SUPPLEMENTARY INFORMATION

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1 Structure of the Ebola virus glycoprotein bound to a human survivor antibody Jeffrey E. Lee, Marnie L. Fusco, Ann J. Hessell, Wendelien B. Oswald, Dennis R. Burton and Erica Ollmann Saphire* Department of Immunology and Microbial Science, The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA USA *Corresponding author (erica@scripps.edu) SUPPLEMENTAL MATERIALS Mechanism of Ebola virus infection The first step of viral-host membrane fusion requires triggering of the metastable, prefusion GP to release the GP1 clamp on GP2. However, the fusogenic trigger of EBOV GP is not well understood. The low ph of the endosome is necessary for infection of GPpseudotyped VSV and required for GP-mediated cell-cell fusion 1. However, it has been suggested that low ph dependence is not required for triggering, but rather, for optimal functioning of endosomal proteases 2. These results are consistent with the EBOV GP structure, as the GP1-GP2 interface is primarily non-polar and hydrophobic, and does not contain the array of histidine or acidic residues usually associated with ph-dependent conformational change in viral glycoproteins 3-8. Furthermore, recognition of GP by the conformational KZ52 antibody is independent of ph (Supplemental Fig. S11). It has been suggested that cleavage by cathepsins L and B triggers the fusogenic conformation 9 or primes EBOV GP for an additional cellular factor, such as a reductase 2. This structure suggests that a third triggering mechanism is also possible, in which proteolytic removal of the mucin-like domain and glycan cap activates or enhances the receptor-binding potential of EBOV GP. Subsequent post-receptor binding interactions with an endosomal cofactor may induce conformational change and trigger the fusogenic state. Release of constraints imposed on GP2 by GP1 will allow the heptad repeat regions of GP2 to undergo significant conformational change (Supplemental Fig. S9). Comparison of pre-fusion EBOV GP and post-fusion GP2 fragment structures 10,11 reveals 1

2 SUPPLEMENTAL METHODS Ebola virus GP infectivity and neutralization. Physiological relevance of the Ebola virus T42V/T230V GP muc crystallization construct was established using a pseudotyped vesicular stomatitis virus (VSV) neutralization assay 13. Constructs encoding the EBOV full-length GP, GP muc, and T42V/T230V GP muc were subcloned into the pcaggs mammalian expression vector. Six-well plates coated with 0.001% (w/v) poly-l-lysine were inoculated with 9 X 10 5 HEK293T cells at 37 C, 5% CO 2 in Opti-MEM media (Invitrogen, Carlsbad, CA) and grown overnight at 37 C and 5% CO 2 to 90% confluency prior to transfection of GP-encoding vectors using Lipofectamine Cells were again grown overnight at 37 C with 5% CO 2. The resulting GP-, GP muc-, and T42V/T230V GP muc-expressing HEK293T cells were infected with 9.0 X 10 6 transducing units of VSV-pseudovirions (MOI=3) with a GFP reporter gene replacing the native envelope G protein (VSV- G). Cells were incubated in Opti-MEM with 2% (v/v) FBS (Opti-MEM-FBS) for 1 hour at 37 C, washed five times with Opti-MEM-FBS and incubated overnight in a solution of Optidoi: /nature07082 that, during this process, the kink between the pre-fusion HR1 A and HR1 B segments straightens and HR1 C undergoes a loop to helix transition to extend HR1 D into a single 44-residue helix encompassing all of HR1 (Supplemental Fig.S5). HR1 A and HR1 B undergo a rotational and translational movement of ~40 Å to position the internal fusion loops at the top of the trimeric GP2 (Supplemental Fig. S9 and Supplemental Movie 1). The disulfide bond and antiparallel strands appear critical for maintenance of the overall loop structure and importantly, may provide a path to physically translate forces of conformational change from HR1 to the fusion loop. The residues from the hydrophobic fusion peptide insert into the host membrane and probably adopt a 3 10 helix conformation, as suggested by an NMR model of the fusion loop determined in the presence of a detergent-resistant membrane fraction 12. Subsequently, HR2 swings from the viral membrane-proximal surface towards the host membrane-proximal surface, ultimately packing with HR1 to form the post-fusion GP2 six-helix bundle. As a result, both the internal fusion loop and transmembrane domain become juxtaposed, facilitating fusion of the host and viral membranes and viral entry. 2

