Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

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.