Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Structural & Molecular Biology volume 17, pages 1453–1460 (2010)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Single-molecule FRET assay for probing the structural dynamics of the initiation complex.
Figure 2: RNA-dependent DNA polymerase activity of RT correlates with its binding orientation in the initiation complex.
Figure 3: The stem-loop structure upstream of the PBS causes the major pauses during initiation and governs the initiation-to-elongation transition.
Figure 4: Disruption of the stem-loop structure upstream of the PBS occurs upon addition of the sixth nucleotide to the tRNA primer.
Figure 5: HIV-1 NC destabilizes the stem-loop structure.
Figure 6: Structural dynamics of the HIV-1 initiation complex regulate the early phases of reverse transcription.

Similar content being viewed by others

References

  1. Telesnitsky, A. & Goff, S.P. Reverse transcriptase and the generation of retroviral DNA. in Retroviruses (eds. Coffin, J.M., Hughes, S.H. & Varmus, H.E.) 121–160 (Cold Spring Harbor Laboratory Press, 1997).

  2. Marquet, R., Isel, C., Ehresmann, C. & Ehresmann, B. tRNAs as primer of reverse transcriptases. Biochimie 77, 113–124 (1995).

    Article  CAS  Google Scholar 

  3. Cobrinik, D., Soskey, L. & Leis, J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J. Virol. 62, 3622–3630 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Cordell, B., Swanstrom, R., Goodman, H.M. & Bishop, J.M. tRNATrp as primer for RNA-directed DNA polymerase: structural determinants of function. J. Biol. Chem. 254, 1866–1874 (1979).

    CAS  PubMed  Google Scholar 

  5. Harrich, D. & Hooker, B. Mechanistic aspects of HIV-1 reverse transcription initiation. Rev. Med. Virol. 12, 31–45 (2002).

    Article  CAS  Google Scholar 

  6. Götte, M., Li, X. & Wainberg, M.A. HIV-1 reverse transcription: a brief overview focused on structure-function relationships among molecules involved in initiation of the reaction. Arch. Biochem. Biophys. 365, 199–210 (1999).

    Article  Google Scholar 

  7. Le Grice, S.F. “In the beginning”: initiation of minus strand DNA synthesis in retroviruses and LTR-containing retrotransposons. Biochemistry 42, 14349–14355 (2003).

    Article  CAS  Google Scholar 

  8. Tisné, C. Structural bases of the annealing of primer tRNA3Lys to the HIV-1 viral RNA. Curr. HIV Res. 3, 147–156 (2005).

    Article  Google Scholar 

  9. Abbink, T.E. & Berkhout, B. HIV-1 reverse transcription initiation: a potential target for novel antivirals? Virus Res. 134, 4–18 (2008).

    Article  CAS  Google Scholar 

  10. Isel, C., Ehresmann, C. & Marquet, R. Initiation of HIV reverse transcription. Viruses 2, 213–243 (2010).

    Article  CAS  Google Scholar 

  11. Isel, C. et al. Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. EMBO J. 15, 917–924 (1996).

    Article  CAS  Google Scholar 

  12. Lanchy, J.M., Ehresmann, C., Le Grice, S.F., Ehresmann, B. & Marquet, R. Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription. EMBO J. 15, 7178–7187 (1996).

    Article  CAS  Google Scholar 

  13. Lanchy, J.M. et al. Contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of HIV-1 reverse transcription. J. Biol. Chem. 273, 24425–24432 (1998).

    Article  CAS  Google Scholar 

  14. Goldschmidt, V. et al. Structural variability of the initiation complex of HIV-1 reverse transcription. J. Biol. Chem. 279, 35923–35931 (2004).

    Article  CAS  Google Scholar 

  15. Wilkinson, K.A. et al. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. PLoS Biol. 6, e96 (2008).

    Article  Google Scholar 

  16. Liang, C. et al. Mechanistic studies of early pausing events during initiation of HIV-1 reverse transcription. J. Biol. Chem. 273, 21309–21315 (1998).

