PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication.
PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication.
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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。
DOI：
10.1186/s12985-022-01848-5
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发表时间：
2022-07-14
期刊：
影响因子：
4.8
通讯作者：
Zhou, Yuanping
中科院分区：
文献类型：
--
作者：
Anwar, Muhammad Ikram;Li, Ni;Zhou, Qing;Chen, Mingxiao;Hu, Chengguang;Wu, Tao;Chen, Haihang;Li, Yi-Ping;Zhou, Yuanping
丙型肝炎病毒（HCV）感染增加肝细胞癌的风险。确定HCV感染所需的宿主因子将有助于揭示HCV的发病机制。能够复制HCV感染克隆的适应性突变可能提示携带突变的病毒蛋白可能特异性地与HCV生命周期所必需的一些细胞因子相互作用。之前，我们发现HCV NS5B （RNA依赖性RNA聚合酶）中的D559G突变对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。HCV基因型2a克隆J6cc的野生型ns5b和D559G-NS5B在肝癌Huh7.5细胞中异位表达，并提取ns5b结合蛋白，用质谱法鉴定。通过基因敲除或敲低、互补、共免疫沉淀（Co-IP）、共定位、病毒感染与复制、酶活性等实验，探讨所选细胞蛋白对HCV感染的必要性和作用方式。质谱鉴定出许多细胞蛋白，其中蛋白磷酸酶2调节亚基B 'delta （PPP2R5D， PP2A调节B亚基）是d559g - ns5b拉下的蛋白之一，并被选中进行进一步的研究。Co-IP证实PPP2R5D与HCV NS5B特异性相互作用，但不与HCV Core和NS3蛋白相互作用，D559G略微增强了相互作用。NS5B也在内质网中与PPP2R5D共定位。敲低PPP2R5D和敲除PPP2R5D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的短暂和稳定表达使HCV感染恢复到接近野生型Huh7.5细胞的水平。复制子实验显示PPP2R5D促进HCV复制，但PP2A的磷酸酶活性和催化亚基不受NS5B的影响。PPP2R5D与HCV NS5B相互作用，通过促进HCV复制，是培养肝癌细胞感染HCV所必需的。
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. 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. 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. PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.
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影响因子：
5
作者：
Ranieri A;Kemp E;Burgoyne JR;Avkiran M
通讯作者：
Avkiran M
DOI：
10.1080/07391102.2018.1491419
发表时间：
2019-06-13
影响因子：
4.4
作者：
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作者：
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影响因子：
4.8
作者：
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通讯作者：
Moradpour, D
影响因子：
5.4
作者：
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通讯作者：
Lai, MMC