*International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, 110 067 New Delhi, India.
†Institute of Liver and Biliary Sciences (ILBS), D-1, Vasant Kunj, New Delhi, India.
Biochem J. 2014 Sep 1;462(2):347-58. doi: 10.1042/BJ20140316.
The host-mediated RNAi pathways restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show in the present study that the siRNA-mediated silencing of Drosha, Dicer and Ago2 [argonaute RISC (RNA-induced silencing complex) catalytic component 2] transcripts in Huh7 cells resulted in elevated levels of HBV (hepatitis B virus)-specific RNAs and, conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in CHB (chronic hepatitis B) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RSS (RNA-silencing suppressor) properties. Using GFP-based reversion of silencing assays, in the present study we found that HBx is an RSS protein. Through a series of deletions and substitution mutants, we found that the full-length HBx protein is required for optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer-mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defence, in particular by abrogating the function of Dicer. The present study may have implications in the development of newer strategies to combat HBV infection.
宿主介导的 RNAi 途径限制了植物、无脊椎动物和脊椎动物系统中病毒的复制。然而，人们对 RNAi 与哺乳动物宿主中各种病毒感染之间的相互作用知之甚少。本研究表明，在 Huh7 细胞中， Drosha、Dicer 和 Ago2[Argonaute RISC（RNA 诱导沉默复合物）催化成分 2]转录物的 siRNA 介导沉默导致 HBV（乙型肝炎病毒）特异性 RNA 的水平升高，而相反，我们观察到 HepG2 细胞中感染 HBV 后相同 RNAi 成分的 mRNA 和蛋白质水平降低。在 CHB（慢性乙型肝炎）患者中也可以检测到类似的降低。对具有高血清 HBV DNA 载量（>log108 IU/ml）的 CHB 肝活检样本进行分析，揭示了 Drosha、Dicer 和 Ago2 的 mRNA 和蛋白质水平降低。CHB 患者样本中关键 RNAi 途径成分的低表达水平以及肝细胞建立了 HBV 复制与 RNAi 成分之间的联系。还检查了 HBV 蛋白是否具有 RSS（RNA 沉默抑制子）特性。在本研究中，我们使用 GFP 为基础的沉默逆转测定发现 HBx 是一种 RSS 蛋白。通过一系列缺失和取代突变，我们发现全长 HBx 蛋白是 RSS 活性的最佳需要。体外切割测定表明，HBx 蛋白抑制了人 Dicer 介导的 dsRNA 加工成 siRNA。总之，我们的研究结果表明，HBx 蛋白可能作为 RSS 来操纵宿主 RNAi 防御，特别是通过削弱 Dicer 的功能。本研究可能对开发针对 HBV 感染的新策略具有重要意义。