    Article  CAS  Google Scholar 

  17. Thrall, S.H. et al. Pre-steady-state kinetic characterization of RNA-primed initiation of transcription by HIV-1 reverse transcriptase and analysis of the transition to a processive DNA-primed polymerization mode. Biochemistry 37, 13349–13358 (1998).

    Article  CAS  Google Scholar 

  18. Lanchy, J.M. et al. Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis. J. Biol. Chem. 275, 12306–12312 (2000).

    Article  CAS  Google Scholar 

  19. Rong, L. et al. HIV-1 nucleocapsid protein and the secondary structure of the binary complex formed between tRNALys.3 and viral RNA template play different roles during initiation of (–) strand DNA reverse transcription. J. Biol. Chem. 276, 47725–47732 (2001).

    Article  CAS  Google Scholar 

  20. Abbondanzieri, E.A. et al. Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453, 184–189 (2008).

    Article  CAS  Google Scholar 

  21. Liu, S., Abbondanzieri, E.A., Rausch, J.W., Le Grice, S.F. & Zhuang, X. Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 322, 1092–1097 (2008).

    Article  CAS  Google Scholar 

  22. DeStefano, J.J., Mallaber, L.M., Fay, P.J. & Bambara, R.A. Determinants of the RNase H cleavage specificity of human immunodeficiency virus reverse transcriptase. Nucleic Acids Res. 21, 4330–4338 (1993).

    Article  CAS  Google Scholar 

  23. Stryer, L. & Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

    Article  CAS  Google Scholar 

  24. Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl. Acad. Sci. USA 93, 6264–6268 (1996).

    Article  CAS  Google Scholar 

  25. Iwatani, Y., Rosen, A.E., Guo, J., Musier-Forsyth, K. & Levin, J.G. Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions. J. Biol. Chem. 278, 14185–14195 (2003).

    Article  CAS  Google Scholar 

  26. Huang, H., Chopra, R., Verdine, G.L. & Harrison, S.C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675 (1998).

    Article  CAS  Google Scholar 

  27. Sarafianos, S.G. et al. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. EMBO J. 20, 1449–1461 (2001).

    Article  CAS  Google Scholar 

  28. Isel, C. et al. Structural basis for the specificity of the initiation of HIV-1 reverse transcription. EMBO J. 18, 1038–1048 (1999).

    Article  CAS  Google Scholar 

  29. Paillart, J.C. et al. First snapshots of the HIV-1 RNA structure in infected cells and in virions. J. Biol. Chem. 279, 48397–48403 (2004).

    Article  CAS  Google Scholar 

  30. Suo, Z. & Johnson, K.A. Effect of RNA secondary structure on the kinetics of DNA synthesis catalyzed by HIV-1 reverse transcriptase. Biochemistry 36, 12459–12467 (1997).

    Article  CAS  Google Scholar 

  31. Thomas, J.A. & Gorelick, R.J. Nucleocapsid protein function in early infection processes. Virus Res. 134, 39–63 (2008).

    Article  CAS  Google Scholar 

  32. Rein, A., Henderson, L.E. & Levin, J.G. Nucleic-acid-chaperon activity of retroviral nucleocapsid proteins: significance for viral replication. Trends Biochem. Sci. 23, 297–301 (1998).

    Article  CAS  Google Scholar 

  33. Herschlag, D. RNA chaperones and the RNA folding problem. J. Biol. Chem. 270, 20871–20874 (1995).

    Article  CAS  Google Scholar 

  34. Rodríguez-Rodríguez, L., Tsuchihashi, Z., Fuentes, G.M., Bambara, R.A. & Fay, P.J. Influence of human immunodeficiency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro. J. Biol. Chem. 270, 15005–15011 (1995).

    Article  Google Scholar 

  35. Wu, W. et al. Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. J. Virol. 70, 7132–7142 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ji, X., Klarmann, G.J. & Preston, B.D. Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro. Biochemistry 35, 132–143 (1996).

