Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.
Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.
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DOI：
10.1371/journal.ppat.1003589
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Suzuki T
中科院分区：
文献类型：
--
作者：
Suzuki R;Matsuda M;Watashi K;Aizaki H;Matsuura Y;Wakita T;Suzuki T
丙型肝炎病毒 (HCV) 非结构蛋白 2 (NS2) 是一种疏水性跨膜蛋白，不仅是 NS2-NS3 裂解所必需的，而且也是感染性病毒产生所必需的。为了鉴定与 NS2 相互作用并对 HCV 传播重要的细胞因子，我们使用全长 NS2 作为诱饵，通过分裂泛素膜酵母双杂交测定筛选人肝脏 cDNA 文库，并鉴定了信号肽酶复合物亚基 1 (SPCS1)，它是微粒体信号肽酶复合物的组成部分。内源性SPCS1的沉默导致传染性HCV的产生显着减少，而结构蛋白的加工、细胞进入、RNA复制以及病毒从细胞中的释放都没有受到损害。日本脑炎病毒的传播不受 SPCS1 敲除的影响，表明 SPCS1 并未广泛调节黄病毒科的病毒生命周期。 SPCS1被发现与NS2和E2相互作用。免疫共沉淀试验证明，细胞中形成了 NS2、E2 和 SPCS1 的复合物。 SPCS1 的敲低削弱了 NS2 与 E2 的相互作用。我们的研究结果表明，SPCS1 通过与 NS2 和 E2 相互作用，在膜相关 NS2-E2 复合物的形成中发挥关键作用，从而导致结构蛋白和非结构蛋白之间的协调相互作用，并促进感染性颗粒组装的早期步骤。病毒劫持宿主细胞并利用宿主衍生的蛋白质进行病毒传播。就丙型肝炎病毒 (HCV) 而言，已鉴定出许多基因组复制所需的宿主因子；然而，对于与 HCV 蛋白相互作用且对病毒组装过程很重要的细胞蛋白知之甚少。非结构蛋白 2 (NS2) 的 C 端一半和 NS3 的 N 端三分之一形成 NS2-3 蛋白酶，可裂解 NS2/3 连接。 NS2 在独立于蛋白酶活性的病毒组装过程中也发挥着关键作用。我们进行了分裂泛素酵母双杂交筛选，并鉴定了信号肽酶复合物亚基 1 (SPCS1)，它是微粒体信号肽酶复合物的一个亚基。在这项研究中，我们提供的证据表明 SPCS1 与 NS2 和 E2 相互作用，导致 E2-SPCS1-NS2 复合物形成，并且在感染性 HCV 颗粒的组装中具有关键作用。据我们所知，SPCS1 是第一个参与 HCV 生命周期调节的 NS2 相互作用细胞因子。
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles. Viruses hijack host cells and utilize host-derived proteins for viral propagation. In the case of hepatitis C virus (HCV), many host factors have been identified that are required for genome replication; however, only a little is known about cellular proteins that interact with HCV proteins and are important for the viral assembly process. The C-terminal half of nonstructural protein 2 (NS2), and the N-terminal third of NS3, form the NS2-3 protease that cleaves the NS2/3 junction. NS2 also plays a key role in the viral assembly process independently of the protease activity. We performed split-ubiquitin yeast two-hybrid screening and identified signal peptidase complex subunit 1 (SPCS1), which is a subunit of the microsomal signal peptidase complex. In this study, we provide evidence that SPCS1 interacts with both NS2 and E2, resulting in E2-SPCS1-NS2 complex formation, and has a critical role in the assembly of infectious HCV particles. To our knowledge, SPCS1 is the first NS2-interacting cellular factor that is involved in regulation of the HCV lifecycle.
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