Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Nucleic Acids Res. 2019 Jul 9;47(12):6411-6424. doi: 10.1093/nar/gkz278.
The liver-specific microRNA, miR-122, is an essential host factor for replication of the hepatitis C virus (HCV). miR-122 stabilizes the positive-strand HCV RNA genome and promotes its synthesis by binding two sites (S1 and S2) near its 5' end in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using an unbiased quantitative proteomics screen, we identified the TNRC6 protein paralogs, TNRC6B and TNRC6C, as functionally important but redundant components of the miR-122/Ago2 host factor complex. Doubly depleting TNRC6B and TNRC6C proteins reduced HCV replication in human hepatoma cells, dampening miR-122 stimulation of viral RNA synthesis without reducing the stability or translational activity of the viral RNA. TNRC6B/C were required for optimal miR-122 host factor activity only when S1 was able to bind miR-122, and restricted replication when S1 was mutated and only S2 bound by miR-122. TNRC6B/C preferentially associated with S1, and TNRC6B/C depletion enhanced Ago2 association at S2. Collectively, these data suggest a model in which TNRC6B/C regulate the assembly of miR-122/Ago complexes on HCV RNA, preferentially directing miR-122/Ago2 to S1 while restricting its association with S2, thereby fine-tuning the spatial organization of miR-122/Ago2 complexes on the viral genome.
肝脏特异性 microRNA，miR-122，是丙型肝炎病毒 (HCV) 复制的必需宿主因子。miR-122 稳定正链 HCV RNA 基因组，并通过与 Ago2 结合在其 5' 端附近的两个位点 (S1 和 S2) 促进其合成。Ago2 对于宿主因子的这两种活性都是必需的，但是否涉及其他宿主蛋白尚不清楚。使用无偏定量蛋白质组学筛选，我们鉴定了 TNRC6 蛋白的同源物 TNRC6B 和 TNRC6C，它们是 miR-122/Ago2 宿主因子复合物的功能重要但冗余的组成部分。TNRC6B 和 TNRC6C 蛋白的双重缺失降低了人肝癌细胞中的 HCV 复制，抑制了 miR-122 对病毒 RNA 合成的刺激，而不降低病毒 RNA 的稳定性或翻译活性。只有当 S1 能够与 miR-122 结合时，TNRC6B/C 才需要最佳的 miR-122 宿主因子活性，并且当 S1 发生突变且仅 S2 被 miR-122 结合时，TNRC6B/C 会限制复制。TNRC6B/C 优先与 S1 结合，并且 TNRC6B/C 缺失增强了 Ago2 在 S2 处的结合。总之，这些数据表明了一个模型，即 TNRC6B/C 调节 miR-122/Ago 复合物在 HCV RNA 上的组装，优先将 miR-122/Ago2 引导至 S1，同时限制其与 S2 的结合，从而精细调节 miR-122/Ago2 复合物在病毒基因组上的空间组织。
