Structure of the Envelope Protein Domain III of Omsk Hemorrhagic Fever Virus☆

Virology 351 (2006) 188–195 www.elsevier.com/locate/yviro

Structure of the envelope protein domain III of Omsk hemorrhagic fever virus☆

David E. Volk a,d, Leonard Chavez h, David W.C. Beasley b,c,f,g, Alan D.T. Barrett c,e,f,g, ⁎ Michael R. Holbrook c,e,f,h,g, David G. Gorenstein a,d,

a Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1157, USA b Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1157, USA c Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-1157, USA d Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-1157, USA e Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555-1157, USA f Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-1157, USA g Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555-1157, USA h Department of Pathology, University of New Mexico, Albuquerque, NM 87131, USA Received 3 February 2006; accepted 6 March 2006 Available online 2 May 2006

Abstract

We have solved the NMR solution structure of domain III from the Omsk hemorrhagic fever virus envelope protein and report the first sequencing of the Guriev strain of this virus. Important structural differences between tick-borne flaviviruses, such as OHFV and TBE, and mosquito-borne flaviviruses, such as West Nile virus, are discussed. © 2006 Elsevier Inc. All rights reserved.

Introduction complex includes Russian-Spring Summer encephalitis (RSSE), Central European TBE (CE-TBE), Powassan (POW), Omsk hemorrhagic fever (OHF) is caused by Omsk Langat (LGT), louping-ill (LI), OHFV, Alkhurma (ALK) (Zaki, hemorrhagic fever virus (OHFV), which was first isolated 1997), and Kyasanur Forest disease (KFD) viruses (Work, from human serum in 1947 and is endemic to a small region 1958). OHF, ALK, and KFD viruses are the only tick-borne near Omsk, Russia (Kharitonova and Leonov, 1985). The virus flaviviruses known to cause hemorrhagic disease. Unlike ALK is a tick-borne member of the genus Flavivirus of the family and KFD viruses, OHF virus causes a hemorrhagic disease in Flaviviridae, and it is a NIAID Category C priority pathogen humans with few neurological effects (Burke and Monath, and a potential biothreat agent. The flaviviruses are typically 2001). In the mouse model, OHF virus also causes disease with transmitted by either mosquitoes or ticks and include major few neurological signs compared to neurotropic tick-borne human pathogens such as dengue (DEN), West Nile (WN), flaviviruses and also demonstrated significantly different tissue yellow fever (YF), Japanese encephalitis (JE), and members of localization indicating potential differences in host cell interac- the tick-borne encephalitis virus (TBE) complex. The TBE tions (Holbrook et al., 2005). The flavivirus E-protein is the major virion surface protein ☆ The atomic coordinates of the NMR solution structure (1Z3R) have been and is also the primary immunogen. It is thought to play a deposited with the Protein Data Bank, Research Collaboratory for Structural central role in virus–host cell receptor binding and membrane Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). fusion (Heinz and Allison, 2000, 2001, 2003). The flavivirus E- NMR Chemical shifts (6309) have been deposited at the BioMagResBank, protein has 0–3 glycosylation sites and is approximately 500 University of Wisconsin-Madison (http://www.bmrb.wisc.edu/). amino acids in length, of which the N-terminal 400 amino acids ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, UT Medical Branch, Route 1157, Galveston, TX 77555-1157, USA. Fax: +1 contains three distinct domains (domains I, II, and III; ED1, 409 747 6850. ED2, and ED3, respectively) that have been identified both E-mail address: [email protected] (D.G. Gorenstein). immunologically (Mandl et al., 1989; Rey et al., 1995; Roehrig,

