Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription

Reverse transcriptase is an essential enzyme for HIV replication. Single-molecule studies reveal for the first time the structural dynamics of the reverse transcription initiation complex in real time. Reverse transcriptase can bind its tRNA–vRNA substrate in two opposite orientations and flip between these two states. Human immunodeficiency virus (HIV) initiates reverse transcription of its viral RNA (vRNA) genome from a cellular tRNA 3 Lys primer. This process is characterized by a slow initiation phase with specific pauses, followed by a fast elongation phase. We report a single-molecule study that monitors the dynamics of individual initiation complexes, comprised of vRNA, tRNA and HIV reverse transcriptase (RT). RT transitions between two opposite binding orientations on tRNA–vRNA complexes, and the prominent pausing events are related to RT binding in a flipped orientation opposite to the polymerization-competent configuration. A stem-loop structure within the vRNA is responsible for maintaining the enzyme predominantly in this flipped orientation. Disruption of the stem-loop structure triggers the initiation-to-elongation transition. These results highlight the important role of the structural dynamics of the initiation complex in directing transitions between early reverse transcription phases.