Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Campus de Cantoblanco-UAM, 28049 Madrid, Spain.
Viruses. 2021 Jan 18;13(1):131. doi: 10.3390/v13010131.
HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2 RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1 RT (positions 342-351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342-350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.
HIV 逆转录酶（RT）将病毒基因组 RNA 逆转录成双链 DNA。在逆转录过程中，对 RNase H 切割具有抗性的多聚嘌呤序列（PPT）被用作正链 DNA 合成的引物。非核苷逆转录酶抑制剂（NNRTIs）可以通过增强 PPT 去除来干扰正链 DNA 合成的起始，而 HIV RT 连接亚结构域突变 N348I 和 N348I/T369I 通过改变 RNase H 切割特异性来减轻这种影响。现在，我们证明在已批准的非核苷逆转录酶抑制剂（NNRTIs）中，奈韦拉平和多拉韦林的效果最大。HIV-1 RT 中的 N348I/T369I 组合对 PPT/U3 位点的 RNase H 切割特异性具有显性效应。生化研究表明，野生型 HIV-1 和 HIV-2 RT 能够有效地和准确地处理所有测试的 HIV PPT 序列。然而，HIV-2 RT 在 PPT/U3 连接点的切割准确性在取代 HIV-1 RT 的序列 YQEPFKNLKT（位置 342-351）为 HIV-2 酶的等效残基（HQGDKILKV）后得到进一步提高。我们的结果强调了连接亚结构域中β-片层 17 和 18 及其连接环（残基 342-350）在 HIV RTs 的 RNase H 切割窗口中的作用。
