The initiation of reverse transcription in human immunodeficiency virus-1 (HIV-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 tRNA 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 (smFRET) 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 interac... More
The initiation of reverse transcription in human immunodeficiency virus-1 (HIV-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 tRNA 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 (smFRET) 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 (PBS) 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 (H1) formation and reduces global structural heterogeneity. The stabilization of H1 upon RT binding may serve to destabilize helix 2 (H2)， the first pause site for RT during initiation， during later steps of reverse transcription initiation.
