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

The unprecedented medical and economic impact of the SARS-CoV-2 pandemic represents a global challenge to the scientific community. In the absence of any vaccine or therapeutic agent, it is essential to understand how SARS-CoV-2 interacts with its host cell with the aim of identifying viral vulnerabilities for potential therapeutic intervention. Inhibitors of viral entry have been developed for HIV and HCV and represent an attractive step for therapeutic intervention. In this application we will use our expertise in CRISPR-Cas9 genome-wide forward genetic screens to identify host factors required for virus entry and infection. SARS-CoV-2 cell entry is dependent on its spike protein which mediates both binding to its cognate cell surface receptor, Ace2, and fusion of viral and cellular membranes. The Spike protein is activated by the host cell serine protease TMPRSS2 which promotes viral fusion with the cellular membrane. The role of other host cell proteins in viral entry and infection is less clear, nor do we know how the low oxygen tension of COVID19-infected lungs, affects viral infection. We have developed different models of virus entry, including internalisation of Spike fusion protein, Spike protein pseudovirus infection and, infection with live SARS-CoV-2 virus. Using these tools we will perform genome-wide forward genetic screens to identify host factors required for (i) the regulation of cell surface Ace2 expression; (ii) entry of SARS-CoV-2 into the cell and whether this is affected by low oxygen tension. (iii) Having identified the host factors required for SARS-CoV-2-mediated entry we will validate our results in the context of SARS-CoV-2 infection of primary human bronchial epithelial cells. We will characterise our hits and determine their therapeutic potential. We anticipate that the comprehensive identification of host cell factors essential for SARS-CoV-2 infection will identify targets for the development of novel antiviral agents.