Division of Biochemistry, Department of Biomedical Sciences and Nihon University School of Medicine, Tokyo 173-8610, Japan.
J Lipid Res. 2013 Apr;54(4):881-92. doi: 10.1194/jlr.M026443. Epub 2013 Jan 12.
Lipid droplets (LD) are dynamic storage organelles that are involved in lipid homeostasis. Hepatitis C virus (HCV) is closely associated with LDs. HCV Core and nonstructural (NS) proteins colocalize with LDs and presumably are involved in virion formation at that site. We demonstrated that HCV NS4B, an integral membrane protein in endoplasmic reticulum (ER), strongly targeted LDs. Confocal imaging studies showed that NS4B localized at the margins of LDs. Biochemical fractionation of HCV-replicating cells suggested that NS4B existed in membranes associated with LDs rather than on the LD surface membrane itself. The N- and C-terminal cytosolic domains of NS4B showed targeting of LDs, with the former being much stronger. In both domains, activity was present in the region containing an amphipathic α-helix, in which 10 hydrophobic residues were identified as putative determinants for targeting LDs. JFH1 mutants with alanine substitutions for the hydrophobic residues were defective for virus replication. W43A mutant with a single alanine substitution showed loss of association of NS4B with LDs and severely reduced release of infectious virions compared with wild-type JFH1. NS4B plays a crucial role in virus replication at the site of virion formation, namely, the microenvironment associated with LDs.
脂滴（LD）是一种动态的储存细胞器，参与脂质的动态平衡。丙型肝炎病毒（HCV）与 LD 密切相关。HCV 核心蛋白和非结构（NS）蛋白与 LD 共定位，推测它们参与了该部位的病毒形成。我们证明了 HCV NS4B，一种内质网（ER）中的整合膜蛋白，强烈靶向 LD。共聚焦成像研究表明 NS4B 定位于 LD 的边缘。HCV 复制细胞的生化分离表明 NS4B 存在于与 LD 相关的膜中，而不是在 LD 表面膜本身。NS4B 的 N 端和 C 端胞质结构域显示出靶向 LD 的作用，前者的作用更强。在这两个结构域中，活性存在于包含一个两亲性α螺旋的区域中，其中 10 个疏水性残基被鉴定为靶向 LD 的假定决定因素。对于疏水性残基的丙氨酸取代 JFH1 突变体，病毒复制缺陷。与野生型 JFH1 相比，带有单个丙氨酸取代的 W43A 突变体显示 NS4B 与 LD 的关联丧失，并且传染性病毒粒子的释放严重减少。NS4B 在病毒形成部位的病毒复制中起着至关重要的作用，即与 LD 相关的微环境。
