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Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer

Nature Structural & Molecular Biology volume 24, pages 370–378 (2017)Cite this article

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A Corrigendum to this article was published on 06 June 2017

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Abstract

Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-Å resolution. A single CD4 molecule is embraced by a quaternary HIV-1–Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.

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Figure 1: Cryo-EM structure of the initial interaction between the HIV-1 Env trimer and the CD4 receptor.
Figure 2: Quaternary configuration of the CD4-binding site in the initial interaction with the HIV-1 Env trimer.
Figure 3: Charge inversions in CD4-BS2 reduce the CD4-binding capacity of the BG505 SOSIP.
Figure 4: CD4 surface interacting with CD4-BS2 in the initial contact with Env.
Figure 5: Quaternary interactions of selected antibodies to CD4-BS with the HIV-1 Env trimer.

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Change history

  • 27 April 2017

    In the version of this article initially published, funding information for B.C. and C.S.P. was missing NIH grant S10 OD019994-01. In addition, there was an incorrect comma in the introduction (after "glycoprotein" in the sentence "Upon binding to the primary cellular receptor, CD4, the external gp120 Env glycoprotein undergoes major conformational changes...") that has been removed. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank J.P. Moore and A. Cupo (Weill Medical College of Cornell University, New York, New York, USA) for providing plasmids for expression of the BG505 SOSIP.664 trimer and furin; M. Farzan (Scripps Research Institute, Jupiter, Florida, USA) for providing the plasmid to express human CD4-immunoglobulin; D.R. Burton (Scripps Research Institute, La Jolla, California, USA, and Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA), M. Connors (Laboratory of Immunoregulation, NIAID, NIH, Bethesda, Maryland, USA), J.R. Mascola (Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA), and J.E. Robinson (Tulane University School of Medicine, New Orleans, Louisiana, USA) for anti-gp120 mAbs; E. Berger (Laboratory of Viral Diseases, NIAID, NIG, Bethesda, Maryland, USA) for the full-length CD4 gene and the gene encoding gp160 from BG505-T332N and BaL; D. Ichikawa and Y. Lin for help in performing selected experiments; J. Arthos, E.A. Berger, and R. Cimbro for helpful discussion; P. Gangopadhyay for help with MATLAB and Origin; and the AIDS Reagent Program for providing the reagents indicated in the Online Methods. The GPU-enabled frame-alignment program we used was generously provided by Y. Cheng and X. Li. The cryo-EM work was done at the National Resource for Automated Molecular Microscopy based at the Simons Electron Microscopy Center, which is supported by grants from the NIH (GM103310 and S10 OD019994-01) and the Simons Foundation (349247) to B.C. and C.S.P. This research was supported by the Intramural Programs of the Vaccine Research Center and of the Division of Intramural Research, NIAID, NIH.

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Author notes

  1. Qingbo Liu and Priyamvada Acharya: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA

    Qingbo Liu, Peng Zhang, Christina Guzzo, Jacky Lu, Alice Kwon, Deepali Gururani, Huiyi Miao & Paolo Lusso

  2. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA

    Priyamvada Acharya, Tatsiana Bylund, Gwo-Yu Chuang, Aliaksandr Druz, Tongqing Zhou & Peter D Kwong

  3. National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, New York, USA

    Priyamvada Acharya, William J Rice, Christoph Wigge, Bridget Carragher & Clinton S Potter

  4. Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA

    Michael A Dolan

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Contributions

Q.L. and P.L. conceived the project and designed biological and virological studies; P.A., T.Z., and P.D.K. conceived and designed structural studies; Q.L., J.L., and D.G. mutagenized gp160 from different HIV-1 isolates and produced infectious pseudoparticles; Q.L., C.G., H.M., and A.K. carried out infectivity assays and antigenicity studies of surface-expressed Envs; Q.L., D.G., and P.Z. produced and purified mutants of SOSIP trimer and gp120 monomer; Q.L. characterized the purified proteins and performed immunogenicity studies and CD4-binding and CCR5-binding assays; A.D. expressed DS-SOSIP proteins for SPR and cryo-EM studies; Q.L. and P.A. purified CD4-BS2 mutants of DS-SOSIP trimer and performed SPR analysis of CD4 binding; P.A. purified DS-SOSIP, prepared the DS-SOSIP-CD4-PGT145 complex, and performed cryo-EM experiments, with W.J.R. and C.W. providing assistance with electron microscopy data collection; P.A., T.B., G.-Y.C., and T.Z. analyzed the structure; M.A.D. performed docking experiments and MD simulations; B.C., C.S.P., and P.D.K. supervised cryo-EM experiments and structural analyses; Q.L., P.A., P.D.K., and P.L. analyzed data and wrote the paper; and all coauthors read and commented on the manuscript.

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Correspondence to Paolo Lusso.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM structure determination of the DS-SOSIP trimer–sCD4–PGT145 complex.

(a) Representative micrograph (left and middle) with red circles (middle) showing the particles picked, and its Fourier transformed image (right) showing CTF fitting. (b) Representative 2D class averages from RELION. Red circles show examples of 2D classes with views of the trimer bound to CD4 and PGT145 Fab. (c) Maps of 3D classes from a RELION 3D classification run. Class I was further refined using RELION autorefine. (d) Angular distribution of particles contributing to the final reconstruction. (e) Masks used for RELION autorefine, and corresponding gold standard FSC plots.

Supplementary Figure 2 Density shots of various regions of the DS-SOSIP trimer–sCD4–PGT145 complex.

