Structure, Volume 20
Supplemental Information Mechanism of Dengue Virus Broad Cross-Neutralization by a Monoclonal Antibody Joseph J.B. Cockburn, M. Erika Navarro Sanchez, Nickolas Fretes, Agathe Urvoas Isabelle Staropoli, Carlos M. Kikuti, Lark L. Coffey, Fernando Arenzana Seisdedos, Hugues Bedouelle, and Felix A. Rey Inventory of Supplemental Information Supplemental data Figure S1 Overall features of the complexes between scFv 4E11 and the DENV DIIIs, related to Figure 1 Figure S2 Determinants of Mab 4E11 specificity for DENV amongst the other flaviviruses, related to Figure 2. Table S1A Intermolecular hydrogen bonds by serotype Table S1B Intermolecular Van der Waals contacts by serotype Table S2 Crystallization and cryo-cooling conditions Supplemental Experimental Procedures Preparation cells and viruses Production of recombinant DENV-3 sE in Drosophila cells
Supplemental Figures
Figure S1
Figure S2
Supplemental Figure Legends Figure S1 Overall features of the complexes between scFv 4E11 and the DENV DIIIs, related to Figure 1 (A-B) Proteolytic cleavage of DIII from the DENV-3 sE. SDS-PAGE gels run under reducing conditions, showing the protein samples used to obtain the crystals of scFv 4E11 in complex with (A) DENV-1 DIII (lane 8), and (B) DENV-3 DIII (lane 2) described in this study. In both cases, lane 1 contains markers at the indicated molecular weights (in kDa); the color contrast in the marker lanes has been enhanced to improve the visibility of the lower molecular weight markers. In (B), the full-length DENV-3 sE protein (~50kDa) was used, since recombinant DENV-3 DIII was not available. However, a band at the molecular weight expected for the DIII is clearly visible, whereas the full-length protein is almost entirely absent. The relative strengths of the scFv 4E11 and DIII bands in (A) and (B) are similar, indicating that the DENV-3 DIII is present in stoichiometric amounts. Lanes 2-7 in part (A) are fractions collected from the elution of scFv 4E11 from a gel filtration column. They are included because they were part of the gel, but are of no further interest here. (C) The complexes between scFv 4E11 and DIII for DENV serotypes 2-4 superposed into that for DENV-1 via residues in DIII. The 4E11 variable domains are colored yellow, red, green and blue for DENV-1 through -4, respectively.
Figure S2 Determinants of Mab 4E11 specificity for DENV among the other flaviviruses, related to Figure 2. (A) Superposition of the tick borne encephalitis virus (TBEV) DIII (brown; taken from PDB accession 1SVB) onto that of DENV-1 in complex with scFv 4E11. Note the clashes between TBEV DIII residue His390 and scFv 4E11 residue TyrL28, which would prohibit 4E11 binding. (B) Sequence alignment of flavivirus DIII A- and G-strand residues. Abbreviations used: JE – Japanese encephalitis virus; MVE – Murray Valley encephalitis virus; Kun – Kunjin virus; WN – West Nile virus; SLE – St. Louis encephalitis virus; YF – Yellow fever virus; TBE – Tick-borne encephalitis virus; LI – Louping ill virus; LGT – Langat virus; POW – Powassan virus; APO – Apoi virus; RB – Rio Bravo virus. Asterisks mark DENV-1 DIII residues 308 and 389, and their counterparts in other DENV serotypes and other flaviviruses.
Supplementary Tables Table S1A Intermolecular hydrogen bonds by serotype. Related to Figure 1 Resa DENV1b 305 306 Phe(O)-ArgL50(NH2) 3.0Å/138˚ 307 Lys(NZ)-GluL55(OE1) 2.7Å 308 Leu(N)-GluH97(OE2) 2.8Å/134˚ 309 Glu(OE1)-ArgH94(NH2) 3.0Å Glu(OE2)-ArgH94(NE) 2.9Å 310 Lys(NZ)-AspH52(OD1) 2.7Å Lys(NZ)-LysH30(O) 2.9Å/152˚ 311 Glu(N)-TyrH33(OH) 3.0Å/116˚ Glu(OE2)-ArgL27d(NH1) 2.9Å
DENV2b
DENV3b
