Binding of the Nonnucleoside Reverse Transcriptase Inhibitor Efavirenz to HIV-1 Reverse Transcriptase Monomers and Dimers

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was the first target of antiretroviral therapy in the treatment of AIDS. RT converts single-stranded viral RNA into double-stranded proviral DNA. The enzyme has two activities, DNA polymerase and RNase H. The biologically relevant form is a heterodimer composed of two subunits, p66 and p51. The subunits are products of the same gene and have identical N-terminal amino acid sequences; p51 lacks the C-terminal RNase H domain. In solution RT exists as a complex equilibrium mixture of p66/p51 heterodimer, p66/p66 and p51/p51 homodimers, and p66 and p51 monomers. Two classes of inhibitors have been developed and approved for clinical use, NRTIs and NNRTIs. NNRTIs are highly effective and relatively noncytotoxic small, amphiphilic, noncompetitive inhibitors that nestle into a hydrophobic pocket ∼10 A away from the polymerase active site in the p66 subunit of RT. NNRTIs also have diverse effects on RT subunit dimerization; some enhance dimerization and others weaken dimerization. Efavirenz is an NNRTI capable of affecting several steps in HIV-1 reverse transcription and replication. This work reports two novel functions of the inhibitor: (1) efavirenz, and presumably also other NNRTIs, binds to monomeric forms of RT and (2) efavirenz is a slow binding inhibitor of heterodimer and monomers. In addition, the binding site on p66 and p51 monomers was identified. Five techniques were used to characterize the interaction of efavirenz to all species of RT; equilibrium dialysis, tryptophan fluorescence, native gel electrophoresis, and hydrogen-deuterium exchange and Fourier transform ion cyclotron resonance mass spectrometry. Although the biological significance of efavirenz binding to monomeric forms of RT is not known, this work suggests that monomeric forms of RT may be potential targets for HIV-1 therapeutics.