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Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy

Nature Structural & Molecular Biology volume 20, pages 1352–1357 (2013)Cite this article

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Abstract

The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target-cell membranes. Here, we present the cryo-EM structure, at subnanometer resolution (∼6 Å at 0.143 FSC), of the 'closed', prefusion state of trimeric HIV-1 Env complexed to the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the 'open' quaternary conformation. The architecture of trimeric HIV-1 Env in the prefusion state and in the activated intermediate state resembles the corresponding states of influenza hemagglutinin trimers, thus providing direct evidence for the similarity in entry mechanisms used by HIV-1, influenza and related enveloped viruses.

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Figure 1: Structure of the prefusion state of trimeric HIV-1 Env bound to VRC03.
Figure 2: Detailed view of gp120 and gp41 structural elements.
Figure 3: Molecular structure of soluble trimeric HIV-1 Env in the closed state.
Figure 4: Comparison of structures of trimeric Env in the closed, prefusion and open, activated conformations.
Figure 5: Comparison of the structures of influenza hemagglutinin and HIV-1 Env trimers in different states.

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  • 10 November 2013

    In the version of this article initially published online, the NIH-FEI Living Lab for Structural Biology was not mentioned in the Acknowledgments section. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

This work was supported by funds to S.S. and J.L.S.M. from the Center for Cancer Research at the National Cancer Institute, US National Institutes of Health (NIH), and to S.S. from the NIH Intramural AIDS Targeted Antiviral Program. We thank J. Mascola (Vaccine Research Center, NIH) for providing VRC03 antibodies; K. Kang and W. Olson (Progenics) for providing soluble KNH1144 gp140 trimers; S. Fellini, S. Chacko and their colleagues for continued support with use of the Biowulf cluster for computing at NIH; D. Schauder and H. He for assistance with data collection; P. Rao and the NIH-FEI Living Lab for Structural Biology for assistance with collection of the tilt-pair images; and L. Earl for helpful discussions and comments.

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Authors and Affiliations

  1. Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Alberto Bartesaghi, Alan Merk, Mario J Borgnia, Jacqueline L S Milne & Sriram Subramaniam

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  1. Alberto Bartesaghi
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Contributions

A.B., A.M., M.J.B., J.L.S.M. and S.S. analyzed and interpreted data; S.S. was responsible for data collection; all authors helped compose the manuscript.

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Correspondence to Sriram Subramaniam.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cryo-electron microscopic imaging of the Env-VRC03 complex.

(a, b) Representative images recorded at a dose of ∼10 electrons/Å2 and underfocus values of ∼2.6 m (a) and ∼1.5 m (b) (leftmost panels). Scale bar is 400 Å Band-pass filtered versions of these images allows easier particle visualization (middle panels). Rotationally averaged FFTs of each image show signal from the Thon rings extending to ∼8-Å for micrographs at higher defocus values and to ∼6-Å for micrographs at lower defocus values (rightmost panels). (c) Gallery of selected initial class averages obtained by classification of single particle projection images without any prior translational or rotational alignment reveals the characteristic views of the VRC03-bound gp120 trimer.

Supplementary Figure 2 Map resolution and validation.

(a, b) Maps obtained from FREALIGN20 (a) and RELION21 (b) shown in top view, fitted with PDB 3SE8 (ref. 23) coordinates. (c) Plots of the Fourier Shell Correlation (FSC) coefficient for the structure of the Env-VRC03 complex. The curves show the “gold-standard” curve obtained using RELION and the curve obtained from the correlation of two halves of the data set obtained using FREALIGN, both indicating a resolution value of ∼6-Å as measured by the 0.143 FSC cutoff criterion. (d) Validation of density map using tilt-pair parameter plot as suggested by R. Henderson and colleagues44. The spread in orientational assignments around the known goniometer settings is within ∼12.5° for >62% of the selected particle pairs, consistent with the ∼6-Å resolution reported for the maps.

Supplementary Figure 3 Correspondence between 6-Å and 20-Å density maps.

(a) 20-Å structure of the complex of VRC03 with native trimeric Env obtained using cryo-electron tomography of intact HIV-1 (ref. 14). Side views (left) and top views (right) of the density map, fitted with three copies of the structure of the complex of VRC03 Fab with gp120 (PDB 3SE8 (ref. 23)). The 2D cryo-electron microscopic images in Figure 1 can be recognized as projections of the propeller-shaped 3D structure of the Env-VRC03 complex shown here. (b) Side (left) and top views (right) of the superposition of the density map of soluble trimeric HIV-1 Env-VRC03 Fab complex (obtained using cryo-electron microscopy at ∼6-Å resolution) with density map of the native trimeric HIV-1 Env-VRC03 complex (obtained using cryo-electron tomography at ∼20-Å resolution).

Supplementary Figure 4 Change in appearance of density map with filtering to lower resolutions.

(a-c) Side views of the 6-Å density of map of the complex of VRC03 Fab with soluble trimeric HIV-1 Env, filtered to resolutions of 10-Å (a), 15-Å (b) and 20-Å (c). As the resolution is progressively lowered, the overall shape of the map is unaltered, but the central helices which are prominently resolved at 10-Å resolution, fade into the background at resolutions of 20-Å or lower, resulting in what has the appearance of a central cavity that is observed in the tomographic density maps of unliganded and VRC03-bound trimeric Env.

Supplementary Figure 5 Comparison of the structure of the closed, prefusion conformation of soluble trimeric HIV-1 Env presented in this work with that reported by Mao, Sodorski and colleagues for ligand-free, tail-truncated, trimeric HIV-1 Env.

(a) Side (top) and top view (bottom) of the map presented in this work (identical to that presented in Figure 3). (b) Side (top) and top view (bottom) of map reported by Mao, Sodroski and colleagues (EMD-5447 (ref. 24)).

Supplementary Figure 6 Structure of the open, activated trimeric Env conformation.

Side (top) and top view (bottom) of the 9-Å density map of the open state of trimeric Env stabilized in a complex with the Fab fragment of the 17b monoclonal antibody. The map is fitted with three copies of PDB 3HMG (ref. 39) for the gp41 central densities (cyan) and three copies of gp120 (red) and the Fv fragment of 17b (green) derived from the coordinates for the structure of the gp120-sCD4-17b complex (PDB 1GC1 (ref. 2)).

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Bartesaghi, A., Merk, A., Borgnia, M. et al. Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy. Nat Struct Mol Biol 20, 1352–1357 (2013). https://doi.org/10.1038/nsmb.2711

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