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Obstruction of Dengue Virus Maturation by Fab 1
Fragments of the 2H2 Antibody 2
3
Zhiqing Wanga, Long Lia,b, Janice G. Penningtona,c, Ju Shenga, Moh-Lan Yapa, Pavel 4
Plevkaa, Geng Menga, Lei Suna, Wen Jianga, and Michael G. Rossmanna,# 5
6
aDepartment of Biological Sciences, Purdue University, 240 S. Martin Jischke Drive, West 7
Lafayette, IN 47907 8
bPresent address: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, 9
Boston, MA 02115 10
cPresent address: Department of Botany, 430 Lincoln Drive, University of Wisconsin-Madison, 11
Madison, WI 53706 12
13
Corresponding author: [email protected] 14
15
16
Running Title: Immature DENV complexed with 2H2 Fab 17
18
Abstract Word Count: 234 19
Text Word Count: 2848 20
21
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00472-13 JVI Accepts, published online ahead of print on 5 June 2013
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ABSTRACT 22
The 2H2 monoclonal antibody recognizes the precursor peptide on the immature dengue 23
virus and might, therefore, be a useful tool for investigating the conformational change that 24
occurs when the immature virus enters an acidic environment. During dengue virus 25
maturation, the spiky, immature, non-infectious virions change their structure to smooth-26
surfaced particles in the slightly acid environment of the trans-Golgi network, thereby 27
allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions 28
become fully infectious when they release the cleaved precursor peptide on reaching the 29
neutral pH environment of the extracellular space. Here we report on the cryo-electron 30
microscopy structures of the immature virus complexed with the 2H2 antigen binding 31
fragments (Fab) at different concentrations and varied pH conditions. At neutral pH and 32
high concentration of the Fab molecules, three Fab molecules bind to three precursor-33
membrane proteins on each spike of the immature virus. However, at a low concentration 34
of the Fab molecules and at pH 7.0, only two Fab molecules bind to each spike. Changing to 35
slightly acidic pH caused no detectable change of structure for the high Fab concentration 36
sample, but caused severe structural damage to the low concentration sample. Therefore, 37
the 2H2 Fab inhibits the maturation process of immature dengue virus when the Fab 38
molecules are at high concentration, because the three Fab molecules on each spike hold 39
the precursor-membrane molecules together, thereby inhibiting the normal conformational 40
change that occurs during maturation. 41
42
43
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INTRODUCTION 44
Dengue virus (DENV) is a lipid-enveloped, positive-stranded RNA virus that is a member of 45
Flaviviridae. Mosquitos are the major vector for DENV transmission to humans. Symptoms of 46
primary infection are febrile and non-fatal, whereas secondary infections lead to life threatening 47
symptoms such as hemorrhagic fever or dengue shock syndrome (4). The severity of the 48
secondary DENV infection may be associated with antibody-dependent enhancement of 49
infection (ADE) (3, 7). 50
DENV is assembled initially on the endoplasmic reticulum of cells in an immature non-51
infectious form. The fully infectious mature virus is not formed until it is released from its host 52
(24, 25). Both immature (26) and mature (11) DENV particles have icosahedral symmetry with 53
diameters of about 600Å and 500Å with spiky and smooth surfaces, respectively. The structural 54
proteins of DENV are the capsid protein, the precursor-membrane (prM) protein and the 55
envelope (E) protein. The latter two are membrane anchored and are involved in structural re-56
arrangements during maturation and fusion. The prM molecule is a chaperone protein that helps 57
E to fold and to form a heterodimer with prM. The 180 copies of prM-E heterodimers then 58
assemble into 60 trimeric spikes of the immature virus (26). In this form, the pr peptide is located 59
on the top of each trimeric spike, burying much of the fusion loop underneath it (12). Immature 60
DENV are further processed in the trans-Golgi network where the acidic environment causes the 61
immature particles to change into mature-like particles. This structural re-arrangement makes the 62
furin (a cellular protease) cleavage site on prM accessible, resulting in the cleavage of prM, 63
leaving the M protein anchored in the membrane and the pr peptide protecting the fusion loop. 64
The pr peptide is released when the virus is secreted into the neutral pH extracellular space (24, 65
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25). However, DENV maturation is often incomplete with both immature and partially mature 66
