Ma W P, Crouch R J
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Genes Cells. 1996 Jun;1(6):581-93. doi: 10.1046/j.1365-2443.1996.d01-265.x.
Reverse transcription, which converts an RNA genome into double-stranded DNA, requires both the polymerase and RNase H activities of reverse transcriptase (RT). In vitro, poorly processive RT dissociates from partially copied RNA-DNA hybrids, that are usually extended by a second RT molecule. Despite similar structures, RNase HI of Escherichia coli can degrade RNA-DNA hybrids that are resistant to RNase H of RT. E. coli RNase HI is used to determine the accessibility to and requirement for RNA-DNA hybrids in reverse transcription in vivo and in vitro.
In the presence of E. coli RNase HI, reverse transcription yields incomplete cDNA molecules due to degradation of RNA-DNA hybrids. Delivery of E. coli RNase HI to Ty1 particles via fusion to the capsid protein can reduce retrotransposition by more than 99%, also indicating inhibition of DNA synthesis in vivo.
Inhibition of both reverse transcription in vitro and retrotransposition in vivo by E. coli RNase HI indicates that the poor processivity of RT exposes RNA-DNA hybrids critical for reverse transcription to degradation. Targeting a cellular RNase H to HIV may help define the site(s) of RNA-DNA hybrids that are susceptible to nonretroviral RNase H and may be useful for gene therapy to inhibit retroviral replication.
逆转录是将RNA基因组转化为双链DNA的过程，这需要逆转录酶（RT）同时具备聚合酶和核糖核酸酶H活性。在体外，持续性差的RT会从部分复制的RNA-DNA杂交体上解离，这些杂交体通常由第二个RT分子延伸。尽管结构相似，但大肠杆菌的核糖核酸酶HI能降解对RT的核糖核酸酶H具有抗性的RNA-DNA杂交体。大肠杆菌核糖核酸酶HI用于确定体内和体外逆转录过程中RNA-DNA杂交体的可及性和需求。
在大肠杆菌核糖核酸酶HI存在的情况下，由于RNA-DNA杂交体的降解，逆转录产生不完整的cDNA分子。通过与衣壳蛋白融合将大肠杆菌核糖核酸酶HI递送至Ty1颗粒可使逆转座减少99%以上，这也表明体内DNA合成受到抑制。
大肠杆菌核糖核酸酶HI对体外逆转录和体内逆转座的抑制表明，RT持续性差会使对逆转录至关重要的RNA-DNA杂交体易于降解。将细胞核糖核酸酶H靶向HIV可能有助于确定易受非逆转录病毒核糖核酸酶H影响的RNA-DNA杂交体位点，并且可能对抑制逆转录病毒复制的基因治疗有用。