    Article  CAS  Google Scholar 

  37. Grohmann, D., Godet, J., Mely, Y., Darlix, J.L. & Restle, T. HIV-1 nucleocapsid traps reverse transcriptase on nucleic acid substrates. Biochemistry 47, 12230–12240 (2008).

    Article  CAS  Google Scholar 

  38. Tanchou, V., Gabus, C., Rogemond, V. & Darlix, J.L. Formation of stable and functional HIV-1 nucleocapsid complexes in vitro. J. Mol. Biol. 252, 563–571 (1995).

    Article  CAS  Google Scholar 

  39. Lori, F. et al. Viral DNA carried by human immunodeficiency virus type 1 virions. J. Virol. 66, 5067–5074 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Trono, D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J. Virol. 66, 4893–4900 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhang, H., Dornadula, G., Orenstein, J. & Pomerantz, R.J. Morphologic changes in human immunodeficiency virus type 1 virions secondary to intravirion reverse transcription: evidence indicating that reverse transcription may not take place within the intact viral core. J. Hum. Virol. 3, 165–172 (2000).

    CAS  PubMed  Google Scholar 

  42. Houzet, L. et al. Nucleocapsid mutations turn HIV-1 into a DNA-containing virus. Nucleic Acids Res. 36, 2311–2319 (2008).

    Article  CAS  Google Scholar 

  43. Thomas, J.A., Bosche, W.J., Shatzer, T.L., Johnson, D.G. & Gorelick, R.J. Mutations in human immunodeficiency virus type 1 nucleocapsid protein zinc fingers cause premature reverse transcription. J. Virol. 82, 9318–9328 (2008).

    Article  CAS  Google Scholar 

  44. Beerens, N., Groot, F. & Berkhout, B. Initiation of HIV-1 reverse transcription is regulated by a primer activation signal. J. Biol. Chem. 276, 31247–31256 (2001).

    Article  CAS  Google Scholar 

  45. Wu, T. et al. Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications. Virology 405, 556–567 (2010).

    Article  CAS  Google Scholar 

  46. Hooker, C.W., Lott, W.B. & Harrich, D. Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription. J. Virol. 75, 3095–3104 (2001).

    Article  CAS  Google Scholar 

  47. Rigourd, M. et al. Inhibition of the initiation of HIV-1 reverse transcription by 3′-azido-3′-deoxythymidine. Comparison with elongation. J. Biol. Chem. 275, 26944–26951 (2000).

    CAS  PubMed  Google Scholar 

  48. Le Grice, S.F.J., Cameron, C.E. & Benkovic, S.J. Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Methods Enzymol. 262, 130–144 (1995).

    Article  CAS  Google Scholar 

  49. Rausch, J.W., Sathyanarayana, B.K., Bona, M.K. & Le Grice, S.F. Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking. J. Biol. Chem. 275, 16015–16022 (2000).

    Article  CAS  Google Scholar 

  50. Lapham, J. & Crothers, D.M. RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 2, 289–296 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    Article  CAS  Google Scholar 

  52. Venezia, C.F., Howard, K.J., Ignatov, M.E., Holladay, L.A. & Barkley, M.D. Effects of efavirenz binding on the subunit equilibria of HIV-1 reverse transcriptase. Biochemistry 45, 2779–2789 (2006).

    Article  CAS  Google Scholar 

  53. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    Article  CAS  Google Scholar 

  54. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

  55. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Shixin Liu and Bryan T Harada: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Shixin Liu & Xiaowei Zhuang

  2. Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA

    Bryan T Harada

  3. HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, USA

    Jennifer T Miller & Stuart F J Le Grice

  4. Department of Physics, Harvard University, Cambridge, Massachusetts, USA

    Xiaowei Zhuang

  5. Howard Hughes Medical Institute, Cambridge, Massachusetts, USA

    Xiaowei Zhuang

Authors
  1. Shixin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bryan T Harada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer T Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stuart F J Le Grice
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaowei Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Xiaowei Zhuang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 2282 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1937

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing