Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+.

HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.