Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Antiviral Res. 2010 Mar;85(3):541-50. doi: 10.1016/j.antiviral.2010.01.005. Epub 2010 Jan 25.
Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-beta and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%).
戊型肝炎病毒（HEV）是一种无包膜、单链、正链 RNA 病毒，是水传播性肝炎的主要病因。RNA 干扰（RNAi）是一种由双链 RNA 诱导的序列特异性细胞抗病毒防御机制，我们用它来研究几种基因和 HEV 的 3' 顺式作用元件（CAE）的敲低。在本报告中，针对 HEV 的假定解旋酶和复制酶结构域以及 3'CAE 区域开发了 shRNA。通过 northern 杂交证实了 siRNA 的产生。通过干扰素-β和 2'，5'-寡聚腺苷酸合成酶基因的转录分析验证了由于 shRNA 表达可能引起的先天反应诱导，发现不存在。最初，使用抑制融合病毒亚基因组靶标域-荧光素酶报告基因构建体，针对各自的基因/3'CAE 测试所选 shRNA 的效率。使用 HEV 复制子和报告复制子研究有效的 shRNA 对 HepG2 细胞中 HEV 复制的抑制作用。通过亚基因组靶标-报告构建体和报告复制子中荧光素酶活性的降低证明了 RNAi 介导的沉默。实时 PCR 用于证明转染细胞中天然复制子复制的抑制。设计的 shRNA 被发现能够在不同程度上（45-93%）有效地抑制病毒复制。