3 MEM-FBS with 10% (v/v) Pen/Strep and 10% (v/v) L-glutamine. The supernatant, containing VSV-pseudovirions complemented with EBOV GP, was harvested, clarified and frozen at -80 C in 1 ml aliquots. To test whether our GP variants affect EBOV infectivity or KZ52 neutralization, round-bottom 96-well plates were inoculated with 0.5 X 10 5 Vero E6 cells and grown at 37 C for 28 hours. KZ52 human antibody was incubated in incremental concentrations from 0.1 μg/ml to 316 μg/ml with Opti-MEM and clarified GP-, GP muc-, or T42V/T230V GP muc-containing VSV-pseudovirus supernatant for 1 hour at 37 C. The resulting antibody-pseudovirus mixture was then incubated with uninfected cells, replacing the uninfected cell supernatant. After an hour, DMEM containing 10% (v/v) Pen/Strep, 10% (v/v) L-glutamine and 2% (v/v) FBS was added and cells were grown overnight. Cells were washed with PBS, dissociated from the plate using trypsin and separated using a 25-gauge needle prior to loading on a high-throughput BD FACSCalibur unit (BD Biosciences, San Jose, CA). Data were processed using the FlowJo v program (TreeStar, Ashland, OR) and IC 50 values were calculated in GraphPad Prism4 (GraphPad, San Diego, CA). The number of infected cells in wells with VSV-pseudovirus (no KZ52 antibody) was used as a baseline for calculations of the relative infection percentages. All experiments were performed in triplicate. KZ52 recognition of GP at low ph. Immunoaffinity-purified T42V/T230V GP muc was buffer exchanged using an Ultrafree K cut-off centrifugal concentrator into seven different ph solutions (ph 4.0, 4.6, 5.0, 5.6, 6.0, 6.6 and 7.0), and incubated at room temperature for 2 hours (ph verified using litmus paper). Each solution contained 150 mm NaCl and 10 mm buffer, made by dilution of a 1M Hampton Research ph Screen stock. After ph incubation, samples were neutralized to a final concentration of 100 mm Tris-HCl ph 7.5 (note: conformational changes of class I GP are typically irreversible). Each sample was analyzed by a non-denaturing immunoblot using human IgG KZ52 or mouse IgG 16B12 anti-ha monoclonal primary antibodies (1:2000 dilution) and goat anti-human or anti-mouse peroxidase-conjugated secondary antibody (1:1000 dilution) followed by SigmaFAST BCIP/NBT. 3

4 Table S1. Crystallographic data statistics Data collection Space group H32 Unit cell a=b=273.1 Å, c=409.7 Å, = =90, =120 Wavelength (Å) Resolution (Å) No. of reflections 626,036 No. of unique reflections 151,305 R sym (%) a 14.2 (56.8) b Completeness (%) 96.2 (94.7) b Redundancy 4.1 (3.6) b I/ (I) 6.2 (1.7) b Refinement No. of protein atoms 23,122 No. of glycan atoms 417 Resolution (Å) No. of reflections (work/test) 146,996/4,405 R work /R free (%) c 26.2/30.2 Root mean square deviation Bond length (Å) Angles ( ) 1.1 Cross-validated coordinate error Sigma A/Luzatti (Å) 1.0/0.7 Average B-values (Å 2 ) Fab KZ52 (variable/constant) (108.5/152.4) GP1 (base/head/glycan cap) (106.0/110.8/129.8) GP Glycans Ramachandran plot (%) d Most favoured 76.4 Allowed 20.6 Generously allowed 2.1 Disallowed 1.0 a R sym = j I j - <I> / <I>, where I j and <I> represent the diffraction intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements. b Values given in parentheses refer to reflections in the outer resolution shell: Å c R work = ( hkl F obs - k F calc ) ( hkl F obs ), where F obs and F calc are the observed and calculated structure factors, respectively. For R free, the sum is extended over a subset of reflections (3%) excluded from all stages of refinement. d as defined in Procheck

