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

在进入宿主细胞后，冠状病毒，包括SARS-CoV 2，重塑细胞膜以形成复制细胞器。这种膜重塑的一个特征是形成独特的双膜囊泡（DMV），病毒RNA在其中驻留和复制。我们发现两种SARS-CoV-2蛋白（nsp 3C和nsp 4）的表达足以诱导人细胞系中大量DMV的形成，并相应上调宿主细胞脂肪生成途径，我们的实验数据表明这是DMV形成所必需的。其他病毒的复制依赖于宿主细胞器在DMV外膜与宿主细胞器接触部位的脂质转运。我们已经在SARS-CoV-2感染的肺上皮细胞中发现了DMV与多种宿主细胞器之间广泛的膜接触位点，在本研究中，我们的目标是建立稳定的诱导型SARS-CoV-2 nsp 3C/nsp 4细胞系，作为SARS-CoV-2复制的模型。使用这个模型，我们将阐明介导DMV形成的脂质供应的关键途径，首先探索不同的细胞脂质来源的贡献。我们将通过表达SARS-CoV-2关键候选蛋白和操纵膜接触位点机制，进一步确定DMV驱动的细胞器间脂质转运机制是如何调节的，以确定潜在的治疗干预的新靶点，最后我们将评估脂肪生成/脂质转运抑制对SARS-CoV-2感染细胞中DMV形成和细胞存活的影响。

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.