Abstract
Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIVmac) in complex with neutralizing antibodies. These structures provide residue-level definition for SIV-specific disulfide-bonded variable loops (V1 and V2), which we used to delineate variable-loop coverage of the Env trimer. The defined variable loops enabled us to investigate assembled Env-glycan shields throughout SIV, which we found to comprise both N- and O-linked glycans, the latter emanating from V1 inserts, which bound the O-link-specific lectin jacalin. We also investigated in situ SIVmac-Env trimers on virions, determining cryo-electron tomography structures at subnanometer resolutions for an antibody-bound complex and a ligand-free state. Collectively, these structures define the prefusion-closed structure of the SIV-Env trimer and delineate variable-loop and glycan-shielding mechanisms of immune evasion conserved throughout SIV evolution.
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Data availability
Cryo-ET maps for the ITS90.03-bound spike and the unliganded spike have been deposited at the Electron Microscopy Data Bank (EMDB) with accession codes EMD-25065 and EMD-25064, respectively. Cryo-EM maps and fitted coordinates have been deposited at the Electron Microscopy Data Bank with accession codes EMD-27718, EMD-27735 and EMD-27631 and to the Protein Data Bank with IDs 8DUA and 8DVD.
Code availability
The program GLYCO is available at https://github.com/myungjinlee/GLYCO.
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Acknowledgements
We thank J. Hoxie of the University of Pennsylvania for the SUPT1-CCR5 cell line, J. Stuckey of the Vaccine Research Center for assistance with figures, members of the Structural Biology Section and Structural Bioinformatics Core, Vaccine Research Center for discussions or comments on the manuscript, and the HIV Reagent Program for ARP-829 and ARP-830. Support for this work was provided by the Intramural Research Programs of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health and from the National Cancer Institute, National Institutes of Health under contract nos. HHSN261200800001E and 75N91019D00024. Some of this work was performed at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247) and NIH National Institute of General Medical Sciences (GM103310), with additional support from NYSTAR and the New York State Assembly. Cryo-ET work was performed at the Yale Cryo-EM Resource, which is funded in part by NIH grants nos. 1S10OD023603-01A1 and R01AI150560. This work utilized the computational resources of the NIH HPC Biowulf cluster.
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Contributions
Conceptualization was provided by J.G., T.Z., R.D.M., M.R. and P.D.K., ITS92.02 isolation and neutralization by R.D.M. and H.C.W., protein expression by Y.Y., B.Z. and R.V., protein purification by T.Z. and A.F.N., cryo-EM structure determination and analysis by J.G., molecular dynamics by M.L., negative stain electron microscopy by Y.T., SIV Env divergence by T.B., virion production and characterization by J.W.B., B.F.K. and J.D.L. and cryo-ET structure determination and analysis by C.W. and J.L. The first draft was written by J.G., and all authors provided feedback and edits.
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Nature Structural & Molecular Biology thanks James Hoxie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Beth Moorefield, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Antigenic screening and HRV 3 C purification through capture of co-expression enriches for minority population of trimeric Env.
a, Sequence homology between HIV-1 and several SIV strains is shown. b, The 96-well screening results are presented as red-white heatmap with the top scoring constructs enlarged on the right panel showing their specific values for HIV-1 and SIV antibodies. The bottom-right table summarized the screening antibody epitopes. The ITS92 epitope was unknown during the screening and the accompanying structures revealed it to be a gp41 conformationally dependent epitope. c, Negative stain 2D images show a small population of trimers, highlighted here in the green squares. d, Schematic of the purification strategy is shown with on column binding of the supernatant to ITS92.02 followed by HRV 3 C cleavage, allowing the avoidance of harsh purification buffers. A similar strategy was used for PGT145 but included co-expression with the HRV 3 C IgG.
Extended Data Fig. 2 Cryo-EM analysis of SIVE660.CR54 SOS-2P in complex with ITS92.02 reveals a trimer with high flexibility at the apex.
a, Representative micrograph and CTF of the micrograph are shown. 8,259 micrographs were collected in total. b, Representative 2D class averages are shown. c, The gold-standard Fourier shell correlation resulted in a resolution of 4.32 Å using non-uniform refinement with C3 symmetry. d, The orientations of all particles used in the final refinement are shown as a heatmap. e, The local resolution of the full map is shown generated through cryoSPARC using an FSC cutoff of 0.5. f, A wild-type sequence alignment near the ITS92.02 epitope is shown highlighting residues within a 5 Å footprint in red. We note that non-conserved residue E613 forms a salt bridge with R94 of the ITS92.02 heavy chain. g, The prefusion (magenta) and postfusion (gray, PDB ID 1QBZ) conformations of gp41 protomers are aligned with the prefusion footprint residues colored red and the corresponding residues in the postfusion conformation are shown in blue. Footprint residues between positions 638 and 652 (HXB2 numbering) align well, residues from 608–614 extend away from the helical region of the epitope.
Extended Data Fig. 3 Cryo-EM analysis of SIVmac239 SOS-2P in complex with PGT145 contains a stabilized apex region.
a, Representative micrograph and CTF of the micrograph are shown. 6,255 micrographs total were collected. b, Representative 2D class averages are shown. c, The gold-standard Fourier shell correlation resulted in a resolution of 4.12 Å using non-uniform refinement with C1 symmetry. d, The orientations of all particles used in the final refinement are shown as a heatmap. e, The local resolution of the full map is shown in two contours. Maps were generated through cryoSPARC using an FSC cutoff of 0.5.
