Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.载脂蛋白B（Apolipoprotein B, ApoB）与载脂蛋白E（Apolipoprotein E, ApoE）已被证实可参与丙型肝炎病毒（hepatitis C virus, HCV）的颗粒形成与组织嗜性，但其具体作用机制仍未明确。本研究通过锌指核酸酶（zinc finger nucleases）介导的载脂蛋白B（ApoB）和/或载脂蛋白E（ApoE）基因敲除Huh7细胞，证实载脂蛋白中的两亲性α螺旋结构参与HCV颗粒的形成过程。尽管单独敲除ApoB或ApoE基因的Huh7细胞仅出现颗粒形成轻度下降，但同时敲除ApoB与ApoE基因的Huh7双敲除（DKO）细胞则严重受损感染性HCV颗粒的形成能力，提示ApoB与ApoE在感染性HCV颗粒形成中存在功能冗余。cDNA微阵列（cDNA microarray）分析显示，相较于人类肝组织中高表达的所有可交换载脂蛋白（包括ApoA1、ApoA2、ApoC1、ApoC2与ApoC3），Huh7细胞中主要表达ApoB与ApoE。在外源表达实验中，不仅ApoE，其他可交换载脂蛋白均可挽救DKO细胞中HCV感染性颗粒的形成能力。此外，上述载脂蛋白的表达还可促进基因型1b与3a嵌合病毒的感染性颗粒形成。进一步研究发现，可交换载脂蛋白中的两亲性α螺旋结构可通过与病毒颗粒相互作用，挽救DKO细胞中的颗粒形成过程。本研究结果表明，可交换载脂蛋白中的两亲性α螺旋结构在HCV感染性颗粒形成中发挥关键作用，为阐明HCV的生命周期以及开发靶向病毒组装的新型抗HCV治疗手段提供了理论依据。