Phe(O)-ArgL50(NH2) 3.0Å/146˚ Lys(NZ)-GluL55(OE1) 3.0Å Lys(NZ)-GluL55(O) 3.1Å/162˚ Ile(N)-GluH97(OE2) 3.2Å/129˚ Val(O)-ThrH32(OG1) 2.6Å/138˚
Phe(O)-ArgL50(NH2) 2.7Å/144˚ Leu(N)-GluH97(OE2) 2.8Å/137˚ Lys(O)-ThrH32(OG1) 2.7Å/133˚
Lys(NZ)-AspH52(OD1) 2.6Å Lys(NZ)-LysH30(O) 2.8Å/144˚
Lys(NZ)-AspH52(OD1) 2.7Å Lys(NZ)-LysH30(O) 2.6Å/165˚
Glu(N)-TyrH33(OH) 3.0Å/129˚ Glu(OE2)-ArgL27d(NH1) 2.7Å Glu(OE2)-ArgL27d(NH2) 2.7Å Ile(N)-TyrL28(OH) 2.9Å/125˚
Glu(N)-TyrH33(OH) 3.1Å/132˚ Glu(OE2)-ArgL27d(NH1) 2.9Å Glu(OE2)-ArgL27d(NH2) 3.0Å Val(N)-TyrL28(OH) 2.9Å/128˚
DENV4b Lys(NZ)-AsnL53(OE1) 2.9Å/132˚ Phe(O)-ArgL50(NH2) 3.2Å/137˚ Ile(N)-GluH97(OE2) 3.0Å/132˚ Asp(OD1)-ArgH94(NH2) 3.1Å Asp(OD2)-ArgH94(NE) 3.1Å Lys(NZ)-AspH52(OD1) 2.8Å Lys(NZ)-LysH30(O) 2.7Å/143˚ Lys(NZ)-ThrH32(O) 3.3Å/121˚ Glu(N)-TyrH33(OH) 3.1Å/109˚ Glu(OE2)-ArgL27d(NH1) 3.1Å Glu(OE2)-ArgL27d(NH2) 3.3Å Met(N)-TyrL28(OH) 3.0Å/135˚
312 Val(N)-TyrL28(OH) 3.0Å/126˚ 323 Gln(NE2)-AspH31(O) 2.7Å/118˚ Glu(OE1)-ArgH94(NH2) 2.7Å 325 Lys(NZ)-GluL55(OE1) 2.7Å 327 Thr(O)-TyrH102(OH) 3.1Å/131˚ 361 Asn(OD1)-TyrH102(OH) 2.8Å/134˚ 362 Glu(OE2)-TyrH102(OH) 2.5Å/170˚ Asp(OD1)-TyrH102(OH) 2.5Å/114˚ H94 Glu(O)-Arg (NH2) 3.0Å/144˚ Asn(O)-ArgH94(NH2) 3.1Å/139˚ H94 Glu(O)-Arg (NH1) 3.0Å/153˚ Asn(O)-ArgH94(NH1) 2.7Å/149˚ Asn(ND2)-AspH31(OD1) 3.3Å/111˚ 366 L30 L30 L30 Lys(O)-Asn (ND2) 2.9Å/145˚ Lys(O)-Asn (ND2) 2.8Å/155˚ Thr(O)-AsnL30(ND2) 3.0Å/146˚ 388 Lys(O)-Asn (ND2) 2.9Å/144˚ L28 L28 390 Ser(N)-Tyr (O) 2.9Å/116˚ Asn(N)-Tyr (O) 3.0Å/118˚ Asn(N)-TyrL28(O) 2.9Å/120˚ His(N)-TyrL28(O) 3.0Å/119˚ a DIII residue number (DENV-1 numbering) b Contacts are listed in the following format: RES1(AT1)-RES2(AT2) D(/A), where RES1 and RES2 are DIII and 4E11 residues, respectively, AT1 and AT2 are the atoms making the interaction, and D is the corresponding inter-atomic distance in Å. In the case of hydrogen bonds, A is the donor-acceptor-R angle in degrees. Salt bridges are denoted in red, and other hydrogen bonds in blue.
Table S1B Intermolecular Van der Waals contacts by serotype (calculated with a 4 Å distance cut-off). Related to Figure 1. DIIIa 305
DENV-1 TyrL49
306 307
ArgL50 TyrL49 ArgL50 GluL55 GluH97
308
GluH97 TrpH96 TyrL28 ArgH94 AspH31 ThrH32 TyrH33 TrpH96 LysH30 AspH31 ThrH32 TyrH33 AlaH54 AspH52 TrpH96 TyrH33 TrpH96 TyrL28 ArgL27d TyrL28 AspH31 ArgH94 TyrH102 GluL55
309
310
311
312 323 325 327
360 361 362
TyrH102 LeuH3 ArgH94 PheH27
363 364
AsnH28 ArgH94 AspH31
4E11b DENV-2 DENV-3 TyrL49 TyrL49 L53 Asn ArgL50 ArgL50 L49 Tyr TyrL49 L50 Arg ArgL50 L55 Glu GluH97 GluH97 LeuL46 GluH97 GluH97 H96 Trp TrpH96 PheL32 TyrL28 H31 Asp AspH31 H32 Thr ThrH32 H33 Tyr TyrH33 TrpH96 TrpH96 LysH30 AspH31 ThrH32 TyrH33 AlaH54 AspH52 TrpH96 TyrH33 TrpH96 TyrL28 ArgL27d TyrL28 AspH31 SerL56 LysH2 SerL56 LysH2 TyrH102 ArgH94 LysH2
AspH31
LysH30 AspH31 ThrH32 TyrH33 AlaH54 AspH52 TrpH96 TyrH33 TrpH96 TyrL28 ArgL27d TyrL28 ArgH94 LysH2 TyrL49 GluL55 AlaH101 SerL56
DENV-4 AsnL53 ArgL50 ArgL50 GluH97
GluH97 TrpH96 ArgH94 AspH31 ThrH32 TyrH33 TrpH96 LysH30 AspH31 ThrH32 TyrH33 AlaH54 AspH52 TrpH96 TyrH33 TrpH96 TyrL28 ArgL27d TyrL28 AspH31 ArgH94 TyrH102 TyrL49 GluL55 TyrH102 TyrH102 LeuH3 ArgH94 PheH27 GlyH26 GlyH26 ArgH94 AspH31
AspH31
366 385 387
ArgL50
388
TyrL28 AsnL30 TyrL28
389 390 391 a b
L53
TyrL28 GlyL29 TyrL28
Asn ArgL50 ArgL50
TyrL28 AsnL30 TyrL28 AsnL30 TyrL28 TyrL28
L53
Asn
ArgL50 AsnL30 TyrL28 AsnL30 TyrL28 TyrL28 GlyL29 TyrL28
ArgL50 AsnL30 TyrL28 AsnL30 TyrL28 AsnL30 TyrL28
Residue number in DIII (DENV-1 numbering) 4E11 residues interacting with the residue from DIII of the indicated serotype
Table S2 Crystallization and cryo-cooling conditions DENV1
DENV2
DENV3
Crystallisation
24% PEG 4K 0.1M MES pH 6.5
70% JBS Classic 8 D1a
