Homann M, Tzortzakaki S, Rittner K, Sczakiel G, Tabler M
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Nucleic Acids Res. 1993 Jun 25;21(12):2809-14. doi: 10.1093/nar/21.12.2809.
The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1.
将锤头状核酶的催化结构域整合到一段413个核苷酸长的反义RNA中，该反义RNA靶向人类免疫缺陷病毒1型（HIV-1）的5'-前导序列/ gag区域（位置+222至+634）。结果表明，所得的催化性反义RNA在生理离子强度和温度下能在体外特异性切割其靶RNA。我们将这种催化性反义RNA的抗病毒效果与相应的未修饰反义RNA以及一种在体外不能切割底物RNA的突变催化性反义RNA进行了比较。将这些RNA中的每一种与感染性完整前病毒HIV-1 DNA一起共转染到人SW480细胞中，随后分析HIV-1复制情况。与亲本反义RNA和无活性突变体相比，催化活性结构域的存在导致对HIV-1复制的抑制作用增强了4至7倍。体外进行的动力学和结构研究表明，催化性反义RNA与亲本反义RNA相比，形成双链的能力没有改变。总之，这些数据表明，切割靶RNA的能力是观察到的HIV-1复制抑制作用增强的关键先决条件。
