Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B
Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B
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DOI：
10.1111/1348-0421.12278
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发表时间：
2015-08
影响因子：
2.6
通讯作者：
Imelda Rosalyn Sianipar;Chieko Matsui;N. Minami;X. Gan;Lin Deng;H. Hotta;I. Shoji
中科院分区：
文献类型：
--
作者：
Imelda Rosalyn Sianipar;Chieko Matsui;N. Minami;X. Gan;Lin Deng;H. Hotta;I. Shoji
丙型肝炎病毒（HCV）NS 5A蛋白在病毒RNA复制、病毒组装和病毒致病中起着重要作用。虽然NS 5A没有已知的酶活性，但它通过与细胞蛋白相互作用来调节各种细胞途径。据报道，HCV NS 5A（和其他HCV蛋白）通过泛素-蛋白酶体途径降解;然而，HCV感染中泛素化和去泛素化的生理作用在很大程度上是未知的。为了阐明去泛素化在HCV感染中的作用，我们试图鉴定一种能与NS 5A蛋白相互作用的去泛素化酶（DUB）。卵巢肿瘤蛋白（OTU），去泛素化酶7 B（OTUD 7 B），被鉴定为一种新的NS 5A结合蛋白。免疫共沉淀分析表明，NS 5A与Huh-7和HCV RNA复制子细胞中的OTUD 7 B相互作用。免疫荧光染色显示HCV NS 5A蛋白与OTUD 7 B共定位于细胞质中。此外，发现HCV感染增强OTUD 7 B的核定位。使用一系列NS 5A缺失突变体定位NS 5A上的OTUD 7 B结合结构域。本研究结果表明，NS 5A的结构域I是重要的，从NS 5A的氨基酸121至126的区域是必要的相互作用。NS 5A中的V121 A或V124 A突变破坏NS 5A-OTUD 7 B相互作用。该体内泛素化测定的结果表明HCV NS 5A增强OTUD 7 B DUB活性。总之，这些结果表明HCV NS 5A蛋白与OTUD 7 B相互作用，从而调节其DUB活性。
Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.