Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Paris, France.
CNRS UMR 3569, Paris, France.
PLoS Pathog. 2018 Feb 7;14(2):e1006863. doi: 10.1371/journal.ppat.1006863. eCollection 2018 Feb.
Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle.
近年来，在多种哺乳动物物种中发现了几种与丙型肝炎病毒 (HCV) 具有不同程度遗传相关性的同源物。HCV 的感染性生命周期依赖于其单一多蛋白的第一个关键蛋白水解事件，该事件由非结构蛋白 2 (NS2) 完成，允许复制酶组装和基因组复制。在这项研究中，我们对 HCV 和动物肝炎病毒 NS2 的蛋白水解作用模式和调节机制的保守性进行了特征描述和评估。我们首先证明，来自马、蝙蝠、啮齿动物、新和旧世界灵长类动物肝炎病毒的 NS2 也是半胱氨酸蛋白酶。使用标记的病毒蛋白前体和催化三联体突变体，我们证明了来自马 NPHV 和灵长类 GBV-B 的 NS2 分别作为与 HCV 最密切和最不相关的病毒，像 HCV NS2 一样作为二聚体蛋白酶，具有两个复合活性位点。与 NS3 N 端结构域 (NS3N) 通过 NS3N 关键疏水性表面斑块作为 HCV NS2 蛋白酶辅助因子的报道的重要作用一致，我们通过功能获得/缺失突变研究表明，一些异源肝炎病毒 NS3N 可能作为 HCV NS2 的辅助因子，只要 HCV 样疏水性残基保守。然而，史无前例的是，我们还观察到在所有研究的肝炎病毒中，通过灵活的接头在 C 端标记融合物的背景下，在没有 NS3 部分的情况下，NS2 蛋白酶具有有效的内在蛋白水解活性，以及在 HCV 双顺反子全长基因组的背景下。这些发现表明，NS3N 作为调节因子而不是肝炎病毒 NS2 蛋白酶的必需辅助因子发挥作用。总体而言，NS2 的独特特征，包括作为具有两个复合活性位点的二聚体的酶功能以及额外的 NS3 独立的蛋白水解活性，在肝炎病毒中是保守的，无论它们的遗传距离如何，这突出了它们在肝炎病毒生命周期中的功能意义。
