Mechanisms of coronavirus replication: the role of cellular lipids in the generation of replication organelles

Following entry into the host cell, coronaviruses, including SARS-CoV2, remodel cellular membranes to form replication organelles. A characteristic feature of this membrane remodelling is the formation of distinctive double membrane vesicles (DMVs) in which the viral RNA resides and replicates. DMV generation involves rewiring of host lipid metabolism. We found that expression of two SARS-CoV-2 proteins (nsp3C and nsp4) is sufficient to induce abundant DMV formation in human cell lines, with a corresponding upregulation of host cell lipogenic pathways that our preliminary data suggests is essential for the formation of DMVs. Replication of other viruses has been shown to depend on lipid transport from host cell organelles at sites of contact between the outer DMV membrane and that of the host organelle. We have identified extensive membrane contact sites between DMVs and a variety of host cell organelles in SARS-CoV-2 infected lung epithelial cells. In the proposed study, we aim to generate stable inducible SARS-CoV-2 nsp3C/nsp4 cell lines to use as a model of SARS-CoV-2 replication. Using this model, we will elucidate key pathways that mediate lipid provision for DMV formation, by first exploring the contribution of different cellular lipid sources. We will further establish, by expression of key candidate SARS-CoV-2 proteins and manipulation of membrane contact site machinery, how DMV-driven interorganelle lipid transport mechanisms are regulated, in order to identify novel targets for potential therapeutic intervention. Finally we will assess the effect of lipogenesis/lipid transport inhibition on DMV formation and cell survival in SARS-CoV-2 infected cells.