The blue mesh represents the cryo-EM map. Zoomed-in views are shown for (a) the CDR H3 loop of PGT145 binding to a proteoglycan epitope at the interface of the three gp120 protomers in the Env trimer; (b) the gp120-gp41 interface; and (c) the trimer-CD4 interface showing continuous, well-defined CD4 electron density wedged between two gp120 protomers.

Supplementary Figure 3 Infectivity of HIV-1 BaL pseudotypes with individual and combined mutations in and around the CD4-BS2 region.

The infectivity of each mutant was calculated relative to that of the wild-type (WT). The data shown represent the mean ± range of duplicate wells from a representative experiment out of three performed which yielded similar results.

Supplementary Figure 4 Reactivity with anti-gp120 monoclonal antibodies and electrophoretic mobility of BG505-SOSIP.664 trimers and their mutants.

(a) A panel of anti-gp120 mAbs was used in ELISA to verify the correct folding of purified BG505-SOSIP.664 and DS-SOSIP.664 trimers and their mutants expressed in HEK 293FS cells. Of note, mAb F105, which reacts well with monomeric gp120 but not with the functional trimer, was virtually negative on all trimers, whereas trimer-preferring mAbs (eg, PG9, PGT145, PGT151) showed similar binding levels to the WT and the mutants, indicating the correct folding of the trimers. Optical density (OD) values are shown for the WT (top row), while binding relative to WT is shown for all mutants. (b) The correct size and cleavage of recombinant trimers were assessed by SDS-PAGE under reducing and non-reducing conditions.

Supplementary Figure 5 Effect of mutations in CD4-BS2 on CD4 binding to the HIV-1 Env trimer.

(a) Binding of WT and mutated BG505 SOSIP.664 soluble trimers to sCD4 as determined by ELISA. Purified soluble trimers were captured by MAb 2G12 immobilized to the plate and tested against serial dilutions of sCD4. The table on the right side reports area-under-the-curve (AUC) values and p values for the comparison between each mutant and WT as assessed by unpaired t-test. The data shown represent the mean ± range of duplicate wells from a representative experiment out of three performed which yielded similar results. (b) Binding of WT and mutated native, cell surface-expressed Env trimers to CD4-Ig as determined by flow cytometry. HEK 293T cells were transfected with plasmids encoding WT or mutated full-length gp160 and tested after 48-72 hours for CD4-Ig surface binding. The data shown are from a representative experiment out of two performed which yielded similar results.

Supplementary Figure 6 Effects of CD4-BS2 mutations in monomeric gp120.

(a) Binding of soluble CD4 (sCD4) to WT and mutated monomeric gp120 from BG505-T332N and BaL, as determined by ELISA. The data shown represent the mean ± range of duplicate wells from a representative experiment out of three performed which yielded similar results. (b) Binding of CCR5 (left) and mAb 48d (right) to WT and mutated monomeric BaL gp120, as determined by flow cytometry and ELISA, respectively. The data shown are from a representative experiment out of three performed which yielded similar results. (c) Reactivity of WT and mutated monomeric gp120 from BG505-T332N and BaL with a panel of anti-gp120 mAbs, as determined by ELISA.

Supplementary Figure 7 Spatial orientation of CD4-BS2 residues in the structures of trimeric versus monomeric gp120.

Upper panels: side view with the key residues in CD4-BS2 (K207, E62, E64 and H66) highlighted by stick representation and color code based on charge (blue, positive; red, negative). Lower panels: front view, surface representation. In the trimer, the side chains of CD4-BS2 residues form a continuous, electrostatically-charged surface that is totally exposed to the solvent; in contrast, in monomeric gp120, the side chains and relative charges are partially or completely buried. Of note, the spatial orientation of these residues is nearly identical in all the available monomeric gp120 structures, regardless of the presence and nature of co-crystallized ligands (see Supplementary Table 1).

Supplementary Figure 8 Mutations in the CD4-BS2 contact site of CD4 affect HIV-1 entry.

(a) Full-length human CD4 mutants were expressed in Cf2Th/syn-CCR5 cells and infected with luciferase-expressing pseudoviruses carrying WT BaL or BG505-T332N Env. Relative infectivity values for the mutants were calculated as percent of the value obtained with WT CD4. The data shown represent the mean ± range of duplicate wells from a representative experiment out of two performed which yielded similar results. (b) Reactivity of WT and mutated CD4 with anti-CD4 mAbs tested by flow cytometry. Mean fluorescence intensity (MFI) values are shown for WT (top row), while MFI relative to WT is shown for each mutant.

Supplementary Figure 9 Somatic hypermutation-evolved interaction of human anti-HIV-1 Env mAbs with CD4-BS2.

(a) Structural alignment of VRC01-class antibodies. Negatively-charged residues that interact with CD4-BS2 are highlighted in green. CD4-BS2 interaction through antibody framework 1 (b) and 3 (c). Somatic hypermutation-evolved acidic residues in both antibody framework 1 and 3 show no interaction with gp120 in complexes with monomeric core gp120 (upper row). Models in the trimer context (bottom row) with antibodies docked through gp120 outer-domain superposition indicate interaction with positively-charged CD4-BS2 residues on the neighboring protomer. Electrostatic potential surfaces for negatively-charged tips of framework 1 and 3 and the neighboring gp120 protomer are shown in the panels on the right.

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Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1–3 (PDF 4821 kb)

Supplementary Table 4

BSA for various trimers (XLSX 20 kb)

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Liu, Q., Acharya, P., Dolan, M. et al. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer. Nat Struct Mol Biol 24, 370–378 (2017). https://doi.org/10.1038/nsmb.3382

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