Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.
Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.
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载脂蛋白中的两亲性α-螺旋对于形成感染性丙型肝炎病毒颗粒至关重要。
DOI：
10.1371/journal.ppat.1004534
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发表时间：
2014-12
期刊：
影响因子：
6.7
通讯作者：
Matsuura Y
中科院分区：
文献类型：
--
作者：
Fukuhara T;Wada M;Nakamura S;Ono C;Shiokawa M;Yamamoto S;Motomura T;Okamoto T;Okuzaki D;Yamamoto M;Saito I;Wakita T;Koike K;Matsuura Y
载脂蛋白B（ApoB）和载脂蛋白E（ApoE）参与了丙型肝炎病毒（HCV）的颗粒形成和组织嗜性，但其确切作用尚不清楚。我们用锌指核酸酶介导的载脂蛋白B（ApoB）和/或ApoE基因敲除的Huh 7细胞研究了载脂蛋白中的两亲性α螺旋参与HCV颗粒的形成。尽管ApoB或ApoE基因缺陷的Huh 7细胞表现出轻微的颗粒形成减少，但Huh 7（DKO）细胞中ApoB和ApoE基因的敲除严重损害了感染性HCV颗粒的形成，表明ApoB和ApoE在感染性HCV颗粒的形成中具有冗余作用。cDNA微阵列分析显示，ApoB和ApoE主要在Huh 7细胞中表达，而在人肝组织中，所有可交换的载脂蛋白，包括ApoA 1、ApoA 2、ApoC 1、ApoC 2和ApoC 3均高水平表达。不仅ApoE，而且其他可交换的载脂蛋白的外源表达拯救了HCV在DKO细胞中的感染性颗粒形成。此外，这些载脂蛋白的表达促进了基因型1b和3a嵌合病毒感染性颗粒的形成。此外，可交换载脂蛋白中两亲性α-螺旋的表达通过与病毒颗粒的相互作用促进了DKO细胞中的颗粒形成。这些结果表明，可交换载脂蛋白中的两亲性α-螺旋在HCV感染性颗粒的形成中起着重要作用，并为理解HCV的生命周期和开发以病毒组装为靶点的新型抗HCV治疗药物提供了线索。 体外系统已被开发用于研究丙型肝炎病毒（HCV）感染，并揭示了HCV生命周期的许多细节。载脂蛋白B（ApoB）和ApoE在HCV颗粒形成中起关键作用，这是基于通过siRNA介导的基因敲低和蛋白质过表达获得的数据。然而，载脂蛋白在HCV组装中的确切作用尚未阐明。在这项研究中，我们表明，感染性颗粒形成的HCV在Huh 7细胞中严重受损的敲除载脂蛋白B和载脂蛋白E基因的人工核酸酶，这种减少被取消的表达不仅载脂蛋白E，但也可交换载脂蛋白，包括载脂蛋白A1，载脂蛋白A2，载脂蛋白C1，载脂蛋白C2和载脂蛋白C3。此外，可交换载脂蛋白中两亲性α-螺旋的表达通过与病毒颗粒的相互作用恢复了双敲除细胞中感染性颗粒的形成。这些结果为了解HCV的生命周期和开发新型抗HCV药物提供了线索。
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. In vitro systems have been developed for the study of hepatitis C virus (HCV) infection and have revealed many details of the life cycle of HCV. Apolipoprotein B (ApoB) and ApoE have been shown to play crucial roles in the particle formation of HCV, based on data obtained by siRNA-mediated gene knockdown and overexpression of the proteins. However, precise roles of the apolipoproteins in HCV assembly have not been elucidated yet. In this study, we show that infectious particle formation of HCV in Huh7 cells was severely impaired by the knockout of both ApoB and ApoE genes by artificial nucleases, and this reduction was cancelled by the expression of not only ApoE, but also other exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3. In addition, expression of amphipathic α-helices in the exchangeable apolipoproteins restored the infectious particle formation in the double-knockout cells through an interaction with viral particles. These results provide clues to the understanding of life cycle of HCV and the development of novel antivirals to HCV.
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DOI：
10.1016/j.bpg.2012.09.009
发表时间：
2012-08-01
影响因子：
3.2
作者：
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通讯作者：
Wedemeyer, Heiner
影响因子：
46.9
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影响因子：
5.4
作者：
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影响因子：
158.5
作者：
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