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Download filePhysiological Mg2+ Conditions Significantly Alter the Inhibition of HIV‑1 and HIV‑2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro
journal contribution
posted on 2016-12-12, 00:00 authored by Vasudevan Achuthan, Kamlendra Singh, Jeffrey J. DeStefanoReverse
transcriptases (RTs) are typically assayed in vitro with 5–10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ 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 Mg2+. At low
concentrations of Mg2+, 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 Mg2+, whereas
inhibition by the “translocation-defective RT inhibitor”
EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine) was unaffected
by Mg2+ concentrations. Steady-state kinetic analyses revealed
that the reduced level of inhibition at low Mg2+ concentrations
resulted from a 3–9-fold (depending on the particular nucleotide
and inhibitor) less efficient incorporation (based on kcat/Km) 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 Mg2+ concentrations. Unlike NRTIs,
NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately
4-fold (based on IC50 values) more effective at low than
at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants
also displayed the Mg2+-dependent difference in susceptibility
to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors
under more physiologically relevant low-Mg2+ conditions
yielded results dramatically different from those from measurements
using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other
NRTIs.