2003; Crill and Roehrig, 2001) and via X-ray crystallography of to differences in serologic and potential host cell interactions. In the extramembrane region of the E-protein of CE-TBE (Rey et particular, because ED3 has been reported to be the receptor- al., 1995), DEN2, and DEN3 viruses (Modis et al., 2003, 2004, binding domain of the virus (Hung et al., 2004; Chu et al., 2005). ED1 is the central domain and ED2 is called the 2005), we hypothesized that variations in the structure of ED3 dimerization domain, as it appears to be the principle region of between different flaviviruses may affect host cell receptor interaction between monomers in E-protein dimers on the virion interactions and thereby play a role in determining variations in surface. ED3 is approximately 100 amino acids in length and is viral pathogenesis. the putative receptor-binding domain based on three criteria. First, it is highly immunogenic with a number of virus-type- Results specific epitopes recognized by neutralizing antibodies being mapped to ED3. Neutralizing antibodies that bind to ED3 have Chemical shift assignments been shown to strongly inhibit virus binding to target cells (Crill and Roehrig, 2001). Secondly, the structure of ED3 resembles With the exception of the first three residues, complete that of an IgC-like fold that is found in a number of receptors. assignments (BMRB accession number 6309) were made for Thirdly, some mosquito-borne flaviviruses contain a four amino the OHF-rED3 protein (Fig. 1) as described in the Materials and acid loop that extends out from the ED3 and contains an RGD methods section. Two proton resonances were shifted unusually motif as occurs in some integrin-binding proteins. More recent far upfield from their normal values. The amide proton of studies have shown that ED3 is directly associated with binding glycine G334, which is situated directly over the phenylalanine of DEN2 virus (Hung et al., 2004), WN virus, and the tick- F332 aromatic ring, resonates at 4.37 ppm. This chemical shift borne LGT virus (M. Holbrook, unpublished results) to host is nearly 4 ppm upfield relative to the 8.35-ppm average glycine cells. Cryoelectron microscopy studies of the mosquito-borne amide proton chemical shift reported by the BMRB. Likewise, WN, DEN2, and YF viruses have found that the E-protein is the HD2 proton of asparagine N381 lays near the edges of both arranged as dimers on the surface of the virus (Kuhn et al., the tryptophan W392 and the phenylalanine F393 aromatic 2002; Mukhopadhyay et al., 2003). These reconstructions also rings and resonates at 5.80 ppm. Although the chemical shifts found that ED3 is arranged as a pentamer at the fivefold axis of determined for OHF-rED3 are significantly different than those symmetry of the virus and that the ED3 projects slightly above recently reported (Mukherjee et al., 2004) for the LGT virus the surface of the virion, allowing access to potential receptor rED3 protein (BMRB 5971), similar patterns are observed molecules and antibodies. The fivefold axis is arranged such between the two chemical shift data sets. that there is a “pore” between the ED3 molecules. When virus Resonances from a minor population were observed for binds to the cell, it is internalized into an endosome that is several residues, including L302, T303, Y304, T363, and acidified. The drop in pH causes a dramatic conformational I364. Residues T363 and I364 are close to the aromatic shift in the E-protein that exposes the viral fusion peptide (Modis et al., 2004), allowing the endosomal and viral membranes to fuse and providing for release of the viral RNA into the cell cytoplasm. Serological analyses (Theiler and Downs, 1973) using antibody absorption studies of OHF virus strains identified the presence of two antigenic subtypes (Theiler and Downs, 1973; Clarke, 1964). Subtype I includes the Kubrin and Balangul strains, and subtype II includes the Bogoluvovska and Guriev strains. The recently completed genomic sequences of the Bogoluvovska (GenBank accession no. AY193805; Lin et al., 2003) and Kubrin (GenBank accession no. AY438626; Li et al., 2004) strains of OHF virus identified six nucleotide differences that encoded four amino acid substitutions in the OHF virus polyprotein. Two of these amino acid differences (S349A and I364M) occur in ED3. Analyses based on the structures of this domain of the E-protein from CE-TBE (Rey et al., 1995), WN (Volk et al., 2004), DEN2 (Modis et al., 2003), and DEN3 (Modis et al., 2005) viruses indicate that mutation I364M lies on the upper surface of ED3, in a region previously identified as an epitope for WN virus subtype- specific monoclonal antibodies (Volk et al., 2004; Beasley and Fig. 1. 1H,15N-HSQC spectrum of OHFV-rED3. Two correlations with proton Barrett, 2002). chemical shifts positioned unusually high upfield are not shown. One of the N84 (N381) side-chain amide protons resonates at 5.80 ppm, and the G37 (G334) The objective of this study was to determine structural amide H-N correlation is at 4.37 (H) and 106.7 (N) ppm. Residues 3–99 of the characteristics of the recombinant OHF-ED3 (rED3) compared OHFV-rED3 protein correspond to residues 300-396 in the intact OHFV with other tick- and mosquito-borne flaviviruses that contribute envelope protein. 190 D.E. Volk et al. / Virology 351 (2006) 188–195 ring of tyrosine Y304. The minor population is consistent TBE ED3 structure (Rey et al., 1995), which shares 93 of 99 with a structure in which in the N-terminal residues, which conserved amino acids. are not involved in any beta-sheet structure, have multiple The program PROCHECK (Laskowski et al., 1996) was conformations. used to analyze the quality of the final ensemble. Analysis of the nonglycine, nonproline residues indicated that 98.7% of the Quality of the NMR structures residues are in the two most favored regions of a Ramachan- dran plot. Specifically, 87.5% of the residues are in the most The 15 final structures in the ensemble (Figs. 2A and B) had favored regions, 11.2% of the residues are in the additionally low molecular and restraints energy penalties. The final allowed region, no residues are in the generously allowed structures had 15 ± 2 distance restraint violations over 0.3 Å, regions, and 1.2% of the residues, namely S299, are in the 0–2 violations over 0.5 Å, and no dihedral angle violations over disallowed regions. 10° (Table 1). The RMSD of the distance restraint error was 0.018 ± 0.001 A, and the RMSD dihedral angle error was Structural details of the NMR ensemble 0.27 ± 0.05°. The structural ensemble has a global backbone atom RMSD of 0.42 ± 0.05 Å, and a global heavy atom RMSD The protein structure consists of three beta sheets arranged of 0.67 ± 0.07 Å. The structures also have a very low backbone in a β-barrel configuration (Fig. 2B). The first beta sheet, atom RMSD, 0.98 Å for residues 302–395, relative to the CE- whose outer face is exposed in the viral particle, consists of

