Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket – Targeting P-pocket by fragment screening

Abstract HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structure-based platform provide an opportunity for designing new types of polymerase inhibitors.