Structures of kinetic intermediate states of HIV-1 reverse transcriptase DNA synthesis

Reverse transcription of the retroviral single-stranded RNA into double-stranded DNA is an integral step during HIV-1 replication, and reverse transcriptase (RT) is a primary target for antiviral therapy. Despite a wealth of structural information on RT, we lack critical insight into the intermediate kinetic states of DNA synthesis. Using catalytically active substrates, and a novel blot/diffusion cryo-electron microscopy approach, we captured 11 structures that define the substrate binding, reactant, transition and product states of dATP addition by RT at 1.9 to 2.4 [A] resolution in the active site. Initial dATP binding to RT-template/primer complex involves a single Mg2+ (site B), and promotes partial closure of the active site pocket by a large conformational change in the {beta}3-{beta}4 loop in the Fingers domain, and formation of a negatively charged pocket where a second "drifting" Mg2+ can bind (site A). During the transition state, the -phosphate oxygen from a previously unobserved dATP conformer aligns with the site A Mg2+ and the primer 3'-OH for nucleophilic attack. In the product state, we captured two substrate conformations in the active site: 1) dATP that had yet to be incorporated into the nascent DNA, and 2) an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H- bonds in the active site of RT. This study provides insights into a fundamental chemical reaction that impacts polymerase fidelity, nucleoside inhibitor drug design, and mechanisms of drug resistance.