Structural and Mechanistic Basis of F227C-Mediated Hypersusceptibility to Islatravir in HIV-1 Reverse Transcriptase

Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA)), a first-in-class nucleoside reverse transcriptase (RT) translocation inhibitor (NRTTI), was recently approved by the FDA for the treatment of HIV-1 infection in combination with the non-nucleoside RT inhibitor (NNRTI), doravirine (DOR). Notably, the RT mutation F227C, which confers clinical resistance to multiple NNRTIs, including DOR, unexpectedly increases susceptibility to ISL. To elucidate the mechanistic basis of this hypersusceptibility, we determined a 1.8 angstrom crystal structure of F227C RT in complex with a ddGMP-terminated primer/template and ISL-triphosphate. The structure reveals conformational rearrangements that propagate into a cleft, thereby affecting ATP-mediated unblocking of chain-terminating antivirals. Complementary biochemical assays showed that although F227C does not significantly affect ISL incorporation, it alters RT translocation and impairs ATP-dependent phosphorolytic excision of ISL-terminated primers, thereby enhancing ISL susceptibility. These findings establish direct structural and mechanistic links between NNRTI resistance and ISL hypersusceptibility, providing a structural foundation for rationally designed, resistance-informed combination regimens that exploit this unique collateral sensitivity.