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

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Following entry into the host cell, coronaviruses, including SARS-CoV2, remodel cellularmembranes to form replication organelles. A characteristic feature of this membrane remodellingis the formation of distinctive double membrane vesicles (DMVs) in which the viral RNA residesand replicates.DMV generation involves rewiring of host lipid metabolism. We found that expression of twoSARS-CoV-2 proteins (nsp3C and nsp4) is sufficient to induce abundant DMV formation in humancell lines, with a corresponding upregulation of host cell lipogenic pathways that our preliminarydata suggests is essential for the formation of DMVs. Replication of other viruses has been shownto depend on lipid transport from host cell organelles at sites of contact between the outer DMVmembrane and that of the host organelle. We have identified extensive membrane contact sitesbetween 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 touse as a model of SARS-CoV-2 replication. Using this model, we will elucidate key pathways thatmediate lipid provision for DMV formation, by first exploring the contribution of different cellularlipid sources. We will further establish, by expression of key candidate SARS-CoV-2 proteins andmanipulation of membrane contact site machinery, how DMV-driven interorganelle lipid transportmechanisms 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 cellsurvival in SARS-CoV-2 infected cells.