Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons
Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons
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DOI：
10.1111/j.1742-4658.2005.04986.x
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发表时间：
2005-11-01
期刊：
影响因子：
5.4
通讯作者：
Krüger, M
中科院分区：
文献类型：
--
作者：
Jarczak, D;Korf, M;Krüger, M
慢性丙型肝炎病毒（HCV）感染是一种临床上重要的肝脏疾病，在相当大比例的患者中治疗选择有限。因此，需要新的有效治疗剂。由于HCV的5 '-和3'-非翻译区（UTR）是高度保守的，并且对于HCV复制具有重要的功能，因此它们是RNA切割核酶或小干扰RNA（siRNA）的有吸引力的靶标。在这项研究中，在两种不同的RNA polIII启动子（tRNA（瓦尔），U6）的控制下，从逆转录病毒载体转录物表达靶向HCV 5 '-和3'-UTR序列的发夹核酶（Rz）。在单顺反子、亚基因组I389/hyg-ubi/NS 3 - 3 '/5.1 HCV复制子细胞中评估了核酶作为单一试剂或与针对HCV 5'-或3 '-UTR的SiRNA组合，最近证明可以抑制HCV复制子。另外，产生核酶构建体，其中核酶的3 '末端侧接组成型转运元件（CTE）序列，所述CTE序列是先前已显示增强锤头状核酶的切割活性的RNA基序。在我们的研究中，tRNA（瓦尔）以及U6启动子驱动的Rzs显着降低HCV复制子RNA表达和HCV内部核糖体进入位点（IRES）介导的HCV NS 5 B蛋白从单顺反子亚基因组复制子的翻译。然而，CTE序列与3 '-末端的连接没有显著增强本研究中测试的Rz的活性。有趣的是，我们检测到tRNA（瓦尔）驱动的Rzs和U6衍生的siRNA的组合对HCV的抑制作用，这表明核酶和siRNA的双重策略可能成为特异性沉默HCV RNA复制的强大分子工具。
Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5'- and 3'-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5'- and 3'-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNA(Val), U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3'/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5'- or 3'-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3'-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNA(Val) as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3'-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNA(Val)-driven Rzs and U6-derived siRNAs both directed against highly conserved 5'- and 3'-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.