Requirements for CEACAMs and cholesterol during murine coronavirus cell entry
Requirements for CEACAMs and cholesterol during murine coronavirus cell entry
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DOI：
10.1128/jvi.78.6.2682-2692.2004
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发表时间：
2004-03-01
影响因子：
5.4
通讯作者：
Gallagher, TM
中科院分区：
文献类型：
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作者：
Thorp, EB;Gallagher, TM
先前的报告已经证明，在被包膜冠状病毒小鼠肝炎病毒（MHV）感染期间，补充胆固醇会增加组织培养中的细胞病变效应，并增强体内致病性。为了进一步了解这些现象的机理，我们分别使用富含甲基- β -环糊精或胆固醇的生长培养基来降低或提高细胞膜固醇。根据感染MHV菌株的不同，胆固醇消耗可使斑块的形成减少2- 20倍，而补充胆固醇可使易感性增加2- 10倍。这些不同的胆固醇水平对病毒刺突(S)蛋白与细胞癌胚抗原相关细胞粘附分子（CEACAM）受体的结合没有影响，相反，它们与S蛋白介导的膜融合活性直接相关。我们考虑了胆固醇是否间接参与了膜融合，通过将ceacam凝聚成“脂筏”膜微域，从而创造了同时结合多个S蛋白的机会，这些S蛋白随后在受体触发的膜融合过程中合作。然而，绝大多数ceacam被冷Triton X-100 （TX-100）溶解，表明它们不存在于脂筏中。此外，工程ceacam附着在糖基磷脂酰肌醇锚定物上，锚定物由tx -100抗性脂筏分隔，但携带这些筏相关ceacam的细胞对MHV感染不敏感。这些发现反驳了胆固醇依赖性CEACAM定位到MHV进入膜微域的重要性，相反表明胆固醇具有更直接的作用。事实上，我们发现即使在没有ceacam的情况下，那些罕见的s介导的融合也需要胆固醇。我们得出结论，胆固醇是一种重要的膜融合辅助因子，无论是否有ceacam，都可以促进MHV的进入。
Previous reports have documented that cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during infection by the enveloped coronavirus murine hepatitis virus (MHV). To move toward a mechanistic understanding of these phenomena, we used growth media enriched with methyl-beta-cyclodextrin or cholesterol to reduce or elevate cellular membrane sterols, respectively. Cholesterol depletions reduced plaque development 2- to 20-fold, depending on the infecting MHV strain, while supplementations increased susceptibility 2- to 10-fold. These various cholesterol levels had no effect on the binding of viral spike (S) proteins to cellular carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, rather they correlated directly with S-protein-mediated membrane fusion activities. We considered whether cholesterol was indirectly involved in membrane fusion by condensing CEACAMs into "lipid raft" membrane microdomains, thereby creating opportunities for simultaneous binding of multiple S proteins that subsequently cooperate in the receptor-triggered membrane fusion process. However, the vast majority of CEACAMs were solubilized by cold Triton X-100 (TX-100), indicating their absence from lipid rafts. Furthermore, engineered CEACAMs appended to glycosylphosphatidylinositol anchors partitioned with TX-100-resistant lipid rafts, but cells bearing these raft-associated CEACAMs were not hypersensitive to MHV infection. These findings argued against the importance of cholesterol-dependent CEACAM localizations into membrane microdomains for MHV entry, instead suggesting that cholesterol had a more direct role. Indeed, we found that cholesterol was required even for those rare S-mediated fusions taking place in the absence of CEACAMs. We conclude that cholesterol is an essential membrane fusion cofactor that can act with or without CEACAMs to promote MHV entry.