Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed using optimized Mg 2+ concentrations (~5-10 mM) several-fold higher than physiological cellular free Mg 2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg 2+ . In the current report, lacZα -based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg 2+ , while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg 2+ . In 0.5 mM Mg 2+ , all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg 2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZ α activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg 2+ . These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg 2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT's activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.