5 SUPPLEMENTAL FIGURES Figure S1. KZ52 neutralization of GP-variant pseudoviruses. VSV-bearing wild-type GP, GP with a deletion of the mucin-like domain (residues ), or GP with a deletion of the mucin-like domain and point mutations deleting two N- linked glycosylation sequons (T42V and T230V), were incubated with KZ52 prior to infection of Vero E6 cells. WT and mutant GP-pseudoviruses were approximately equally infectious and equally neutralized by the conformational antibody KZ52, implying that these mutations do not significantly alter the underlying structure or function of regions of GP necessary for recognition by KZ52, cellular attachment or entry. 5

6 Figure S2. Overall structure of the Ebola virus GP. Ribbon diagram of the GP monomer (residues , , and ). The chain is coloured by residue number in a rainbow gradient from red (N terminus of GP1) to blue (C terminus of GP2) to allow sequential tracing of the molecule. 6

7 Figure S3. Ribbon and topology diagram of GP1. strands are represented as arrows and helices by cylinders. Secondary structural elements are coloured and labeled according to Fig. 2a and disulfide bonds are shown as red lines. 7

8 Figure S4. Internal fusion loops and N terminal fusion peptides of viral glycoproteins. Fusion peptides from structures of the pre-fusion conformations of trimeric class I parainfluenza virus (PIV) 5 F (PDB: 2B9B) and influenza virus HA (PDB: 1RUZ) glycoproteins are compared with internal fusion loops from the class II tick borne encephalitis virus (TBEV) E (PDB: 1SVB), class III vesicular stomatitis virus (VSV) G (PDB: 2J6J) and trimeric class I Ebola virus (EBOV) GP. The EBOV GP internal fusion loop is more similar to those observed in class II and III glycoproteins than those observed in trimeric class I pre-fusion viral glycoproteins. For example, the fusion loops of class II and III glycoproteins, in pre-fusion conformations, generally consist of a hydrophobic region formed between two antiparallel strands covalently linked by one or more disulfide bonds 3,6,15,

9 Figure S5. Conformational differences between the pre- and post-fusion GP2 HR1 helix. The EBOV GP2 HR1 helix is coloured according to the HR1 A - HR1 D segments in Fig. 2c. Note that HR2 and the CX 6 CC motif are disordered in the pre-fusion conformation and that the internal fusion loop was not included in constructs used to determine the post-fusion structure 10,

10 Figure S6. Alignment of GP sequences from filoviral species. GP sequences from the Zaire (Mayinga 1976 and Kikwit 1995), Sudan (Boniface 1976 and Gulu 2000), Côte d Ivoire (1994) and Reston (1989) species of the Ebola virus and the Lake Victoria (LV) species (Popp 1967) of the Marburg virus are aligned. Red boxes indicate complete conservation of a given amino acid. N- and O-linked glycosylation sites for Zaire ebolavirus, predicted by the NetNGlyc 17 and NetOGlyc 18 servers, are indicated by black- and red-coloured Y- shaped symbols, respectively. While a N-linked glycan was observed in the structure at N563, this site was not predicted by the NetNGlyc server. The 3-4 periodicity of the GP2 heptad repeat is indicated by the letters a and d representing the appropriate positions of the a-g repeat. The stutter ( x ) is shown between residues Experimentally determined secondary structural elements are shown as arrows for strands and coils for helices. Note: 17 and 18 are tentatively assigned due to disorder in this region and therefore are shown as a dashed double arrow. Residues 1-32, and were excised from the construct used for crystallization so no secondary structure is illustrated for these regions. KZ52-binding residues are marked with an asterisk above the Zaire ebolavirus sequence and residues residing on the putative receptor-binding surface (RBS) are outlined by black boxes. 10

11 11

12 Figure S7. KZ52 epitope. (a) 2D schematic of the interactions between KZ52 and EBOV GP. Residues from GP1 and the N terminus and internal fusion loop of GP2 that contact KZ52 are coloured in green, red and orange, respectively. KZ52 heavy- and light-chain residues are labeled in blue and light blue, respectively. Hydrogen bonds are depicted as dashed lines and van der Waals interactions are shown as red semi-circles. GP residues not conserved among EBOV subtypes are marked with an asterisk. MSeH100D refers to a selenomethionine residue at position 100 of the KZ52 heavy chain. (b) Footprint of Fab KZ52 on the surface of a GP monomer. The molecular surfaces of GP1 and GP2 are coloured white and grey, respectively. GP atoms within 3.9 Å van der Waals distance from any atom in KZ52 are coloured according to the scheme presented in panel (a). Note that Zaire ebolavirus naturally has a threonine at position 42; however, this site was mutated to a valine to remove the N-linked glycosylation site at N