Fig. 2. (A) Stereo view of the NMR-derived OHFV-rED3 backbone atoms. (B) Two orthogonal side views of a ribbon diagram of the OHFV-rED3 NMR structure. The side chains of residues that differ between OHFV, TBE, and LGT are shown as sticks. The left side is in the same orientation as panel A. D.E. Volk et al. / Virology 351 (2006) 188–195 191

Table 1 sequence differs from the subtype II Bogoluvovska strain at Summary of NMR structure constraints and statistics only two amino acids (S349A and I364M). At position 349, Total restraints 2387 which is located at the bottom of ED3 and is not surface NOE restraints 2227 exposed, the subtype II Bogoluvovska and Guriev strains Intra-residue 762 contain hydroxylated amino acids S349 and T349, respective- Sequential 577 Medium range 153 ly, whereas the subtype I Kubrin strain contains A349 at this Long range 735 position. Talos phi/psi dihedral restraints 160 Structural statistics Comparison of ED3 of OHF virus with ED3 of other NOE violations > 0.5 Å 0 to 2 flaviviruses NOE violations > 0.3 Å 15 ± 2 Dihedral angle violation > 10° 0 RMSD from ideal geometry Residues at positions 306, 310, 331, 336, 364, and 366 are Bond lengths (Å) 0.0341 ± 0.0006 near the upper ED3 solvent exposed surface and vary between Bond angles (°) 4.90 ± 0.06 OHFV strains and/or other flaviviruses (Figs. 2B and 3). The Restraint error RMSD most notable feature of the ED3 alignment is that hemorrhagic Distance restraints (Å) 0.018 ± 0.001 Dihedral restraints (°) 0.27 ± 0.05 fever-causing viruses ALK and KFD are similar to each other Atomic RMSD but distinct from OHF, whereas OHF is closely related to CE- Backbone atoms 0.41 ± 0.06 TBEV and RSSE (Lin et al., 2003). The OHF and TBE ED3 All heavy atoms 0.67 ± 0.07 proteins have an M306 whereas LGT, ALK, KFD, and two Ramachandran statistics strains of CE-TBE [ZZ9 and Pan] have a V306. At position 310, Most favored regions (%) 87.5 Additionally allowed regions (%) 11.2 ALK, KFD, TBE-Spain, and LI viruses have serine and the Generously allowed regions (%) 0.0 Guriev (subtype II) OHFV strain, all strains of CE-TBE, RSSE, Disallowed regions (%) 1.2 and LGT ED3 proteins contain a conservative substitution of a threonine whereas both the Bogoluvovska (subtype II) and Kubrin (subtype I) OHF strains and POW virus have an alanine. beta strands β1, β2, β6, and β9 at residue positions 312–320 Amino acids 331, 336, 364, and 366 are very close to each other (β1, with a bulge at 315–318), 326–332 (β2), 356–357 (β6), on the exposed upper surface of the protein. At position 331, the and 371–375 (β9). The small second beta sheet consists of three OHF strains contain an A331 whereas the KFD and ALK beta strands β3andβ7 at residue positions 338–339 (β3) and are T331, POW, and Deer tick viruses are S331 and LGT is 363–364 (β7). The final strand of this beta sheet that is G331; CE-TBE strains have either a serine, threonine or alanine present in the CE-TBE E-protein X-ray structure (β8) was indicating allowed variability at this position. At position 335 only observed in four of the fifteen structures of the NMR ALK, KFD, POW, Deer tick, LI, TBE-Spain, and TBE-Turkey ensemble. A type I turn occurs