Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells
Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells
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DOI：
10.1515/bc.2001.077
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发表时间：
2001-04-01
影响因子：
3.7
通讯作者：
Qi, GR
中科院分区：
文献类型：
--
作者：
Feng, Y;Kong, YY;Qi, GR
乙型肝炎病毒（HBV）的异常多聚腺苷酸（poly(A)）信号不仅控制着所有病毒RNA 3'端的形成，而且对HBV复制至关重要。因此，针对HBVadr亚型多聚腺苷酸信号区域的一种顺式释放型锤头状核酶（RzA）的抗病毒效应得到了研究。在体外，RzA在其靶位点对HBV RNA的切割率高达70%，而在催化位点有一个碱基突变的失活核酶（dRzA）则完全不能切割靶RNA。当将这些核酶与含HBV基因组的质粒p3.611共转染到HepG2细胞中时，野生型核酶RzA能有效降低HBV RNA水平并抑制HBV复制，而其失活形式dRzA的作用则弱得多，这表明锤头状核酶的活性催化结构域能显著提高反义介导的抑制程度。此外，还存在一种效果梯度：释放的核酶量越高，细胞内靶HBV RNA的减少以及子代DNA的减少就越多。这些结果提示了锤头状核酶RzA用于HBV感染基因治疗的可能性。
The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3' end of all the viral RNA, but is also crucial for HBV replication. Hence, a cia-releasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid p3.611, the wild-type ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.