Mechanisms of nucleoside analog resistance and the role of magnesium ions in HIV-1 reverse transcriptase

Effectiveness of antiviral drugs used to treat HIV infections is limited by the evolution of drug resistance. There are two common mechanisms leading to the resistance against nucleoside reverse transcriptase inhibitors (NRTIs): Selective discrimination before incorporation and excision after incorporation. In order to better understand the mechanisms of NRTI resistance, we investigated each step involved in the incorporation and excision of two NRTIs: Azidothymidine (AZT) and Tenofovir (TFV) using two RT mutants, K65R and K66R. Our results showed that the mutation of K65R does not significantly affect the ATP-mediated NRTIs excision, but rather affects the pyrophosphate-mediated NRTIs excision by decreasing the efficiency of pyrophosphate-mediated excision. Our data suggest that K65R may not prefer the ATP- or pyrophosphate-mediated excision as a mechanism to excise NRTIs leading to drug resistance. Rather, the NRTI resistance conferred by K65R is achieved mainly through the discrimination mechanism rather than through the excision mechanism. Furthermore, our results showed that the primary effect of K65R on NRTI discrimination (TFV and AZT) is to decrease the rate of the chemistry step. In addition, our results showed that the drug resistances for TFV and AZT conferred K65R are comparable. Compared with K65R, the resistance conferred by K66R is mild. We also revisited the classic two-metal-ion mechanism from analysis of enzyme kinetics and crystal structure. Our data showed that a tight binding (K [subscript d] is 0.5 μM) of the Mg²⁺ -nucleotide complex induces a conformational change of the enzyme from open to the closed state. The catalytic Mg²⁺ binds (K [subscript d] is 3.7 mM) after the conformational change. The binding of the catalytic Mg²⁺ stabilizes the Mg²⁺ -nucleotide complex in the active site and stimulates the catalysis of the reaction. In addition, our data suggested that changing the free Mg²⁺ from 10 mM to 0.25 mM significantly affects the processivity, but not the fidelity of HIVRT.