Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3′/5′ termini, termed the “zipper” complex, or nucleation through the hairpin loops in a “kissing” complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.