Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 2013 May 3;288(18):12522-32. doi: 10.1074/jbc.M113.458398. Epub 2013 Mar 11.
HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.
HIV-1 逆转录酶（RT）在合成前病毒 DNA 时经常掺入核糖核苷三磷酸（rNTP），特别是在巨噬细胞中发现的有限 dNTP 条件下。我们研究了嵌入 DNA 模板中的 rNMP 对 HIV-1 RT 介导的 DNA 合成的机制影响。我们观察到，模板嵌入的 rNMP 在低巨噬细胞 dNTP 浓度下诱导 RT 暂停和延迟 DNA 合成动力学，但在高 T 细胞 dNTP 浓度下则不会。尽管 RT 与含 rNMP 的模板-引物的结合亲和力没有改变，但 RT 的 dNTP 掺入动力学在 rNMP 位点的上下游一个核苷酸处显著降低，导致暂停位点。最后，HIV-1 RT 在 rNMP 位点变得更容易出错，具有更高的错配延伸能力，但没有增强的错误插入能力。这些数据表明，嵌入 DNA 模板中的 rNMP 可能会影响逆转录动力学，并影响巨噬细胞中的病毒突变。
