Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins | Nature Structural & Molecular Biology

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Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell–cell fusion events to establish the maternal–fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid β-sheet or ‘clamp’ with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions.

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Structural models of ASCT2WT–SYCY1RBD at 2.62 Å and 3.5 Å resolution, ASCT2CO–SUPYNRBD and ASCT2WT–Nb469 complexes have been deposited to the PDB with accession codes 8OUH, 8OUJ, 8OUI and 8OUD, respectively, and the corresponding cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-17192, EMD-17194, EMD-17193 and EMD-17189. Materials are available upon reasonable request. Source data are provided with this paper.

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IECB cryo-EM imaging facility is acknowledged for support in cryo-EM sample screening and initial data acquisition, and the EMBL-Heidelberg Cryo-Electron Microscopy Service Platform for support in image acquisition. R. Assal for preliminary protein production and construct screening. This research was funded by the European Research Council (ERC) under the European Union Horizon 2020 Program (grant no. 771965), with additional contributions from the Institut National du Cancer (INCA grant 2017-44), IDEX Senior Chair Universite de Bordeaux and Region Nouvelle-Aquitaine (grant no. 8166910), all to N.R., Instruct-ERIC (PID6507), as part of the European Strategy Forum on Research Infrastructures (ESFRI), and the Research Foundation-Flanders (FWO) for nanobody discovery. A. Lundqvist for technical assistance during nanobody discovery. The Swiss National Science Foundation (SNSF) (PP00P3_198903 and 310030_207974 to CP) supported the work performed by the group of C.P. F.A.R. and M.B. acknowledge funding from grant ANR-10-LABX-62-IBEID.

These authors contributed equally: Shashank Khare, Miryam I. Villalba, Juan C. Canul-Tec, Arantza Balsebre Cajiao, Anand Kumar.

Fundamental Microbiology and Pathogenicity Unit, CNRS, Université de Bordeaux, IECB, Bordeaux, France

Shashank Khare, Miryam I. Villalba, Juan C. Canul-Tec, Anand Kumar & Nicolas Reyes

Biozentrum, University of Basel, Basel, Switzerland

Arantza Balsebre Cajiao & Camilo Perez

Institut Pasteur, Université Paris Cité, CNRS UMR3569, Unité de Virologie Structurale, Paris, France

Marija Backovic & Felix A. Rey

Structural Biology Brussels, Vrije Universiteit Brussel, VUB, Brussels, Belgium

Els Pardon & Jan Steyaert

VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium

Els Pardon & Jan Steyaert

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA

Camilo Perez

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S.K., J.C.C.-T. and A.K. performed protein purification, cryo-EM sample preparation and image analysis, as well as atomic model building and refinement. S.K. and M.I.V. performed binding assays, and M.I.V. performed cell fusion assays, as well as optimized, screened and produced the HERV-derived proteins. M.I.V. and J.C.C.-T. produced the proteoliposomes. A.B.C. and C.P. performed and analyzed transport and electrophysiology assays with proteoliposomes and binders. M.B. and F.A.R. contributed to the design and development of soluble HERV constructs. E.P. and J.S. designed and performed nanobody development. N.R. analyzed functional and cryo-EM data, as well as structures. All authors contributed to the preparation of the manuscript. N.R. conceived and supervised the project.

Correspondence to Camilo Perez or Nicolas Reyes.

A.K. is currently an employee of the Integrated Drug Discovery, Sanofi R&D, France. All other authors declare no competing interests.

Nature Structural & Molecular Biology thanks Winfried Weissenhorn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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a, Amino acid sequence alignment of ASCT2WT and ASCT2CO. b, Cell-cell fusion assay based on split-GFP. ASCT2CO shows similar levels of syncytin-1 mediated cell-cell fusion than ASCT2WT. Purified SYCY1∆439 and suppressyn constructs recognize ASCT2WT and inhibit syncytin-1 mediated cell-cell fusion. The syncytin-1 mutant C186S precludes cell fusion and it is used as a negative control to account for background fluorescence. c, Reconstituted ASCT2CO yields nearly 5-fold higher radioactive substrate uptake in proteoliposomes compared to ASCT2WT. In b, and c bars depict averages of at least three biologically independent experiments, and error bars represent SEM. Circles represent values from individual experiments.

