Abstract

Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection1 and a common target of antiretroviral drugs2. The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA5 each bound to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer–template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5′ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

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Acknowledgements

We thank A. Frost and L. Stryer for suggesting beta-octyl glucoside as an additive for cryo-EM, R. Kornberg, M. Levitt, P. Geiduschek and W. Sundquist for reading the manuscript, M. Levitt for discussion of alternative tRNA folds and general support, D. Herschlag for discussions, and N. R. Latorraca for discussions and assistance with the Sherlock cluster. Supported by National Institutes of Health grant GM082545 to E.V.P., T32-GM008294 (Molecular Biophysics Training Program) to K.P.L., A.T.C. and K.K., National Science Foundation Graduate Research Fellowship Program (DGE-114747) to A.T.C and K.K., and Gabilan Stanford Graduate Fellowship to K.K. We thank Stanford University and the Stanford Research Computing Center for providing the Sherlock cluster resources. Additional calculations were performed on the Stanford BioX3 cluster, supported by NIH Shared Instrumentation Grant 1S10RR02664701.

Reviewer information

Nature thanks N. Sluis-Cremer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Kevin P. Larsen, Yamuna Kalyani Mathiharan.

Affiliations

  1. Program in Biophysics, Stanford University, Stanford, CA, USA

    • Kevin P. Larsen
    • , Kalli Kappel
    •  & Aaron T. Coey
  2. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    • Kevin P. Larsen
    • , Aaron T. Coey
    • , Dong-Hua Chen
    • , Daniel Barrero
    • , Lauren Madigan
    • , Joseph D. Puglisi
    • , Georgios Skiniotis
    •  & Elisabetta Viani Puglisi
  3. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • Yamuna Kalyani Mathiharan
    •  & Georgios Skiniotis

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Contributions

K.P.L., Y.K.M. and D.-H.C. acquired preliminary cryo-EM data and performed initial cryo-EM map calculations. Y.K.M. acquired cryo-EM data and obtained the 3D reconstructions shown in the main manuscript. K.P.L. acquired Mg2+ cryo-EM data and performed corresponding cryo-EM map calculations. A.T.C. purified the vRNA used for single-molecule experimentation and performed the single-molecule experiments. K.P.L., D.B. and L.M. performed all vRNA and RT sample preparations. K.P.L. performed all α-32P-dTTP incorporation assays. D.B. performed the RT activity assays. K.P.L. designed the purification scheme and purified the RTIC used in all experimentation. K.K. performed the vRNA–tRNA model building with input from K.P.L. K.P.L. and Y.K.M. performed final RTIC model building and refinement. K.P.L., Y.K.M, G.S., J.D.P. and E.V.P. interpreted the data. K.P.L and E.V.P wrote the manuscript with input from J.D.P., K.K., Y.K.M. and G.S.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Elisabetta Viani Puglisi.