between residues 365–368. viruses have a serine, whereas at position 336 there is a lysine This beta sheet is positioned at the end of ED3 that is away for all tick-borne viruses except LGT which is replaced by from ED1 and ED2 in the intact envelope protein, is exposed another basic residue, arginine. POW and Deer tick virus have to solvent, and is located at the “pore” formed at the virion an aspartic acid insertion between residues 335 and 336. fivefold axis by the intersection of five dimeric envelope Residues 364 and 366, which are surface exposed near the top proteins on the viral surface (Mukhopadhyay et al., 2003). The third beta sheet consists of beta strands β4, β5 (in one structure only), β10, and β11 positioned at residues 342–346 Table 2 Secondary structure elements of flavivirus ED3 proteins (β4), 349–353 (β5), 379–385 (β10), and 388–394 (β11). This beta sheet lies between the first sheet and ED1 in the Tick-borne flaviviruses Mosquito-borne flaviviruses mature virus. All three beta sheets are also present in the CE- OHFV TBEV DEN2 DEN3 WNV a b c d TBE structure, and the two structures align to 0.98 Å for the NMR X-ray X-ray X-ray NMR backbone atoms of residues 302–395. Beta strands β5 and β8 β1 312–320 312–320 306–314 304–312 309–317 present in CE-TBE were not generally observed in the NMR β2 326–332 326–332 320–326 318–324 323–329 β β3 338–339 338–339 333–334 331–332 ensemble, although 5 was observed in one of the fifteen β – – – – – β 4 342 346 342 346 347 351 335 338 340 348 structures, and in another structure, strand 9 was extended on β5 349–353 the N-terminal side to include residues 369 and 370 which β6 356–357 356–357 347–351 348–349 359–355 added to the small second beta sheet (Table 2). β7 363–365 356–357 355–356 β8 369–370 363–365 357–358 β – – – – – Genomic sequence of ED3 of the Guriev strain of OHF virus 9 371 375 368 369 365 370 363 368 370 376 β10 379–385 371–375 374–380 373–378 380–388 β11 388–394 379–385 387–393 385–390 391–399 The ED3 protein sequences of the Bogoluvovska and a Rey et al. (1995). Guriev OHF virus strains, which are both members of subtype b Modis et al. (2003). II, differ at five amino acids (Fig. 3: A310T, A317V, S349T, c Modis et al. (2005). I364M and N366S), whereas the subtype I Kubrin strain d Volk et al. (2004). 192 D.E. Volk et al. / Virology 351 (2006) 188–195

Fig. 3. Envelope protein domain III amino acid alignment of the members of the TBE serocomplex. of beta sheet 2, differ among the OHFV strains. The OHF virus strain, not only has residue 364 also been changed to Bogoluvovska strain of OHF, LI, RSSE, POW, and TBE ED3 M364, but position 366 has also been mutated to S366; ALK, proteins contain residue I364 whereas OHF strains Kubrin and POW, Deer tick virus, and CE-TBE strains 4387 and D1283 Guriev, and ALK, KFD, and LGT viruses have M364. The have a conservative a substitution to a threonine at residue 366. Kubrin strain of OHF and LGT viruses also contain residue Although there is no T366 in KFD, position 367 has been N366 but position 364 has changed to M364. In the Guriev changed to T367. Overall, OHF, KFD, CE-TBE/RSSE, and D.E. Volk et al. / Virology 351 (2006) 188–195 193

