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 Mg2+ concentrations (similar to 5-10 mM) several-fold higher than physiological cellular free Mg2+ (similar to 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 Mg2+. In the current report, lacZ alpha-based alpha-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 Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Me. In 0.5 mM Mg2+, 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) Mg2+ on a lacZ alpha template. Unlike alpha-complementation assays which are dependent on LacZ alpha activity, the NGS assay scores mutations at all positions and of every type. Consistent with a-complementation assays, a similar to four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Me 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.