of Ace2 from the cell surface (ii) SARS-CoV-2 pseudovirus, which are virus particles that express full-length Spike protein on their cell surface and readily infect their target cells. We will use these virus entry models together with live SARS-CoV-2 infection to perform CRISPR/Cas9 based genetic screens, in both normal conditions and in low levels of oxygen. We will identify a comprehensive set of host factors required for virus entry into the host cell and validate them in the context of SARS-CoV-2 infection of primary human airway cells. We will characterise the validated hits to determine their therapeutic potential. We anticipate that a comprehensive identification of host factors required for SARS-CoV-2 infection will provide a blueprint for the development of novel antiviral agents.