Source data

a, SEC profile of the ASCT2CO-suppressyn complex. b, SEC profile of the ASCT2WT-SYCY1∆469 complex. c, Binding curves of suppressyn (triangles) and SYCY1∆469 (circles) to ASCT2CO in detergent solution. Empty symbols depict the averages of 3 biologically independent experiments, and error bars represent SEM. Solid lines are fits of the Hill equation with APPKD and Hill coefficient values of 1 μM and 1.7, respectively, for SYCY1∆469, and 0.2 μM and 1.5, respectively, for suppressyn.

Source data

a, Representative EM micrograph. 15,376 micrographs were taken in total. b, Gallery of representative 2D class-averages. c, 3D classes from ab initio classification. d, Local refined map and Fourier Shell Correlation (FSC) plot with FSC threshold at 0.143. e, Viewing direction distribution plot. f, The 2.6 Å map (overall resolution) is coloured based on local resolution with the scale bar in angstroms. g, 3D ab-initio class after reprocessing 640k particles to improve the density of the third tranD. h, Local refined map and corresponding FSC plot with threshold at 0.143. i, Viewing direction distribution plot. j, The 3.5 Å map (overall resolution) is coloured based on local resolution with the scale bar in angstroms.

a, Representative EM micrograph. 8,455 micrographs were taken in total. b, Gallery of representative 2D class-averages. c, 3D classes from ab initio classification. d, 3D class from heterogeneous refinement. e, Local refined map and Fourier Shell Correlation (FSC) plot with FSC threshold at 0.143. f, Viewing direction distribution plot. g, 3D-Flex reconstruction map and corresponding FSC plot with threshold at 0.143. h, The 3.6 Å map (overall resolution) is coloured based on local resolution with the scale bar in angstroms.

a, Cryo-EM density corresponding to individual ASCT2WT and SYCY1RBD structural elements. b, Cryo-EM density corresponding to individual ASCT2CO and SUPYNRBD structural elements.

a, Sequence alignment of the SYCY1RBD and SUPYNRBD. Disulfide bonds (S1, S2, and S3) and the secondary structural elements observed in the cryo-EM structures are marked above (SYCY1RBD) and below (SUPYNRBD) the alignment rows. b, Sequence alignment of the RBDs of simian retrovirus 3 (SRV3RBD), avian reticuloendotheliosis virus (REVRBD), simian type D retrovirus (RD114RBD), avian spleen necrosis virus (SNVRBD), and sycncytin-1. Disulfide bonds (S1, S2, and S3) and the secondary structural elements observed in the ASCT2WT-SYCY1RBD cryo-EM structure are marked.

a, Determination of ASCT2CO orientation in proteoliposomes. ASCT2CO N-terminal fused to GFP was reconstituted in proteoliposomes and incubated in the presence or absence of 3C-protease. In-gel fluorescence shows profiles of technical triplicates of non-cleaved and cleaved (inside−out orientation) GFP-ASCT2CO in proteoliposomes. b, Isothermal titration calorimetry analysis of Nb469 binding to ASTCCO at 25 °C in detergent solutions. The black line in the lower panel is the fit of the quadratic binding equation (“one site binding model”) with the following parameters: Nb469:ASCTCO-protomer=1.1; KD = 360 nM; ∆H = 6.3 kcal/mol; ∆S = 8.0 cal/mol/deg.

Source data

a, Representative EM micrograph with examples of individual picked particles (green circles). 4,344 micrographs were taken in total. b, Gallery of representative 2D class-averages. c, 3D classes from ab initio classification. d, Non-uniform refinement map. e, Non-uniform refinement map is coloured based on local resolution. f, Viewing direction distribution plot. g, Fourier Shell Correlation (FSC) plot of the non-uniform refinement with FSC threshold at 0.143.

a, Cryo-EM density around different structural elements of ASCT2CO. b Cryo-EM density around the complementary-determining regions of the Nb469. c, Cryo-EM density around the bound sodium ions (purple spheres), structural waters (red spheres), and L-ala substrate (green sticks). The tranD is shown as ribbon.

Supplementary discussion, Fig. 1 and Tables 1 and 2.

Experimental data points.

Unmodified gel.

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Khare, S., Villalba, M.I., Canul-Tec, J.C. et al. Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins. Nat Struct Mol Biol 31, 1368–1376 (2024). https://doi.org/10.1038/s41594-024-01295-6

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Received: 26 June 2023

Accepted: 26 March 2024

Published: 26 April 2024

Issue Date: September 2024

DOI: https://doi.org/10.1038/s41594-024-01295-6

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