Dynamics of the HIV Reverse Transcription Initiation Complex

Abstract

Reverse transcription of the HIV genome begins from a ternary complex containing a tRNA primer, the highly-structured viral RNA template, and the reverse transcriptase (RT) enzyme. Previous work on RT revealed that RT binds its nucleic acid substrates in a variety of different modes and dynamically switches between these modes, for example, flipping between different binding orientations and sliding between alternate positions on the substrate. To assess RT's dynamics on more complicated substrates and to understand how these dynamics influence the initiation of reverse transcription, we employ a single molecule FRET assay to monitor the interactions between RT and the initiation tRNA/viral RNA complex. These measurements define the binding configuration of RT at each stage of the extension of the tRNA primer. We find that RT can bind the initiation complex in two orientations, corresponding to a productive orientation and an inactive orientation, and spontaneously flip between the two orientations. Both the composition of the primer's 3′ end and the secondary structure of the template mediate RT's binding orientation. As RT extends the tRNA primer, the amount of time it spends in the productive binding orientation first decreases, then increases as more nucleotides are added to the end of the tRNA primer. These results mirror ensemble primer extension assays showing that RT acts slowly and distributively during the addition of the first few nucleotides, then transitions to a fast, processive mode. These results demonstrate that RT's binding dynamics regulate the initiation of reverse transcription and provide a mechanistic explanation for the changes in RT activity during initiation.