Bolland William, Marechal Inès, Petiot Chloé, Porrot Françoise, Guivel-Benhassine Florence, Brelot Anne, Casartelli Nicoletta, Schwartz Olivier, Buchrieser Julian
Virus and Immunity Unit, Institut Pasteur, Université Paris Cité, CNRS UMR3569, Paris, France.
Université Paris Cité, Paris, France.
J Virol. 2025 Jan 31;99(1):e0182324. doi: 10.1128/jvi.01823-24. Epub 2024 Nov 21.
Membrane fusion occurs at the early stages of SARS-CoV-2 replication, during entry of the virus, and later during the formation of multinucleated cells called syncytia. Fusion is mediated by the binding of the viral Spike protein to its receptor ACE2. Lipid rafts are dynamic nanodomains enriched in cholesterol and sphingolipids. Rafts can act as platforms for entry of different viruses by localizing virus receptors, and attachment factors to the same membrane domains. Here, we first demonstrate that cholesterol depletion by methyl-beta-cyclodextrin inhibits Spike-mediated fusion and entry. To further study the role of ACE2 lipid raft localization in SARS-CoV-2 fusion and entry, we designed a GPI-anchored ACE2 construct. Both ACE2 and ACE2-GPI proteins were similarly expressed at the plasma membrane. Through membrane flotation assays, we show that in different cell lines, ACE2-GPI localizes predominantly to raft domains of the plasma membrane while ACE2 is non-raft associated. We then compare the ability of ACE2 and ACE2-GPI to permit SARS-CoV-2 entry, replication, and syncytia formation of different viral variants. We find little difference in the two proteins. Our results demonstrate that SARS-CoV-2 entry and fusion are cholesterol-dependent and raft-independent processes.IMPORTANCERafts are often exploited by viruses and used as platforms to enhance their entry into the cell or spread from cell to cell. The membrane localization of ACE2 and the role of lipid rafts in SARS-CoV-2 entry and cell-to-cell spread are poorly understood. The function of lipid rafts in viral fusion is often studied through their disruption by cholesterol-depleting agents. However, this process may have off-target impacts on viral fusion independently of lipid-raft disruption. Therefore, we created an ACE2 construct that localizes to lipid rafts using a GPI anchor. Conversely, wild-type ACE2 was non-raft associated. We find that the localization of ACE2 to lipid rafts does not modify the fusion dynamics of SARS-CoV-2.
膜融合发生在严重急性呼吸综合征冠状病毒2（SARS-CoV-2）复制的早期阶段，即病毒进入细胞的过程中，以及之后称为合胞体的多核细胞形成期间。融合是由病毒刺突蛋白与其受体血管紧张素转换酶2（ACE2）结合介导的。脂筏是富含胆固醇和鞘脂的动态纳米结构域。脂筏可通过将病毒受体和附着因子定位到同一膜结构域，作为不同病毒进入细胞的平台。在此，我们首先证明，甲基-β-环糊精消耗胆固醇可抑制刺突蛋白介导的融合和进入。为了进一步研究ACE2脂筏定位在SARS-CoV-2融合和进入中的作用，我们设计了一种糖基磷脂酰肌醇（GPI）锚定的ACE2构建体。ACE2和ACE2-GPI蛋白在质膜上的表达相似。通过膜浮选试验，我们表明，在不同细胞系中，ACE2-GPI主要定位于质膜的脂筏结构域，而ACE2与非脂筏相关。然后，我们比较了ACE2和ACE2-GPI允许不同病毒变体的SARS-CoV-2进入、复制和合胞体形成的能力。我们发现这两种蛋白之间几乎没有差异。我们的结果表明，SARS-CoV-2的进入和融合是胆固醇依赖性和非脂筏依赖性过程。
重要性
病毒经常利用脂筏，并将其用作增强进入细胞或在细胞间传播的平台。人们对ACE2的膜定位以及脂筏在SARS-CoV-2进入和细胞间传播中的作用了解甚少。脂筏在病毒融合中的功能通常通过用消耗胆固醇的试剂破坏它们来研究。然而，这一过程可能对病毒融合产生非靶向影响，与脂筏破坏无关。因此，我们创建了一种使用GPI锚定定位于脂筏的ACE2构建体。相反，野生型ACE2与非脂筏相关。我们发现ACE2定位于脂筏不会改变SARS-CoV-2的融合动力学。
