Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion
复制标题
DOI：
10.1371/journal.ppat.1007471
复制
发表时间：
2018-12-01
期刊：
影响因子：
6.7
通讯作者：
Moradpour, Darius
中科院分区：
文献类型：
--
作者：
Gouttenoire, Jerome;Pollan, Angela;Moradpour, Darius
戊型肝炎病毒（HEV）是一种正链RNA病毒，编码3个开放阅读框（ORF）。戊型肝炎病毒ORF 3蛋白是一种小蛋白，迄今为止其特征尚不清楚，参与病毒颗粒分泌和可能的其他功能。在这里，我们表明，戊型肝炎病毒ORF 3蛋白形成膜相关的寡聚体。免疫印迹分析表明ORF 3蛋白在无细胞与细胞系统中表达的翻译后修饰。进一步的分析表明，戊型肝炎病毒ORF 3蛋白是棕榈酰化的半胱氨酸残基在其N-末端区域，证实了H-3-棕榈酸标记，半胱氨酸到丙氨酸取代突变体的调查和治疗棕榈酰化抑制剂2-溴棕榈酸酯（2-BP）。通过定点突变或2-BP处理取消棕榈酰化改变了ORF 3蛋白的亚细胞定位，降低了蛋白的稳定性，并强烈削弱了感染性颗粒的分泌。此外，选择性膜透化加上免疫荧光显微镜显示，HEV ORF 3蛋白完全暴露于细胞溶质侧的膜，允许提出一个模型，其膜拓扑结构和病毒生命周期中所需的相互作用。总之，棕榈酰化决定了HEV ORF 3蛋白在HEV生命周期中的亚细胞定位、膜拓扑结构和功能。
Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by H-3-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.