Extended Data Fig. 4 SIVmac239 sequence and structural comparisons.
a, The SIVmac239 trimer aligns well to the CD4-bound SIVmac239 core (PDB: ID 6TYB). The glycan shield shows strong similarity for most glycans available in the core with notable differences at positions 47 and 464 where sequons were not glycosylated in the core and position 88 where the gp41 glycan at position N625 overlaps in space with N88 of the core. b, A sequence alignment of HIV-1 (BG505) and SIV (mac239) are shown with secondary structure shown below. Notable positions are indicated for SOSIP mutations. c, The Cα residue-by-residue distances are shown for HIV-1 and SIVcpz compared to residues of SIVmac239 indicating areas of close structural alignment and highlighting regions that diverge significantly.
Extended Data Fig. 5 CD4 binding details in SIVmac239 and HIV-1.
a, (left) SIVmac239 gp120 from the trimer structure (magenta) is shown aligned to the CD4-bound SIVmac239 gp120 core (gray). (right) SIVmac239 gp120 from the trimer structure (magenta) is shown aligned to an HIV-1 BG505 gp120 from a trimer (light blue). b, (left) The CD4 (yellow) binding site is shown corresponding to the structural alignments in panel a. W427 adjusts out of the pocket that the F43 of CD4 inserts into. (right) Residue 375, used to confer rhCD4 binding to SHIV, is shown in red. Relative positions of W427 in SIV and HIV-1 are shown proximal to the F43 pocket. c, Conformations of the region from residues 50–80 are shown. The region adopts various conformations in SIV and HIV-1 and undergoes a switch upon CD4 binding.
Extended Data Fig. 6 Details of the primate immunodeficiency viruses conserved features.
a, An additional disulfide was observed in one branch which placed cys at residues 165 and 168, stabilizing the turn between the B and C strands of V1V2. b, Disulfides observed in the hypervariable V1 loop are highlighted in purple. Due to poor alignment confidence in this region the sequences are shown from residue cys157 conserved in all viruses in the tree. c, The HIV-2 glycan shield is modeled as in Fig. 5e with minor deviations from SIVmac239. d, The fusion peptide region of SIVmac239 adopts a helical structure and abuts the core of the Env contrary to the flexible conformations observed in HIV-1 Env (BG505, PDB ID: 5FYL). We note that the FP is one residue longer in the viruses more proximal to HIV-1.
Extended Data Fig. 7 Impact of O-linked saccharides on glycan shielding.
a, Glycan shielding overlaid on SIVmac239 surfaces viewing from side (top panel) and top (bottom panel). b, Glycan coverage of epitope CD4bs (CD4 binding site), PGT145 and V3 regions of SIVmac239. The epitope regions are shown (top middle and top right panels) with the same color code as the plot (top left).
Extended Data Fig. 8 N-linked glycan conservation across diverse primate immunodeficiency viruses.
(top) Bar graph depicting the percent conservation of N-linked glycan sequon positions (HXB2 numbering from 1–683) for the 34 viruses depicted in the phylogenetic tree of Fig. 4. Hypervariable regions extending beyond HXB2 numbering are excluded due to high variability and low confidence alignments. * at positions 143 and 185 denotes artificially high bars due to multiple glycans in inserts. Dotted horizontal lines are shown at 50% and 75% conservation and locations above these thresholds are labeled. (bottom) A detailed plot of sequon locations for all 34 sequences in the phylogenetic tree of Fig. 4. Locations are colored as in Fig. 4b.
Extended Data Fig. 9 Cryo-ET density fit to the model with MPER region extended from the membrane.
a, A slice through a representative tomogram shows multiple SIV virions with Env spikes, black scale bar in the bottom right represents 50 nm. 55 tomograms of SIVmac239 and 105 tomograms of SIVmac239 complex with ITS90.03 were collected. b, FSC curves of the ITS90.03-bound (blue) and unliganded SIV (black) sub-tomogram averages. c, Superposition of the ITS90.03 bound crystal structure with that of the SIV trimer based on the gp120 alignment. d, Glycan N625 of the SIVmac239 trimer must re-orient to accommodate the binding of ITS90.03. e, The V1 insertion region is observed in the ITS90.03-bound and unliganded cryo-ET density. f, Side view of the gp41 density with the gp41 built from the soluble map fit to the density. g, Side view to panel d is shown. h, The pedestal at the base of the gp41 region in HIV-1 Bal is shown in yellow, segmented from density of EMDB map EMD-21412. Coordinates are shown for BG505 SOSIP.664. i, The side of gp41 is shown with the MPER region density shown in gold. j, The fit of the MPER region in relation to the membrane surface density. k, The pedestal at the base of the gp41 region in HIV is shown in yellow with membrane density shown.
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Gorman, J., Wang, C., Mason, R.D. et al. Cryo-EM structures of prefusion SIV envelope trimer. Nat Struct Mol Biol 29, 1080–1091 (2022). https://doi.org/10.1038/s41594-022-00852-1
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DOI: https://doi.org/10.1038/s41594-022-00852-1
