Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA.
Department of Internal Medicine I, Goethe University Hospital, Frankfurt/Main, Germany.
J Virol. 2019 Dec 12;94(1). doi: 10.1128/JVI.00906-19.
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly. Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.
丙型肝炎病毒（HCV）非结构蛋白 2（NS2）是一种多功能蛋白，涉及 HCV RNA 复制和病毒颗粒组装。NS2 编码的半胱氨酸蛋白酶负责 NS2-NS3 前体的自体加工，这是 HCV RNA 复制的一个重要步骤。NS2 还通过将包膜蛋白 2（E2）募集到位于去污剂抗性膜（DRM）上的病毒组装位点，促进 HCV 颗粒组装。然而，调节 NS2 多种功能的基本机制尚不清楚。在这项研究中，我们发现当 NS2 自身在细胞中表达和感染性 HCV 复制时，其第 113 位半胱氨酸残基（NS2/C113）被棕榈酰化。通过引入 NS2/C113S 突变来阻断 NS2 棕榈酰化，降低了 NS2-NS3 自体加工，并损害了 HCV RNA 复制。通过在 NS2 和 NS3 之间插入脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES），将 NS2/C113S 突变体的复制分离出来，使两种蛋白质独立于 NS2 介导的自体加工，从而恢复了复制。这些结果表明，NS2 棕榈酰化通过促进 NS2-NS3 自体加工，对 HCV RNA 复制至关重要。NS2/C113S 突变也损害了感染性 HCV 的组装、NS2 和 E2 在 DRM 上的定位以及 NS2 与 Core 和内质网脂筏相关蛋白 2（Erlin-2）的共定位。总之，我们的研究表明，涉及 HCV RNA 复制和病毒组装的 NS2 的两个主要功能，即 NS2-NS3 自体加工和 E2 募集到 DRM，受 NS2/C113 的棕榈酰化调节。由于 S-棕榈酰化是可逆的，因此 NS2 棕榈酰化可能使 NS2 能够微调 HCV RNA 复制和感染性颗粒组装。慢性丙型肝炎病毒（HCV）感染是导致每年近 40 万人死亡的严重肝脏疾病的主要原因。HCV NS2 蛋白是一种多功能 HCV 复制调节剂，参与病毒基因组复制和感染性病毒组装。然而，使该蛋白能够参与 HCV 复制的多个步骤的潜在机制尚不清楚。在这项研究中，我们发现 NS2 棕榈酰化是其多种功能的主要调节因子，包括 NS2 介导的自身切割和 HCV 包膜蛋白募集到病毒组装位点，进而分别促进 HCV RNA 复制和感染性颗粒组装。这一新发现表明，NS2 棕榈酰化可能成为抑制 HCV RNA 复制和病毒组装的有希望的靶点，为针对 HCV 感染的宿主靶向策略提供了新的途径。
