Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.
Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.
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锤头核酶对丙型肝炎病毒 RNA 进行细胞内切割并抑制病毒蛋白翻译。
DOI：
10.1172/jci119097
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Wu,GY
中科院分区：
文献类型：
--
作者：
Sakamoto,N;Wu,CH;Wu,GY
为研究抗丙型肝炎病毒(HCVRNA)锤头状核酶对病毒蛋白翻译的影响，构建了含有丙型肝炎病毒5‘-非编码区(5’-NCR)和部分核心区的荧光素酶报告基因载体pCMV/T7-NCRCDelta-Luc。设计了4个核酶RZ1-RZ4，分别在136-160、313-337、496-520和373-388位核苷酸上切割。在无细胞条件下，每个核酶在预期的位置切割目标RNA。RZ2和RZ4显著抑制NCRC Delta-Luc RNA的翻译，分别为71%和49%。没有病毒成分的对照荧光素酶的翻译不受核酶的影响。此外，当NCRCDelta-Luc RNA和核酶共转染细胞时，RZ2和RZ4分别显著抑制了73%和56%的表达。相反，锤头结构域点突变的切割缺陷核酶没有显著影响。为了确定内源性核酶的作用，构建了RZ2和RZ4的真核表达载体。与CMV/T7-NCRC-Delta-Luc共转染后，荧光素酶活性分别被抑制50%和61%。此外，将pCMV/T7-NCRCDelta-Luc转入稳定的RZ2和RZ4产生细胞，也显示了荧光素酶活性的显著抑制。针对丙型肝炎病毒基因组的核酶可以在细胞内条件下实质上和特异性地抑制病毒基因的表达。
To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.