subtype I Kubrin strain sequence differs from the subtype II Bogoluvovska strain at only two amino acids (S349A and I364M). Clearly, further genomic sequencing and antigenic comparisons are required to investigate subtype and strain variation in OHF virus. Two major structural differences occur between the tick- borne and mosquito-borne ED3 structures (Fig. 4). With only one exception (YF virus), all of the mosquito-borne flavivirus ED3 proteins have a highly conserved tyrosine (WN virus nomenclatureY329) located twenty amino acids downstream from a phenylalanine. Tyrosine 329 is associated with a tyrosine corner motif (i.e., a structural feature found in many β- sandwich domains (Hemmingsen et al., 1994)) that has been found in a number of other enveloped viruses (Beasley et al., in preparation). This tyrosine hydrogen bonds with a moderately Fig. 4. A comparison of the tick-borne OHFV and mosquito-borne West Nile conserved asparagine residue (WN virus D333) three positions virus ED3 structures. The backbone of residues involved in the tyrosine corner downstream in loop 3 (Fig. 4, gold loop). Thus, in WN virus in West Nile virus, as well as those analogous in OHFV, are colored orange. The RGE integrin-binding loop of West Nile virus (R388-Q391) is colored green. the hydroxyl group of tyrosine Y329 forms a hydrogen bond with the backbone carboxyl oxygen of asparagine D333. In contrast, most members of the TBE complex encode a POW viruses are different at these surface-exposed residues in phenylalanine in place of the tyrosine, except for ALK, KFD, this region that could lead to differential antibody interactions POW, and deer tick viruses that have the tyrosine. This change and potential variation in receptor specificity or receptor- maintains the hydrophobic packing, which stabilizes the first binding kinetics. beta sheet, but it disrupts the tyrosine corner. Thus, in the tick- borne OHF-ED3, residues F312 and F332 interact to stabilize Discussion the interaction between the N-terminal end of β1 and the C- terminal end of β2. However, the lack of the tyrosine corner in The chemical shift data and the solution structure of the the tick-borne viruses allows loop 3 to be positioned in such a recombinant domain III of the OHF virus envelope protein way as to alter the beta sheet structure at the exposed surface, (OHF-rED3) represent the first NMR data and structures for relative to the mosquito-borne viruses. In the context of the this virus, which is a potential biothreat agent due to its high viral surface, this area of ED3 is the most exposed to the outer pathogenicity. The structure is nearly identical to that of the environment, and numerous studies (Holbrook et al., 2005; Li western subtype CE-TBE ED3 obtained by X-ray crystallog- et al., 2004; Volk et al., 2004; Oliphant et al., 2005) have raphy (Rey et al., 1995). Of the 99 residues in OHF-rED3, 93 shown that residues critical for antibody binding are located in residues in a stretch of 97 (residues 300–396) are identical to this immediate area. those in the CE-TBE-ED3 protein. The residue differences Another major structural difference between the tick-borne (OHF to TBE) are A310T, A331T/A, D353N, and K389S. and many of the mosquito-borne viral ED3 structures is the Excluding strain variation, OHF virus differs from other TBE presence or absence of an RGD/E integrin-binding motif. The complex viruses at 18 residues (Fig. 3). All 18 of these second beta sheet in these mosquito-borne viruses contains an residues are either on the exposed upper surface of the viral additional four residues (R-G-D/E-X), which extends the length particle or they line the pore formed at the mature virion's of the beta sheet. In the WN-ED3 (Fig. 4), these residues are fivefold axis by five ED3 proteins (Kuhn et al., 2002). In the R388-G389-E390-Q391. Outside of these four residues, the context of the mature virion, OHFV residues A331, K336, and amino acids in this beta sheet are moderately to highly I364 are surface exposed residues that lie at the top of the ED3 conserved among the tick- and mosquito-borne viruses. The protein near the opening of the fivefold pore. These residues absence of these four residues in the tick-borne viruses and might be expected to interact with antibodies or host cell some mosquito-borne viruses changes the position of the N- proteins. Within the fivefold pore, residues M306 and A310 lie terminal loop, which is somewhat lower (relative to the outer near the top, residues D387 and K389 lie in the middle, and viral surface). residues A349, D351, and D353 lie near the bottom of the Overall, the structure of OHF ED3 is very similar to that of pore. It is significant to note that this region varies between CE-TBE with differences in beta sheets β8 and β11 only. LGT, POW, ALK, KFD, CE-TBE, and OHF viruses and may Nonetheless, the comparisons of amino acid sequences of therefore play a role in the specificity of binding of these members of the TBE complex show that amino acid variation viruses to host cell receptors. among members of the complex is predominantly restricted to We were surprised that the ED3 protein sequences of the surface exposed residues or those that line the pore formed at Bogoluvovska and Guriev OHF virus strains, which are both the mature virion's fivefold axis by five ED3 proteins consistent members of antigenic subtype II, differ at five amino acids with potential roles in antigenic variation and interactions with (A310T, A317V, S349T, I364M, and N366S), whereas the host receptor molecules. 194 D.E. Volk et al. / Virology 351 (2006) 188–195

