Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Sandra Vergara, Xiaohong Zhou, Ulises Santiago, Mounia Alaoui-El-Azher, James F. Conway, Nicolas Sluis-Cremer () and Guillermo Calero ()
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Sandra Vergara: University of Pittsburgh School of Medicine
Xiaohong Zhou: University of Pittsburgh School of Medicine
Ulises Santiago: University of Pittsburgh School of Medicine
Mounia Alaoui-El-Azher: University of Pittsburgh School of Medicine
James F. Conway: University of Pittsburgh School of Medicine
Nicolas Sluis-Cremer: University of Pittsburgh School of Medicine
Guillermo Calero: University of Pittsburgh School of Medicine

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.

Date: 2024
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DOI: 10.1038/s41467-024-54618-y

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