Department of Virology, National Institute of Infectious Diseasesgrid.410795.e, Musashimurayama, Tokyo, Japan.
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseasesgrid.410795.e, Shinjuku, Tokyo, Japan.
mBio. 2022 Dec 20;13(6):e0169822. doi: 10.1128/mbio.01698-22. Epub 2022 Nov 8.
The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry by using CRISPR/Cas9 genome-wide knockout screening, and we found sphingomyelin synthase 1 (SMS1), encoded by the gene, as a candidate. RuV growth was strictly suppressed in -knockout cells and was completely recovered by the overexpression of enzymatically active SMS1 and partially recovered by that of SMS2, another member of the SMS family, but not by that of enzymatically inactive SMS1. An entry assay using pseudotyped vesicular stomatitis virus possessing RuV envelope proteins revealed that sphingomyelin generated by SMSs is crucial for at least RuV entry. In -knockout cells, lipid mixing between the RuV envelope membrane and the membrane of host cells occurred, but entry of the RuV genome from the viral particles into the cytoplasm was strongly inhibited. This indicates that sphingomyelin produced by SMSs is essential for the formation of membrane pores after hemifusion occurs during RuV entry. Infection with rubella virus during pregnancy causes congenital rubella syndrome in infants. Despite its importance in public health, the detailed mechanisms of rubella virus cell entry have only recently become somewhat clearer. The E1 protein of rubella virus is classified as a class II fusion protein based on its structural similarity, but it has the unique feature that its activity is dependent on calcium ion binding in the fusion loops. In this study, we found another unique feature, as cellular sphingomyelin plays a critical role in the penetration of the nucleocapsid into the cytoplasm after hemifusion by rubella virus. This provides important insight into the entry mechanism of rubella virus. This study also presents a model of hemifusion arrest during cell entry by an intact virus, providing a useful tool for analyzing membrane fusion, a biologically important phenomenon.
宿主细胞膜的脂质组成是决定包膜病毒进入细胞的关键因素之一。为了阐明风疹病毒（RuV）等包膜病毒进入细胞的详细机制，我们使用 CRISPR/Cas9 全基因组敲除筛选寻找参与该进入过程的宿主因子，发现编码 基因的神经酰胺合成酶 1（SMS1）是候选因子之一。 -敲除细胞中 RuV 的生长受到严格抑制，而过表达具有酶活性的 SMS1 可完全恢复，SMS1 的另一个家族成员 SMS2 部分恢复，但无酶活性的 SMS1 则不能恢复。使用具有 RuV 包膜蛋白的假型水疱性口炎病毒进行的进入试验表明，SMS 产生的神经酰胺对于至少 RuV 的进入是至关重要的。在 -敲除细胞中，RuV 包膜与宿主细胞膜之间发生了脂质混合，但病毒粒子中的 RuV 基因组进入细胞质受到强烈抑制。这表明，SMS 产生的神经酰胺是 RuV 进入时发生半融合后形成膜孔所必需的。风疹病毒感染孕妇会导致婴儿先天性风疹综合征。尽管其对公共卫生很重要，但风疹病毒进入细胞的详细机制直到最近才变得更加清楚。风疹病毒的 E1 蛋白根据其结构相似性被归类为 II 类融合蛋白，但它具有独特的特征，即其活性依赖于融合环中的钙离子结合。在这项研究中，我们发现了另一个独特的特征，即细胞神经酰胺在风疹病毒半融合后对核衣壳进入细胞质起关键作用。这为风疹病毒的进入机制提供了重要的见解。该研究还提出了一种完整病毒进入时半融合阻滞模型，为分析膜融合这一重要的生物学现象提供了有用的工具。
