Haddad Juliano G, Rouillé Yves, Hanoulle Xavier, Descamps Véronique, Hamze Monzer, Dabboussi Fouad, Baumert Thomas F, Duverlie Gilles, Lavie Muriel, Dubuisson Jean
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019, UMR 8204, Centre d'Infection et d'Immunité de Lille, Lille, France.
Laboratoire Microbiologie Santé et Environnement, Ecole Doctorale en Sciences et Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Liban.
J Virol. 2017 Mar 29;91(8). doi: 10.1128/JVI.00048-17. Print 2017 Apr 15.
Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis. Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.
丙型肝炎病毒（HCV）包膜糖蛋白复合物由E1和E2亚基组成。E2是受体结合蛋白，也是中和抗体的主要靶标，而E1的功能仍不清楚。在此，我们利用最近公布的E1胞外域N端区域的结构，通过在感染性克隆背景下对这个79个氨基酸区域内的残基进行突变，来探究这种糖蛋白的功能。对突变体的表型进行了表征，以确定突变对病毒进入、复制和组装的影响。此外，还采用生化方法来表征E1E2异二聚体的折叠和组装。19个突变中有13个导致病毒减毒或失活。有趣的是, 两个减毒突变体T213A和I262A在Huh-7细胞中进入细胞时对紧密连接蛋白-1的依赖性较低。相反，这些病毒依赖紧密连接蛋白-6，这表明这两个突变体在靶细胞系中受体依赖性发生了转变。还观察到突变体D263A出现了意外的表型, 它不再具有感染性，但仍显示出较高水平的核心蛋白分泌。此外，这些非感染性病毒颗粒中没有基因组RNA, 这表明D263A突变导致了缺乏基因组RNA的病毒颗粒的组装和释放。最后，在D263A突变体中观察到HCV RNA和E1之间亚细胞共定位的变化。这一独特的观察结果首次突出了HCV形态发生过程中HCV糖蛋白E1与基因组RNA之间的相互作用。丙型肝炎病毒（HCV）感染是全球主要的公共卫生问题。它编码两种包膜蛋白E1和E2，它们在该病毒的生命周期中起主要作用。E2已得到广泛表征，而E1仍了解甚少。在此，我们在病毒生命周期背景下利用定点诱变研究E1的功能。我们的结果确定了独特的表型。出乎意料的是，两个突变体在细胞进入时明显显示出受体依赖性的转变，突出了E1在调节HCV颗粒与细胞受体相互作用中的作用。更重要的是，另一个突变体导致了缺乏基因组RNA的病毒颗粒的组装和释放。对这一独特的表型进行了进一步表征，并且我们观察到HCV RNA和E1之间亚细胞共定位的变化。这一独特的观察结果首次突出了形态发生过程中病毒包膜蛋白与基因组RNA之间的相互作用。
