Identification of host cell components essential for the SARS-CoV-2 life cycle

所有病毒在其生命周期的各个方面都依赖于宿主细胞机制。我们计划鉴定SARS-CoV-2病毒生命周期所需的非必需宿主（人类）细胞成分。如果我们能够识别它们，这些将为开发预防病毒感染或阻碍/阻止病毒复制的药物提供关键的干预点。我们将使用遗传方法，使基因组中的2万个基因发生突变，并测试病毒是否能够感染细胞、复制并产生新的感染性颗粒。这些结果将为病毒生命周期的各个步骤（包括进入、复制、生物合成和释放）提供宿主-病原体分子相互作用的见解。在实验上，我们将尝试识别不支持病毒生命周期方面的突变细胞。然后，我们将寻求识别能够提供这种保护的潜在突变基因。从技术上讲，这将通过杀死/移除已被成功感染的细胞来实现，使“耐药”细胞存活下来。该项目的初始阶段将在能够成功感染SARS-CoV-2的人类细胞系中建立敏感和特异性的测定方法。然后将使用靶向基因组中所有基因的CRISPR文库以及专门针对“药物靶向”宿主基因的文库进行遗传筛选。如果在“可药物”库中识别出命中的目标，那么结果就可以快速翻译。例如，服用相应的药物并在培养的细胞上进行测试。基因组宽屏幕识别出的突变需要更长的时间来翻译，这取决于所识别的基因和先前对该基因的研究。我们正在开发的报告细胞系支持这个项目，以及我们确定的任何目标都应该支持与其他实验室和工业伙伴的合作努力，将这些发现转化为宿主导向的治疗方法。

All viruses are dependent on host cell machinery for aspects of their lifecycle. We plan to identify non-essential host (human) cell components that are required for the life cycle of the SARS-CoV-2 virus. If we are able to identify them, these will provide critical intervention points for developing drugs that could prevent virus infection or hamper/stop viral replication.We will used a genetic approach, mutating each of the 20,000 genes in the genome and testing whether the virus is able to infect the cell, replicate and produce new infectious particles. The results will provide insights into molecular host-pathogen interactions at various steps of the virus life cycle including entrance, replication, biosynthesis, and release. Experimentally, we will try to identify mutant cells which do not support aspects of the viral life cycle. We will then seek to identify the underlying mutant gene(s) which are able provide this protection. Technically this will be achieved by killing/removing cells which have been successfully infected, allowing the "resistant" ones to survive. The initial stage of the project will be to establish sensitive and specific assays in human cell lines which can be successfully infected with SARS-CoV-2. Genetic screens would then be conducted with CRISPR libraries that target all the gene in the genome as well as a library specialised on "drug targetable" host genes. If hits are identified in a "druggable" library, the results could be rapidly translated. For instance by taking the corresponding drug and testing this on cells in culture. Hits identified by genome wide screens will take longer to translate, depending on the gene that is identified and prior work on the gene. The reporter cell lines we are developing to support this project as well as any targets we identify should support collaborative efforts with other laboratories and industrial partners, to translate these findings into host-directed therapies.