20% PEG 8K 0.1M HEPES pH 7.5
Cryo
24% PEG 4K 0.1M MES pH 6.5 20% glycerol
70% JBS Classic 8 D1 30% ethanolb
20% PEG 8K 0.1M HEPES pH 7.5 25% glycerol
a
DENV4 1.7M ammonium sulphate 12.5% glycerol 0.1M TRIS pH 8.5 1.7M ammonium sulphate 0.1M TRIS pH 8.5 20% glycerol
Jena Biosciences Classic Screen 8, condition D1, consisting of 30% ethanol, 12% PEG 6K and 0.1M sodium acetate, according to the manufacturer’s formulation. b The cryoprotectant drop was supported in a sitting drop well under a layer of paraffin oil.
Supplemental experimental procedures Preparation cells and viruses Mosquito AP61 cells were cultured in Leibovitz-15 medium supplemented with 10% bovine foetal calf serum (FCS), non-essential amino acids, tryptose phosphate buffer, 1% penicillin and 1% streptomycin, at 28 ˚C in 150 cm2 flasks. Human hepatocyte (Huh 7.5) cells were prepared for virus neutralization assays as follows. Cells were cultured in 162 cm2 cell culture flasks containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1% penicillin and 1% streptomycin at 37 ˚C in a 5% CO2 incubator, to 80% confluency. The adherent cells were washed in phosphate buffered saline (PBS), trypsinized, and resuspended in DMEM (supplemented as above) to a density of ~3105 cells/ml. 1 ml volumes of this suspension were dispensed into 12-well cell culture plates and incubated overnight at 37 ˚C in a 5% CO2 incubator. Virus neutralization tests were performed the following day, by which time each well contained ~ 4.5105 cells at ~80% confluency. The cells were washed twice in DMEM containing 2% FCS, 1% penicillin and 1% streptomycin immediately prior to infection. Virus stocks were prepared as follows. AP61 mosquito cells were infected with samples of the relevant parental DENV strain from the collection at the Pasteur Institute at a multiplicity of infection (m.o.i.) of 0.5, and incubated at 28 ˚C. Supernatants were harvested 4-5 days post-infection and loaded onto a 20 % sucrose cushion in 10 mM Tris, 100 mM NaCl and 1 mM EDTA (TNE). The virus was pelleted by ultracentrifugation for 90 minutes at 26000 rpm at 4 ˚C. Supernatants were poured off and the pellet resuspended in TNE buffer. Infectious titers were determined by plaque reduction assays on C6/36 mosquito cells.
Production of recombinant DENV-3 sE in Drosophila cells A DNA fragment containing the DENV-3 genomic region coding for the prM-sE (1680 nt, including all of prM and ending at E codon 394) was amplified by PCR using specific primers
and inserted into the plasmid pt351. This is a shuttle vector containing selection markers for yeast and E..coli, as well as a metallotheionein-inducible expression cassette for Drosophila cells. In the construct, called pT351/DENV-3 sE, the prM-sE sequence is in frame with the Drosophila BiP signal peptide, which directs the recombinant protein to the secretory pathway, an enterokinase cleavage site, and a C-terminal StrepTag (www.iba.com) for affinity
purification.
Drosophila
S2
cells
(invitrogen)
were
co–transfected
with
pT351/DENV-3 sE and a vector conferring resistance to puromycin using the effectene transfection reagent (Qiagen). The selected cells were adapted to serum-free growth medium and grown to high density before induction with 500 M CuSO4. The supernatant was collected 10 days later and concentrated in a flow concentration system using a PES membrane with a 10 kDa molecular weight cutoff (Vivascience). DENV-3 sE was purified by affinity chromatography using a streptactin column. The eluate was concentrated and further purified by size-exclusion chromatography, using a superdex 200 10/300 column (GE healthcare) with 0.5 M NaCl and 50 mM Tris-HCl (pH 8.0).