virus particles then released into the extracellular space (9, 15). 67
The 2H2 mouse monoclonal antibody was developed (8) and characterized (8, 17) as a 68
highly cross-reactive, low neutralizing antibody that binds to immature DENV. Many other 69
immature DENV antibodies are isolated from patient serum and are agents that can cause ADE 70
(2, 5, 16). Here we report the cryo-electron microscopy (cryoEM) structures of 2H2 Fab 71
fragments complexed with immature virus at different concentrations and pH. We also report the 72
crystal structure of the 2H2 Fab fragment and use it to interpret the structures of the virus-Fab 73
complexes. 74
75
MATERIALS AND METHODS 76
Immature DENV preparation. C6/36 mosquito cells were grown in modified Eagle medium 77
(MEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino 78
acids and 20mM HEPES at 28ºC. Infection of DENV-2 (strain P16681) was carried out when the 79
cells were 50-60% confluent at a MOI of 0.5. Culture medium with 20mM NH4Cl was applied to 80
cells 20 hours post infection to stop the virus maturation process. Supernatant was collected, 81
clarified 48 hours post infection and precipitated with 8% PEG overnight. The PEG precipitant 82
was collected, re-suspended in NTE buffer (NaCl 120mM, Tris 20mM EDTA 1mM, pH 8.0) and 83
further purified by a potassium tartrate step gradient centrifugation. The visible virus band at 20 84
to 25% potassium tartrate concentration was extracted and the buffer was exchanged with NTE 85
buffer five times using a Millipore Centricon (100 KDa MW cut-off). The final volume of 86
immature DENV preparation was 100 to 150 μl. The quality of the virus was evaluated by 87
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inspection of the structural protein bands on a SDS PAGE gel. The concentration of the virus was 88
estimated by comparison of the protein band intensities with BSA standards. 89
2H2 Fab production, crystallization and structure determination. Hybridoma cells 90
expressing the 2H2 antibody were obtained from the American Tissue Culture Collection 91
(ATCC) and grown in BD Cell Mab Basal Medium (BD Biosciences) supplemented with 25% 92
fetal bovine serum. The BD CELLine 1000 culture system (BD Biosciences) was used for 93
antibody production. Antibody containing medium was collected every 7 days for 3 weeks. 94
The 2H2 antibody was first purified with a protein A affinity column. Later, the Fab 95
fragment was generated by papain digestion at 37˚C for 6 hrs and separated from the Fc fragment 96
by using a protein A affinity column. The 2H2 Fab was finally purified with a Superdex 75 97
(16/60) column. 98
Purified 2H2 Fab at 10mg/ml was used to set up crystallization screens using the Emerald 99
Wizard I to IV kits (Emerald Biosystems). Crystals were further optimized using the hanging 100
drop method with 25% PEG 6000, 0.1M MES pH 6.0, 0.2M NH4Cl. X-ray diffraction data were 101
collected at the APS beamline 23ID-D using a wavelength of 1.03Å. The diffraction data was 102
indexed and scaled using the HKL2000 program (14). The crystals had a space group of P22121 103
with cell dimensions a=51.55 Å, b= 87.71 Å, c=85.31 Å and diffracted to 1.8Å resolution. The 104
variable and constant domains derived from an HIV Fab (PDB accessing number: 3OZ9) were 105
used as search models to determine the structure by molecular replacement using the program 106
MOLREP in the CCP4 suite of programs (20-22). This HIV antibody was used for the search 107
procedure because it was also a mouse antibody that belonged to the IgG2a family. The 2H2 108
antibody amino acid sequences of the light and heavy chain variable domains were determined 109
by Syd Labs, Inc. The structure was refined (Table 1) using the Phoenix program (1). The final 110
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Rworking and Rfree values are 17.9% and 23.4% using the data to 2.3 Å resolution. A total of 155 111
water molecules were included. 112
Virus Fab complex formation. The 2H2 Fab was added to ~100µl of immature virus at 113
pH 7.0 in phosphate buffer at 4˚C. High (~50 x) and low (~1x) ratios of the Fab molecule relative 114
to each prM molecule in the virus were used for incubating the virus-Fab complex for several 115
hours. The approximate relative ratios of these concentrations were estimated from the intensity 116
of stain on a SDS PAGE gel (Fig. 1). Aliquots of these samples were flash-frozen on Quantifoil 117
holey carbon grids by hand blotting in a biosafety cabinet. The pH of these samples was then 118
lowered in steps of 0.2 to pH 6.0 and aliquots were again used to prepare cryoEM grids. Finally 119
the pH was returned to pH 7.0 in one step and used to prepare cryoEM grids. Each pH change 120
