Physiological Mg 2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg , whereas the free Mg concentration in cells is much lower. Artificially high Mg concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg . At low concentrations of Mg , NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg , whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on k /K ) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC values) more effective at low than at high Mg concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg -dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg conditions yielded results dramatically different from those from measurements using commonly employed high-Mg in vitro conditions. These results also emphasize differences in Mg sensitivity between the translocation inhibitor EFdATP and other NRTIs.