Extended data figures and tables

  1. Extended Data Fig. 1 Purification and activity of RTIC.

    a, Initial anion-exchange purification of the RTIC away from free RT and vRNA–tRNA. This purification was repeated for each sample (>10) used in the manuscript, with only slight variations in the chromatogram. b, Polishing step using size-exclusion chromatography purification of the RTIC after anion exchange. This purification was repeated for each sample used in the manuscript (>10), with only slight variations in the chromatogram. c, A final 10% native TBE gel on the purified components. RT barely enters the gel under these running conditions. The RTIC runs as a single band, but trace amounts of free vRNA and/or vRNA–tRNA complex are sometimes present. This native gel is a representative result that was repeated independently for all purified RTIC samples used in the paper (>10). d, Autoradiograph image illustrating that the RTIC is capable of incorporating an incoming α-32P-dTTP nucleotide when extended and purified using dCTP instead of ddCTP. This gel is a representative result that was repeated independently for crosslinked and uncrosslinked samples (>6 independently prepared samples) used in dTTP incorporation assays. e, The RTIC incorporates α-32P-dTTP at roughly 89% efficiency compared to the free components after reaching a plateau. Values are mean ± s.d. (n = 3 independent experiments) with normalization to total incorporation of free RT + vRNA–tRNA reactions. f, Autoradiograph image showing that the incorporation of dTTP is inhibited in the presence of nevirapine (NNRTI). Images have been adjusted to allow identification of the NNRTI-inhibited band. This gel is a representative result that was repeated independently for crosslinked and uncrosslinked samples (3 samples each). g, Relative activities, judged by primer usage, of wild-type, Q258C, and Q258C/E478Q reverse transcriptase mutants used in this study. Values are mean ± s.d. (n = 3 independent experiments) with normalization to the primer usage of wild-type RT. h, RTIC (triangles), RTIC with NNRTI (circles) or vRNA–tRNA + excess RT (squares) reactions were initiated by addition of α-32P-dTTP and quenched at different time points. Data were fit using the relationship for the free vRNA–tRNA + RT reaction: \({\rm{Intensity}}=A\left(1-{e}^{-{k}_{{\rm{pol}}}t}\right)+B\left(1-{e}^{-{k}_{{\rm{slow}}}t}\right)\). Data were fit using the relationship for the RTIC (with or without NNRTI) reaction: \({\rm{Intensity}}=B\left(1-{e}^{-{k}_{{\rm{slow}}}t}\right)\) where A and B represent the amplitude of the fast and slow processes, respectively, kpol is the apparent extension rate constant, and kslow is the rate of the slow process. The second relationship was used for the RTIC data, as the slow process appears to dominate incorporation when the vRNA–tRNA substrate is crosslinked to RT. The best fits were obtained with: A = 0.7166 AU, kpol = 0.1078 s−1, B = 0.2754, kslow = 0.01002 s−1 for the vRNA–tRNA + excess RT; B = 0.9808, kslow = 0.003140 s−1 for the RTIC; and B = 1.095, kslow = 0.0001714 s−1 for the RTIC with NNRTI. kslow is about 3.19 times slower for crosslinked RTIC than for un-crosslinked components. Assays were independently repeated three times to ensure reproducibility.

  2. Extended Data Fig. 2 Representative negative-stain EM images, cryo-EM images, and 2D averages of the RTIC.

    a, Representative negative-stain EM image of HIV RTIC reveals a mono-disperse sample that is free of aggregates. Approximately a dozen images were taken of each sample before cryo-EM grid preparation to ensure sample quality. b, Cryo-EM image of RTIC without β-OG. The long chains correspond to RNA from the complex with very few particles resembling the protein. Results are reproducible in the absence of β-OG (>10 samples tested). c, Cryo-EM image of RTIC with β-OG. Single particles corresponding to the complex appear similar to the negative-stain visualization. All 5,107 images used in both cryo-EM datasets have a similar appearance with slight differences in particle density. d, Representative 2D averages of RTIC complex from the cryo-EM data collected with β-OG. Both datasets exhibit very similar 2D classes.

  3. Extended Data Fig. 3 Data processing workflow for RTIC complex.

    a, Data processing workflow for the 8.0 Å global and 4.5 Å core maps. b, Gold standard FSC curve of RTIC core and global maps. c, The final 4.5 Å map is coloured according to local resolution estimated by Relion. d, Angular distribution of particle projections. The length of each projection direction is proportional to the number of assigned particles. e, Data processing workflow for the 8.2 Å global Mg2+ map. f, Gold standard FSC curve of RTIC Mg2+ global map.

  4. Extended Data Fig. 4 Quality of the cryo-EM density for the core RTIC map.

    a, View of HIV-1 RT from the front. The subdomains of RT are coloured. Underneath the main RTIC view, each subdomain of RT, plus the p51 subunit, is shown fit into the 4.5 Å map. b, View of HIV-1 RT from the polymerase active site side. The subdomains of RT are coloured. Underneath the main RTIC view, each subdomain of RT, plus the p51 subunit, is shown fit into the 4.5 Å map. In a, b, regions of protein, namely loops and linkers, that lacked sufficient density were removed after comparison with previously published structures of RT. These regions are indicated by dotted lines and are most commonly found in the finger and palm subdomains. c, Representative regions of 4.5 Å map fitted with protein secondary structure that display densities for side chains. A view of the PBS helix fit into the 4.5 Å map is also shown; phosphates of the RNA backbone are partially resolved. Regions are coloured with respect to the main text models.

