Volkmann S, Dannull J, Moelling K
Max-Planck-Institute für Molekulare Genetik, Berlin, Germany.
Biochimie. 1993;75(1-2):71-8. doi: 10.1016/0300-9084(93)90027-p.
Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.
逆转录病毒RNA复制成双链DNA前病毒涉及逆转录酶（RT）在多聚嘌呤序列（PPT）处起始正链DNA合成。PPT在已知的HIV-1逆转录病毒分离株中高度保守。它出现两次，一次在整合酶的编码区内，另一次与3' LTR相邻。所呈现的数据表明，两条与PPT互补的反义寡核苷酸，一条20聚体和一条40聚体，可完全阻断DNA合成，而PPT外的反义寡核苷酸仅有轻微抑制作用。此前已有多个研究小组使用多聚嘌呤序列形成三链体。在复制过程中，HIV的多聚嘌呤序列（PPT）存在于RNA-DNA杂交体中。因此，这里通过体外抗RNase H切割试验检测了由RNA-DNA和覆盖PPT区域的第三条DNA链组成的三链体形成情况。与PPT-RNA平行方向的嘧啶寡核苷酸孵育显示出一定的保护作用。GT-嘧啶-嘌呤混合寡核苷酸（25聚体）无论其方向如何，均可导致高达50%的抗RNase H保护作用。数据表明体外可能与PPT形成了三链体。因此，这种高度保守的结构可能在基于核酸的反义或三链体方法抗病毒治疗中有用。此外，还报道了HIV-1核衣壳（NC）蛋白NCp15对逆转录的影响。数据显示NCp15对RNA指导的DNA合成有两到三倍的刺激作用。
