Sakamoto N, Wu C H, Wu G Y
Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut School of Medicine, Farmington 06030, USA.
J Clin Invest. 1996 Dec 15;98(12):2720-8. doi: 10.1172/JCI119097.
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.
为了确定锤头状核酶对丙型肝炎病毒（HCV）RNA的作用对病毒蛋白翻译的影响，构建了一个荧光素酶报告基因载体pCMV/T7-NCRCdelta-luc，其包含HCV的5'-非编码区（5'-NCR）和部分核心区。设计了四种核酶Rz1-Rz4，分别在核苷酸位置136-160、313-337、496-520和373-388处进行切割。在无细胞条件下，每种核酶均在预期位置切割靶RNA。Rz2和Rz4分别显著抑制NCRCdelta-luc RNA的翻译达71%和49%。缺乏病毒元件的对照荧光素酶mRNA的翻译不受核酶影响。此外，当将NCRCdelta-luc RNA和核酶共转染到细胞中时，Rz2和Rz4分别显著抑制表达达73%和56%。相比之下，在锤头结构域有一个点突变的切割缺陷型核酶没有显著作用。为了确定内源性产生的核酶的作用，构建了Rz2和Rz4的真核表达载体。将这些载体与CMV/T7-NCRCdelta-luc共转染显示荧光素酶活性分别抑制到50%和61%。此外，将pCMV/T7-NCRCdelta-luc转染到稳定的Rz2和Rz4产生细胞中也显示出对荧光素酶活性的显著抑制。针对HCV基因组的核酶在细胞内条件下可显著且特异性地抑制病毒基因表达。