13 13

14 Figure S8. Glycosylation profile of EBOV GP. Non-denaturing immunoblot analysis of wild-type EBOV GP , GP muc, as well as PNGaseF- and EndoH-treated GP muc, each probed for binding with anti-ha IgG 16B12. Comparison of the theoretical molecular weight of non-glycosylated GP muc (52.4 kda) and expressed WT GP muc (~75 kda; lane 3) suggests the presence of ~20 kda of attached carbohydrates on our crystallized construct, consistent with the likely molecular weight of the seven expected N-linked structures. WT GP muc is sensitive to PNGaseF deglycosylation (lane 4) but not EndoH (lane 5), suggesting that glycans outside the mucin-like domain are likely to be complex-type in structure. Note that monomeric (~75 kda), dimeric (~150 kda) and trimeric (~225 kda) species are observed for GP muc, whereas only a monomeric (~150 kda) species of wild-type GP tm is observed, as multimeric species are too large to enter the gel. 14

15 . Figure S9. Model of EBOV GP-mediated membrane fusion. (a) Cartoon of the entire pre-fusion EBOV GP on the viral surface. Cryo-tomography, electron microscopy, and X-ray crystallography have visualized pre-fusion viral glycoproteins in one of two arrangements: an open tripod-like state 6,19,20 or a single stalk structure 5,21,22. As the CX 6 CC hinge motif 11 and the putative EBOV HR2 region are disordered in these crystals, it is as yet unclear which arrangement is favoured by EBOV. However, it is possible the two disulfide bonds formed by CX 6 CC in GP2 may prevent a straight stalk-like vertical drop to the virion surface, instead favouring a kink so that the GP2 adopts a more open or tripod-like conformation, as shown here. Here, all three GP1 subunits are shown in grey, while each GP2 subunit is coloured separately in red, cyan and green. (b) In the endosome, cathepsin L and/or B cleavage of EBOV GP removes the mucin-like domain and glycan cap, leaving GP2 and an ~18 kda fragment of GP1 2,9,23. The precise trigger to activate the fusogenic state is unknown; however, cathepsin cleavage 9, a cellular reductase 2 or another cofactor(s) have been proposed to release GP1 from GP2. (c) Comparison between the pre-fusion GP trimer (PDB: 3CSY) and post-fusion GP2 fragments (PDB: 1EBO and 2EBO) reveal that the four HR1 segments (HR1 A-D ) reorganize to form a single HR1 helix. As a result, the internal fusion loops (IFL) are translated towards the host membrane. Upon insertion into the host membrane, the hydrophobic fusion peptide adopts a 3 10 helix 12. (d) Expected similarity with influenza virus HA suggests that more than one EBOV GP trimer may be required to form the fusion pore 24,25 and that the glycoprotein may assume a tilted orientation upon insertion into the host membrane 26. (e) The formation of the low energy post-fusion six-helix bundle (6HB) requires HR2 and MPER to swing from the viral membrane-proximal surface towards the host membrane, ultimately packing against HR1. These rearrangements juxtapose the internal fusion loop and transmembrane domain, thus facilitating the fusion of the host and viral membranes. 15

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17 Figure S10. Electron density maps. Representative A -weighted 2F o -F c composite omit electron density map contoured at 1, superimposed with the final refined model in the vicinity of the (a) GP1 base subdomain, (b) the N257 N- linked glycan of the GP1 glycan cap subdomain, (c) GP2 HR1 D helix and (d) the N563 N-linked glycan of GP2. Mannose and N-acetylglucosamine sugars are labeled as MAN and GlcNAc, respectively. 17