  5. Extended Data Fig. 5 Mg2+ global map views and structure comparison.

    a, Side and top views of the 8.2 Å global map at different density thresholds. The orientation of the peripheral vRNA and tRNA elements is within the variability seen among the different RTIC conformers. b, A model of the RTIC built into the Mg2+ density using the main text global RTIC model. vRNA and tRNA helices were treated as rigid bodies derived from main text model (see Extended Data Fig. 6 and Methods). c, Comparison of the global RTIC model RNA (grey) with the Mg2+ model RNA (coloured). All three regions of RNA structure (H1, H2, and tRNA) differ in the Mg2+ model, but are adequately described by rigid body movements of the RNA helical elements taken from the global RTIC model. Both H1 and H2 represent a substantial structural barrier to initiation. d, Partial accommodation of H1 into high monovalent salt classes 3, 4 and 7.

  6. Extended Data Fig. 6 Low-resolution tRNA density and fold comparison.

    a, Top and side views of the elongated helical tRNA density observed in the low-resolution global map of the RTIC. b, Top and side views of the vRNA–tRNA model generated using the hypothesized elongated tRNA helical fold. The tRNA model fits the long helical density well. Corresponding secondary structure is in d. c, Top and side views of the vRNA–tRNA model generated using previously hypothesized tRNA secondary structures that have the anticodon and D-stem loops independently folded. Corresponding secondary structure is in e. d, Secondary structure depiction of the new vRNA–tRNA and canonical clover-leaf fold of the tRNA. The different domains are coloured and correspond with the models in panels b and c. e, Secondary-structure depiction of the old vRNA–tRNA fold with independent anticodon and D-stem loops. The domains are coloured and correspond with the model in c and clover-leaf fold of the tRNA in d.

  7. Extended Data Fig. 7 Peripheral RNA heterogeneity of the RTIC conformers.

    a, Tiled views of eight conformations emerging from 3D classification of RTIC. Each class is numbered and class 7 was used for the global RTIC reconstruction. b, Superposition of the eight classes from a. The main areas of RNA heterogeneity are focused on the orientations of vRNA H2, H1 and the connection loop, and the tRNA. With no stabilizing protein contacts, vRNA H2, H1, and the tRNA sample a wide range of conformations, limiting the resolution of the global map. c, Additional RTIC models built into classes 3 (tan) and 4 (blue). The models for the tRNA, vRNA H1, and vRNA H2 were all derived from the global RTIC model and treated as rigid bodies for model building. The connecting loop was not built in these models as the density for this region was not clear in these maps, though there is reasonable density to model a loop near H1. Junctions between the helices serve as hinges that allow movement of the independent domains. The main text global RTIC model (grey) is included as a comparison. d, The vRNA and tRNA helices treated as rigid bodies for modelling are shown in bold. Hinge points for each helix are highlighted with grey circles and serve as points of flexibility for the RTIC.

  8. Extended Data Fig. 8 Single-molecule experimentation and analysis.

    a, Secondary structure depiction of the vRNA–tRNA construct used for single-molecule experiments. The labelling scheme is shown, with the Cy3 dye located on the 5′ end of the vRNA helix 1 and Cy5 dye located on an oligonucleotide positioned near the 5′ end of helix 1. The vRNA–tRNA complex was crosslinked to RT for the experiments. b, Ninety-five per cent of the RTIC complexes are in the high FRET, helix 1 formation, state (480 traces analysed, see Methods). c, Example trace of the ones used for final FRET analysis. The high FRET state of the RTIC complex, which is attributed to helix 1 formation. Photobleaching events for both Cy5 and Cy3 are indicated. d, Examples of traces removed from final FRET analysis. Traces exhibit the presence of multiple molecules (multiple single-dye photobleaching events) or poor dye behaviour (blinking and quenching).

  9. Extended Data Fig. 9 Comparison with NNRTI bound and active RT–nucleic acid complexes in the cryo-EM map.

    All alignments between structures and the RTIC were done using the p51 subunit. a, Comparison of an active conformation RT–nucleic acid structure (pink, 1RTD) with the RTIC core (RT, purple; tRNA primer, red; vRNA template, yellow). The EM map overlay shows the poor fit of the 1RTD model in the fingers, thumb, and primer grip of RT. Deviations of the nucleic acid primer and template of 1RTD away from the RTIC density are also apparent. b, Comparison of an NNRTI-bound RT–nucleic acid structure (dark grey, 3V81) with the RTIC core. The EM map overlay shows the closer fit of the fingers and primer grip regions of RT in the 3V81 model. The thumb region also overlays well, but with slight deviations. Most noticeably, the nucleic acid primer/template in the 3V81 model deviates, although not as dramatically as in 1RTD, from the RTIC core EM density.

  10. Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics

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