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PPP2R5D 通过与病毒 NS5B 结合并增强病毒 RNA 复制来促进丙型肝炎病毒感染。

PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication.

机构信息

Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology and Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, China.

Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou, 510080, China.

出版信息

Virol J. 2022 Jul 14;19(1):118. doi: 10.1186/s12985-022-01848-5.

DOI:10.1186/s12985-022-01848-5

PMID:35836293

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9284890/

Abstract

BACKGROUND

Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection.

METHODS

Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. RESULTS: Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B'delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B.

CONCLUSIONS

PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

摘要

背景

丙型肝炎病毒（HCV）感染增加了肝细胞癌的风险。鉴定 HCV 感染所需的宿主因素将有助于揭示 HCV 发病机制。使 HCV 感染性克隆复制的适应性突变可能提供线索，表明携带突变的病毒蛋白可能与 HCV 生命周期中某些必需的细胞因子特异性相互作用。此前，我们鉴定了 HCV NS5B（RNA 依赖的 RNA 聚合酶）中的 D559G 突变，该突变对于不同基因型克隆的复制很重要。在这里，我们寻找可能与 NS5B 相互作用的因素，并研究其在 HCV 感染中的作用。

方法

将 HCV 2a 型克隆 J6cc 的野生型 NS5B 和 D559G-NS5B 异位表达在肝癌 Huh7.5 细胞中，并用质谱法拉下并鉴定 NS5B 结合蛋白。通过基因敲除或敲低、互补、共免疫沉淀（Co-IP）、共定位、病毒感染和复制以及酶活性等实验，探讨了所选细胞蛋白对 HCV 感染的必要性和作用模式。

结果

质谱法鉴定了许多细胞蛋白，其中蛋白磷酸酶 2 调节亚基 B'delta（PPP2R5D，即 PP2A 调节 B 亚基）是 D559G-NS5B 拉下的蛋白之一，并被选为进一步研究。Co-IP 证实 PPP2R5D 特异性与 HCV NS5B 相互作用，但不与 HCV Core 和 NS3 蛋白相互作用，而 D559G 则略微增强了相互作用。NS5B 也与 PPP2R5D 在内质网中共定位。PPP2R5D 的敲低和敲除分别降低和消除了 Huh7.5 细胞中的 HCV 感染，而 PPP2R5D 敲除细胞中 PPP2R5D 的瞬时和稳定表达将 HCV 感染恢复到接近野生型 Huh7.5 细胞的水平。复制子测定显示 PPP2R5D 促进了 HCV 复制，但 NS5B 不影响 PP2A 的磷酸酶活性和催化亚基。