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

冠状病毒（包括SARS-CoV2）进入宿主细胞后，会重塑细胞膜，形成复制细胞器。这种膜重构的一个特征是形成独特的双膜囊泡（dmv），病毒RNA在其中居住和复制。DMV的产生涉及宿主脂质代谢的重新布线。我们发现两种osars - cov -2蛋白（nsp3C和nsp4）的表达足以在人类细胞系中诱导大量的DMV形成，并相应上调宿主细胞脂质生成途径，我们的初步数据表明这是DMV形成所必需的。其他病毒的复制已被证明依赖于宿主细胞器在dmv外膜和宿主细胞器接触部位的脂质转运。我们在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。在这项研究中，我们的目标是产生稳定的可诱导的SARS-CoV-2 nsp3C/nsp4细胞系，作为SARS-CoV-2复制的模型。使用该模型，我们将通过首先探索不同细胞脂质来源的贡献来阐明介导DMV形成的脂质供应的关键途径。我们将进一步通过表达关键候选SARS-CoV-2蛋白和操纵膜接触位点机制来确定dmv驱动的细胞器间脂质转运机制是如何被调节的，从而确定潜在治疗干预的新靶点。最后，我们将评估抑制脂质生成/脂质转运对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.