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Inositol phosphates are assembly co-factors for HIV-1

An Author Correction to this article was published on 29 August 2018

This article has been updated

Abstract

A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.

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Fig. 1: IP6 induces assembly of HIV-1 Gag in vitro.
Fig. 2: IP6 interacts with Lys290 and Lys359 in the immature HIV-1 Gag hexamer.
Fig. 3: IP6 induces mature CA assembly by interacting with Arg18.
Fig. 4: Model.

Change history

  • 29 August 2018

    In this Letter, the Protein Data Bank (PDB) accessions were incorrectly listed as ‘6BH5, 6BHT and 6BHS’ instead of ‘6BHR, 6BHT and 6BHS’; this has been corrected online.

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Acknowledgements

We thank J. Briggs for discussions and reading of the manuscript. This work was supported by the National Institutes of Health (NIH) grants R01-GM107013 (V.M.V.), R01-GM105684 (G. W. Feigenson), P30-GM110758 and P50-GM082251 (J.R.P.), R01-AI129678 (O.P. and B.K.G.-P.), U54-GM103297 (O.P.), and R01-GM110776 (M.C.J.). F.K.M.S. was supported by Deutsche Forschungsgemeinschaft grant BR 3635/2-1 awarded to J. A. G. Briggs. J.M.W. was supported by NIH postdoctoral fellowship grant F32-GM115007. Anton computer time was provided by the Pittsburgh Supercomputing Center (PSC) through NIH grant R01-GM116961. The Anton machine at PSC was generously made available by D. E. Shaw Research. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation (NSF) grant number OCI-1053575. Specifically, it used the Bridges system, which is supported at PSC by NSF award number ACI-1445606. Some of The EM work was conducted at the Molecular Electron Microscopy Core facility at the University of Virginia.

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Nature thanks E. Freed and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations

Authors

Contributions

R.A.D. performed protein purification and in vitro assembly. F.K.M.S. did comparative analyses of cryo-EM and crystal structure data. K.K.Z, J.M.W., B.K.G.-P. and O.P. carried out crystallization trials and structure determination. B.K.G.-P. performed 2D cryo-EM. J.R.P. and C.X. performed all-atom MD simulations. T.D.L., C.L.R. and M.C.J., performed cell biology and virology. The manuscript was written primarily by R.A.D., J.R.P., B.K.G.-P., O.P. and V.M.V. The project was originally conceived by R.A.D., with input from all authors throughout experimentation and manuscript preparation.

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Correspondence to Robert A. Dick or Owen Pornillos.

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Extended data figures and tables

Extended Data Fig. 1 Effect of acidic molecules on immature s-CANC assembly.

a, Representative negative-stain electron microscopy images. Scale bars, 200 nm. The experiment was repeated twice with similar results. b, Number of immature VLPs per 55 µm2. n = 5, mean shown above box plots; centre lines show medians; box limits indicate 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values.

Extended Data Fig. 2 s-CANC and s-CASP1 VLPs.

ac, Representative negative-stain electron microscopy images of s-CANC (a), s-CASP1 (b) and s-CA (c) proteins assembled in the absence of GT25 and in the presence of the indicated IP6 concentrations. Scale bars, 200 nm. d, Diameters of immature VLPs; mean diameter above plot; n below plot. Centre lines show medians; box limits indicate 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values.

Extended Data Fig. 3 Comparison of the HIV-1 Gag cryo-EM structure with the CACTDSP1–IP6 crystal structure.

a, The crystal structure of CACTDSP1 bound to IP6 (cyan) was superimposed on a previously described model of the CA-SP1 segment build into cryo-EM densities of immature HIV-1 particles (PDB 5L93, orange2). Note the close correspondence in K359 rotamers, which were modelled independently in the two structures. For visualization purposes, only one of the six possible IP6 conformations is displayed. b, RMSD calculations of the crystal structure and PDB 5L93. For full-length (residues 149–237) and CA-SP1 (residues 223–237), the RMSDs were calculated only for the atoms that were modelled in both maps. If a sidechain was not modelled, the entire residue was omitted from the calculation. The overall agreement of the models is very high, indicating that the crystal structure corresponds well with conformations found in the virus. c, The CACTDSP1 bound to IP6 (orange and red, respectively) was fitted into two previously published cryo-EM densities2 from VLPs collected from cells (EMD-2706 and EMD-4017). Both maps are shown at 8.8 Å, which is the resolution of the lower resolved map, EMD-2706. In the zoomed insets, only the density corresponding to IP6 is shown. Matching of models and maps and RMSD calculations were performed in Chimera.