Materials and methods amino acids. After several rounds of refinement, dihedral angle restraints that were inconsistent with the NOE data were Protein expression and purification changed to the dihedral angles suggested for the minority of the selected proteins in the TALOS protein data bank. Uniformly 15N-labeled, 15N,13C-labeled, and unlabeled OHF-rED3 proteins (Kubrin strain) were expressed as a Molecular dynamics calculations maltose-binding protein (MPB) fusion using the pMal-c2x vector as previously described (Yu et al., 2004; White et al., One hundred random structures were generated by anneal- 2003). Briefly, the fusion protein was purified on a maltose ing the protein at 700 K, obtaining the coordinates every 5 psi affinity column (New England Biolabs) and digested with factor and minimizing the structures obtained. The structures were Xa (Novagen). Subsequently, the OHF-rED3 protein was then subjected to dihedral angle restraints (Table 1) followed by separated from the fusion protein on a Superdex75 column, the application of all restraints at 300 K. Finally, the structures concentrated, further purified on a Sephacryl HR S-100 column, were energy minimized for 2000 steps. Fifteen structures with concentrated, and finally dialyzed into NMR buffer. The final low restraint penalties were then chosen for the structural OHF-rED3 protein contained residues 300–396 of the envelope ensemble. The SANDER module within AMBER6.0 (Case et protein and two additional residues on the N-terminus, Ile and al., 1999) was used for all NMR structure calculations and Ser, remaining from the fusion cleavage site. MIDAS (Ferrin et al., 1988) and MOLMOL (Koradi et al., 1996) were used to visualize the structures. Coordinates for the ED3 protein gene sequence of the Guriev strain of OHFV NMR structures of OHF-rED3 have been deposited with the Protein Data Bank (PDB ID 1Z3R) and the chemical shifts The sequencing of the region encoding the envelope protein have been deposited with the BioMagResBank (BMRB of the Guriev strain of OHF virus was performed using methods accession code 6309). described previously (Lin et al., 2003). Acknowledgments NMR spectroscopy and the generation of NMR restraints We thank Bo Xu and coworkers of the University of Texas The NMR samples contained 0.7–0.9 mM protein in 25 mM Medical Branch Protein Expression Core Facility for the deuterated Tris (pH 4.0) and 100 mM NaCl in 90% H2O and expression and purification of OHFV-rED3. This work was 10% D2O. All experiments were acquired on Varian UnityPlus supported by grants U90CCU618754 from the Centers for 600 or 750 MHz spectrometers at 25 °C. Sequence-specific Disease Control, a grant from the Pediatric Dengue Vaccine chemical shifts for the backbone were obtained from three- Initiative, a grant from NIH (U01 AI054827), P42296LS0000 dimensional HNCACB (Sattler et al., 1999), CBCANH from the Defense Advanced Research Projects Agency, contract (Grzesiek and Bax, 1992), and HNCO (Ikura et al., 1990) W911 SR-04-C-0065 with the Edgewood Chemical Biological experiments. The backbone experiments were verified by Center in connection with the Countermeasures to Biological sequential NOEs in an 15N-edited NOE experiment (Marion and Chemical Threats Program at the Institute for Advanced et al., 1989) with a 120-ms mixing time acquired at 750 MHz. Technology of the University of Texas at Austin, 004952-0038- Side chain assignments were derived from HCCH-TOCSY 2003 from the State of Texas Advanced Technology Program, a (Vuister and Bax, 1992), H(CCO)NH-TOCSY (Clore and fellowship from the James W. McLaughlin Fellowship Fund to Gronenborn, 1994), and CC(CO)NH-TOCSY (Clore and D.W.C.B. Gronenborn, 1994) experiments. Aromatic proton chemical shifts were assigned from resonances in a CT-1H,13C-HSQC References spectrum and/or NOE spectra. Stereospecific assignments for most of the side-chain protons were obtained after initial rounds Beasley, D.W., Barrett, A.D., 2002. Identification of neutralizing epitopes within of structure calculations using ambiguous restraints. All spectra structural domain III of the West Nile virus envelope protein. J. Virol. 76, 13097–13100. were processed using VNMR v6.1b (Varian, Inc.) or Felix2000 Burke, D.S., Monath, T.P., 2001. Flaviviruses, In: Knipe, D.M., Howley, P.M. (Accelrys, Inc.) software. (Eds.), Field's Virology, 4th ed. 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