NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles.
NS5A domain I antagonises PKR to facilitate the assembly of infectious hepatitis C virus particles.
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DOI：
10.1371/journal.ppat.1010812
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发表时间：
2023-02
期刊：
影响因子：
6.7
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中科院分区：
文献类型：
--
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丙型肝炎病毒NS5A是由三个结构域(DI、DII和DIII)组成的多功能磷蛋白。DI和DII已被证明在基因组复制中起作用，而DIII在病毒组装中起作用。我们以前证明了2a型DI(JFH1)也在病毒组装中发挥作用，例如P145A突变体阻止了传染性病毒的产生。在这里，我们将这一分析扩展到P145(C142和E191)附近的另外两个保守的和表面暴露的残基，它们在基因组复制中没有缺陷，但削弱了病毒的生产。进一步分析发现，与野生型相比，感染这些突变体的细胞中dsRNA的丰度、脂滴(LD)的大小和分布以及NS5A和LDS之间的共存发生了变化。同时，为了研究DI的这种作用的机制(S)，我们评估了干扰素诱导的双链RNA依赖的蛋白激酶的参与。在PKR沉默的细胞中，C142A和E191A显示出与野生型难以区分的感染性病毒产量、LD大小和NS5A与LD之间的共定位。免疫共沉淀和体外下拉实验证实野生型NS5A结构域I(而不是C142A或E191A)与PKR相互作用。我们进一步证明，通过去除PKR下游效应因子干扰素调节因子-1(IRF1)，C142A和E191A的组装表型得以恢复。这些数据表明，NS5A DI和PKR之间存在一种新的相互作用，这种作用可以逃避通过IRF1阻止病毒组装的抗病毒途径。丙型肝炎病毒的非结构5A蛋白(NS5A)在病毒基因组复制和感染性病毒颗粒的组装中起着至关重要的作用。NS5A是有效和高效的直接作用抗病毒药物的靶点，广泛用于丙型肝炎病毒的治疗。NS5A由3个结构域组成。在这里，我们证明了N-末端结构域I(DI)在病毒组装中发挥作用。DI中的突变既能阻止病毒组装，又能阻止作为丙型肝炎病毒感染标志的脂滴形态的扰动。值得注意的是，这种表型被细胞胞质双链RNA传感器PKR的沉默所废除，PKR是一个关键的抗病毒因子。因此，这些突变揭示了一种迄今尚未确定的由PKR控制的抗病毒途径，该途径具有阻止传染性病毒颗粒组装的功能。
Hepatitis C virus NS5A is a multifunctional phosphoprotein comprised of three domains (DI, DII and DIII). DI and DII have been shown to function in genome replication, whereas DIII has a role in virus assembly. We previously demonstrated that DI in genotype 2a (JFH1) also plays a role in virus assembly, exemplified by the P145A mutant which blocked infectious virus production. Here we extend this analysis to identify two other conserved and surface exposed residues proximal to P145 (C142 and E191) that exhibited no defect in genome replication but impaired virus production. Further analysis revealed changes in the abundance of dsRNA, the size and distribution of lipid droplets (LD) and the co-localisation between NS5A and LDs in cells infected with these mutants, compared to wildtype. In parallel, to investigate the mechanism(s) underpinning this role of DI, we assessed the involvement of the interferon-induced double-stranded RNA-dependent protein kinase (PKR). In PKR-silenced cells, C142A and E191A exhibited levels of infectious virus production, LD size and co-localisation between NS5A and LD that were indistinguishable from wildtype. Co-immunoprecipitation and in vitro pulldown experiments confirmed that wildtype NS5A domain I (but not C142A or E191A) interacted with PKR. We further showed that the assembly phenotype of C142A and E191A was restored by ablation of interferon regulatory factor-1 (IRF1), a downstream effector of PKR. These data suggest a novel interaction between NS5A DI and PKR that functions to evade an antiviral pathway that blocks virus assembly through IRF1. The non-structural 5A protein (NS5A) of hepatitis C virus (HCV) plays a critical role in both virus genome replication and the assembly of infectious virus particles. NS5A is a target for potent and highly efficacious direct acting antivirals used extensively for HCV treatment. NS5A comprises 3 domains. Here, we show that the N-terminal domain I (DI) plays a role in virus assembly. Mutations in DI block both virus assembly and the perturbation of lipid droplet morphology that is a hallmark of HCV infection. Strikingly, this phenotype is abrogated by silencing of the cellular cytoplasmic double-stranded RNA sensor, PKR, a key antiviral factor. These mutations have therefore uncovered a hitherto uncharacterised antiviral pathway controlled by PKR which functions to block assembly of infectious virus particles.
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