Abstract
Human immunodeficiency virus (HIV) initiates reverse transcription of its viral RNA (vRNA) genome from a cellular tRNA3Lys 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.
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Acknowledgements
We thank J. Wu and E. Abbondanzieri for helpful discussions and R. Gorelick (National Cancer Institute (NCI), Frederick, Maryland, USA) for providing NC proteins. This work is supported in part by the US National Institutes of Health (NIH; GM 068518 to X.Z.) and the Intramural Research Program of the Center for Cancer Research, NCI (to S.F.J.L.G.). B.T.H. was supported by a NIH/National Institute of General Medical Sciences Molecular Biophysics Training Grant (GM008313 to the Harvard Biophysics Program). X.Z. is a Howard Hughes Medical Institute investigator.
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S.L., B.T.H. and X.Z. designed the experiments; S.L. and B.T.H. performed the experiments and analyzed the data; S.L., B.T.H. and X.Z. interpret the data and wrote the paper; J.T.M. made the enzyme and some of the tRNA constructs; S.F.J.L.G. contributed to discussion, data interpretation and manuscript preparation.
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Liu, S., Harada, B., Miller, J. et al. Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 17, 1453–1460 (2010). https://doi.org/10.1038/nsmb.1937
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DOI: https://doi.org/10.1038/nsmb.1937