18 Figure S11. Lack of irreversible GP conformational change in response to acidic ph. EBOV GP was incubated in various buffers between ph 4-7 and probed by non-denaturing Western blot using KZ52 as the primary antibody. Positive and negative controls were performed on an untreated (ph 7.5) and disulfide-reduced GP, respectively. Control for total protein was monitored using an antibody directed against the linear HA tag. Note that low ph alone does not result in conformational change sufficient to disrupt binding of the conformational, predominantly GP2-contacting antibody, KZ

19 Supplemental Movie 1. Model of EBOV GP-mediated viral entry. This movie is based on the mechanism proposed in Supplemental Fig. S9. Conformational changes of GP2 were created as a linear extrapolation between the pre-fusion (PDB code: 3CSY) and post-fusion GP2 states (PDB code: 1EBO and 2EBO) 10,11 using RigiMOL/PyMOL 27. The movie can be downloaded from

20 References for Supplemental Information 1. Bar, S., Takada, A., Kawaoka, Y., & Alizon, M. Detection of cell-cell fusion mediated by Ebola virus glycoproteins. J. Virol. 80, 2815 (2006). 2. Schornberg, K. et al. Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein. J. Virol. 80, 4174 (2006). 3. Modis, Y., Ogata, S., Clements, D., & Harrison, S. C. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313 (2004). 4. Bullough, P. A., Hughson, F. M., Skehel, J. J., & Wiley, D. C. Structure of influenza haemagglutinin at the ph of membrane fusion. Nature 371, 37 (1994). 5. Wilson, I. A., Skehel, J. J., & Wiley, D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366 (1981). 6. Roche, S., Rey, F. A., Gaudin, Y., & Bressanelli, S. Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G. Science 315, 843 (2007). 7. Roche, S., Bressanelli, S., Rey, F. A., & Gaudin, Y. Crystal structure of the lowph form of the vesicular stomatitis virus glycoprotein G. Science 313, 187 (2006). 8. Kampmann, T., Mueller, D. S., Mark, A. E., Young, P. R., & Kobe, B. The role of histidine residues in low-ph-mediated viral membrane fusion. Structure 14, 1481 (2006). 9. Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P., & Cunningham, J. M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643 (2005). 10. Malashkevich, V. N. et al. Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-Å resolution. Proc. Natl. Acad. Sci. USA 96, 2662 (1999). 11. Weissenhorn, W., Carfi, A., Lee, K. H., Skehel, J. J., & Wiley, D. C. Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol. Cell. 2, 605 (1998). 12. Freitas, M. S. et al. Structure of the Ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts. J. Biol. Chem. 282, (2007). 20

21 13. Takada, A. et al. A system for functional analysis of Ebola virus glycoprotein. Proc. Natl. Acad. Sci. USA 94, (1997). 14. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283 (1993). 15. Lescar, J. et al. The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph. Cell 105, 137 (2001). 16. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C., & Harrison, S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375, 291 (1995). 17. Gupta, R., Jung, E., & Brunak, S., Prediction of N-glycosylation sites in human proteins, Available at Julenius, K., Molgaard, A., Gupta, R., & Brunak, S. Prediction, conservation analysis and structural characterization of mammalian mucin-type O- glycosylation sites. Glycobiol. 15, 153 (2005). 19. Zhu, P. et al. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441, 847 (2006). 20. Forster, F., Medalia, O., Zauberman, N., Baumeister, W., & Fass, D. Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. Proc. Natl. Acad. Sci. USA 102, 4729 (2005). 21. Zanetti, G., Briggs, J. A., Grunewald, K., Sattentau, Q. J., & Fuller, S. D. Cryoelectron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog. 2, e83 (2006). 22. Yin, H. S., Wen, X., Paterson, R. G., Lamb, R. A., & Jardetzky, T. S. Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439, 38 (2006). 23. Kaletsky, R. L., Simmons, G., & Bates, P. Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity. J. Virol. 81, (2007). 21

22 24. Danieli, T., Pelletier, S. L., Henis, Y. I., & White, J. M. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J. Cell Biol. 133, 559 (1996). 25. Harrison, S. C. Mechanism of membrane fusion by viral envelope proteins. Adv. Virus Res. 64, 231 (2005). 26. Tatulian, S. A., Hinterdorfer, P., Baber, G., & Tamm, L. K. Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy. EMBO J. 14, 5514 (1995). 27. DeLano, W. L., The PyMol Molecular Graphics System (2002). 22