Measurements of the Functional Dynamics of Wild Type and K103N HIV-1 Reverse Transcriptase Reveal the Mechanism of Efavirenz Resistance

Abstract

HIV-1 reverse transcriptase (RT) is a primary target for drug development since it converts viral RNA into double stranded DNA that is subsequently integrated into the human genome. Nonnucleoside RT Inhibitors (NNRTIs) are routinely included in many first-line combination antiretroviral therapies and are also used to prevent mother-to-child transmission of HIV-1. Of note, the mechanisms by which NNRTI inhibit HIV-1 RT remain unclear despite the wealth of available structural and biochemical data. Furthermore, the structural mechanisms by which mutations in HIV-1 RT confer NNRTI resistance are also inadequately understood. To elucidate these mechanisms, we combined several complementary tools that included a novel fluorescence anisotropy-based assay of RT mobility on a didoexy-terminated Template/Primer (T/P) substrate, single-molecule fluorescence measurements of RT shuttling kinetics, and molecular dynamics simulations. We find that efavirenz, an NNRTI, does not significantly alter RT-T/P binding affinity but substantially increases RT mobility on the T/P substrate, reducing the time spent in the polymerase-competent position. Furthermore, we show that the drug resistance K103N mutation in RT does not affect efavirenz binding affinity, but instead eliminates the increased shuttling we typically observe upon NNRTI addition. Taken together with our experiments probing the effect of the cognate dNTP on these dynamics, we provide compelling evidence that NNRTIs act in part by loosening the thumb and fingers clamp of RT on its T/P substrate, permitting highly dynamic T/P translocation and/or dissociation, and that NNRTI-resistant mutations in RT likely confer a structural resistance to this dynamic change.