Extended Data Fig. 4 Interpretation of the IP6 density in the immature CACTDSP1 hexamer structure.

a, Top and side views of the unbiased mFoDFc difference density (blue mesh, 2σ) ascribed to the bound IP6. Shown are six IP6 molecules docked in six rotationally equivalent positions, consistent with the six-fold rotational symmetric density. b, Top view of the docked IP6 molecules within the CACTDSP1 hexamer. Unbiased mFoDFc difference densities (blue mesh) are also shown for both the bound IP6 and sidechains of Lys290 (green) and Lys359 (cyan). Density for Lys359 is more pronounced, which we interpret to mean that this residue adopts a more restricted range of rotamers for binding IP6.

Extended Data Fig. 5 Quantification of wild-type and mutant HIV s-CANC assembly at pH 6 and pH 8.

a, c, Number of immature (purple) and mature (orange) VLPs per 55 μm2 without (−) and with (+) 10 μM IP6 at pH 6 and pH 8. Mean above and n below box plots. Centre lines show medians; box limits indicate 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values. b, d, Representative negative stain electron microscopy images of wild-type and mutant s-CANC assembly in the absence (−) and presence (+) of 10 μM IP6 at pH 6 and pH 8. Scale bar, 400 nm. Repeated three times with similar results. e, Infectivity relative to wild-type virus of IP6 binding residues mutated to alanine and CA residue numbering in parenthesis. Error bars represent s.d., individual data points represented as dots; from four independent experiments.

Extended Data Fig. 6 IP6 modulates the stability of the 6HB.

a, Structural changes observed after 2 μs of molecular dynamics simulations of CACTDSP1 with and without bound IP6. b, RMSDs of the ligand-bound and unbound forms of the CACTDSP1 hexamer. c, RMSFs of the central hexamer during the simulation. The RMSF was averaged over the six central monomers; dashed line shows the s.d. for each residue.

Extended Data Fig. 7 Quantification of mature HIV-1 CA assembly and VLP diameter at pH 6.

a, Example of CA assembly in the absence of IP6 or mellitic acid. b, c, Representative negative-stain electron microscopy images of assemblies induced by IP6 (b) and mellitic acid (c). Scale bars, 200 nm. Tubes (T), cones (C), and other (O) morphologies are marked by coloured arrowheads. ac, Repeated four times with similar results. d, Number of assembled CA tubes (blue), cones (orange) and other (green) per 55 μm2 at increasing IP6 concentrations. Mean shown above plots, n = 5. e, Number of assembled tubes (blue), cones (orange) and other (green) per 55 μm2 at increasing mellitic acid concentrations. Mean shown above and n below box plots. f, Representative images of mature VLPs assembled with IP5 and IP6 at 50 mM NaCl. Scale bars, 100 nm. Repeated three times with similar results. g, Number of CA VLPs per 10 µm2 without and with IP3, IP4, IP5, and IP6. Mean shown above, n = 5. d, e, g, Centre lines show medians; box limits indicate 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values.

Extended Data Fig. 8 Crystal structure of IP6 bound to the mature CA hexamer.

a, b, Top view (a) and side view (b) of a second CA hexamer crystal structure (P212121 space group) showing the protein in yellow ribbons and unbiased mFoDFc difference density in blue mesh, contoured at 2.5σ. c, Close-up view showing IP6 densities both above and below the ring of Arg18 residues (magenta).

Extended Data Table 1 Crystallographic statistics

Supplementary information

Supplementary Figure 1

FACs gating strategy. a, Events were plotted along forward scatter (FSC) and side scatter (SSC) axises using FlowJo. Events with the right morphology were gated as "Live" cells and the position of the gate was copied onto all samples. b, Events from the "Live" gate were isolated and plotted along GFP and RFP intensity axes. The "Non-Fluorescent" gate was created based on a HEK293FT fluorescence negative sample (plot not shown) and the gate was copied onto all isolated "Live" samples. The "GFP-Positive" gate was created based on a HEK293FT GFP positive sample (plot not shown) and the gate was copied onto all isolated "Live" samples. Representative comparison of two cell types transduced with HIV Env deficient virus with a GFP reporter (HEK293FT = WT and IPPK KO = HEK293FT with inositol-pentakisphosphate 2-Kinase knocked out).

Reporting Summary

Video 1: 2-μs trajectories of IP6-unbound (left) and bound (right) CACTDSP1 models.

The protein hexamer is shown in cartoon representation and IP6 molecule is in stick representation.

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Dick, R.A., Zadrozny, K.K., Xu, C. et al. Inositol phosphates are assembly co-factors for HIV-1. Nature 560, 509–512 (2018). https://doi.org/10.1038/s41586-018-0396-4

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