Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites
Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites
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DOI：
10.1053/j.gastro.2016.11.047
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发表时间：
2017-03-01
期刊：
影响因子：
29.4
通讯作者：
Cosset, Francois-Loic
中科院分区：
文献类型：
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作者：
Boson, Bertrand;Denolly, Solene;Cosset, Francois-Loic
背景和目的：Daclatasvir 是一种直接作用的抗病毒剂和 NS5A 的有效抑制剂，NS5A 可能通过膜网成形参与丙型肝炎病毒 (HCV) 基因组的复制，并可能通过 HCV RNA 基因组转移到病毒颗粒组装位点参与新病毒体的组装。达卡他韦抑制新膜网结构的形成，并最终抑制复制复合体囊泡的形成，但也抑制早期组装步骤。我们研究了达卡他韦诱导的 HCV 蛋白聚集、病毒 RNA 的细胞内定位和病毒颗粒组装抑制之间的关系。方法：在达卡他韦存在下短时间从 Huh7.5 肝癌细胞中产生细胞培养物衍生的 HCV 颗粒。对物理颗粒的感染性和产生进行定量，并对生产细胞进行亚细胞分级分离。通过共聚焦显微镜和结构照明显微镜定量分析核心、E2、NS5A、NS4B 蛋白和病毒 RNA 之间的细胞内共定位。结果：HCV 感染细胞短暂暴露于达卡他韦可减少病毒组装，并诱导结构蛋白与非结构 HCV 蛋白聚集，包括核心、E2、NS4B 和 NS5A。这些簇状结构似乎是不活跃的组装平台，可能是由于与复制复合体失去了功能连接。 Daclatasvir 大大减少了病毒基因组向这些核心簇的传递，而不改变 HCV RNA 与 NS5A 的共定位。相比之下，达卡他韦既不诱导簇状结构，也不抑制感染达卡他韦抗性突变体（NS5A-Y93H）的细胞中的HCV组装，这表明达卡他韦的目标是抑制这两个过程的NS5A的共同的、特定的功能。结论：除了抑制复制复合物的生物发生外，达拉他韦还通过阻止病毒基因组转移到组装位点来防止病毒组装。这导致 HCV 蛋白聚集，因为病毒颗粒和复制复合体囊泡无法形成或流出。达卡他韦的这种双重作用模式可以解释其在阻断培养细胞中 HCV 复制和治疗 HCV 感染患者方面的功效。
BACKGROUND & AIMS: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. METHODS: Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. RESULTS: Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. CONCLUSIONS: In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.