Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex

The initiation of reverse transcription in human immunodeficiency virus-1 is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNALys3 as a primer for initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription initiation kinetics. Using single-molecule Förster resonance energy transfer spectroscopy, we demonstrate that the vRNA–tRNA initiation complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid–RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA–tRNA primer binding site helix from 18 base pairs to 22 base pairs stabilizes RT binding to the complex and that the tRNA 5′ end has a role in modulating RT binding. RT occupancy on the complex stabilizes helix 1 formation and reduces global structural heterogeneity. The stabilization of helix 1 upon RT binding may serve to destabilize helix 2, the first pause site for RT during initiation, during later steps of reverse transcription initiation.