was performed by centrifuging the sample using a Millipore Centricon (100 KDa MW cut-off) at 121
10,000 rpm for 10 min at 4˚C. 122
Data collection and single particle reconstruction. Images of each sample were taken 123
on a CM200 FEG transmission electron microscope (Philips/FEI) at a magnification of 51,000 124
under low-dose conditions (~20 e/Å2) and recorded on Kodak SO-163 films. The micrographs 125
were digitized using a Nikon 9000 scanner with 6.35μm step size. Particles were selected 126
manually with the boxer program in EMAN (13, 19). The microscope contrast transfer function 127
parameters for each micrograph were first determined using an automated fitting method (23) 128
and then manually verified and corrected using the EMAN ctfit graphic program. To avoid model 129
bias, 5 “random” initial models per dataset were generated by random particle orientations 130
assignment. Iterative refinement processes including 2-D particle alignments and 3-D 131
icosahedral reconstructions were performed using the program jspr.py (6) with the 132
EMAN/EMAN2 program (13, 19). The Fourier shell correlation (FSC) between structures built 133
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from the five independent models was then calculated to evaluate the convergence. Among the 134
converged datasets, particles with stable orientations and centers were kept for further 135
refinement. 136
For the immature DENV complexed with high concentration Fabs at pH 7.0, pH 6.0 and 137
back neutralized to pH 7.0, 676, 694 and 680 particles were used for the initial image 138
reconstructions, respectively. The final reconstructions utilized 378 (Fig. 2a to c; Fig. 3a and b), 139
152 (Fig.2g to i), 344 (map not shown) particles, respectively. The resolutions of these maps 140
were estimated to be 21Å, 25Å and 21Å (respectively) based on the resolution at which the FSC 141
became less than 0.5. For the immature DENV complexed with a low Fab concentration at pH 142
7.0, two datasets with 2518 and 4635 particle images were used for the initial reconstruction. The 143
final reconstruction used 802 (map not shown) and 2253 (Fig. 2d to f; Fig. 3c and d) of these 144
particles that produced maps whose resolution was estimated to be 23Å and 21Å, respectively. 145
Structure analysis. The 12.5Å resolution, cryoEM map of the immature DENV (EMDB 146
accessing number: 5422) had been interpreted using the prM-E heterodimer crystal structure 147
(PDB accession number: 3C6D) in terms of 60 trimeric spikes per viron. The structure of the 148
prM-E trimer (12) was used as a single rigid body to fit into the cryoEM map of the various 149
complexes using the EMfit program (18). The density of these cryoEM maps was then set to zero 150
at all grid points that were within 3Å of any atom in the fitted structure. The resultant maps were 151
used to fit the crystal structure of the 2H2 Fab fragments into each of the three independent 152
positions on the glycoprotein spike within the icosahedral asymmetric unit using the EMfit 153
program. The three positions were identified as green, blue and magenta, with the blue position 154
being between the green and magenta positions. The surface areas between neighboring Fab 155
molecules was calculated by the program PISA (10). 156
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Protein structure and cryo-EM density map accession numbers. The structure factors 157
and atomic coordinates of 2H2 Fab were deposited with the Protein Data Bank, accesssion num-158
ber 4KVC. The cryo-EM density maps of the 2H2 Fab complexed with immature dengue virus at 159
different pH conditions and Fab concentrations were deposited with the EM Data Bank, acces-160
sion numbers EMD-5674, EMD-5675, EMD-5676, and EMD-5677. The fitted atomic coordi-161
nates complex has been deposited with the Protein Data Bank, accession number XXXX. 162
163
RESULTS 164
The 2H2 Fab fragments were complexed with immature virus at neutral pH for cryoEM data 165
collection. Complexes were formed both when the Fab was at high and at low concentration. 166
These complexes were then moved to a low pH environment for cryoEM studies. The complexes 167
were back neutralized for the final cryoEM studies. However, the low concentration Fab virus 168
complexes disintegrated when the pH was lowered and could therefore be studied only at the 169
initial neutral pH. 170
The cryoEM reconstruction showed that one 2H2 Fab molecule bound to each prM 171
molecule per trimeric spike of the immature virus when a high concentration of Fab molecules 172
was used at neutral pH. In this structure, three Fab densities (green, blue, and magenta) were 173
clearly resolved and were oriented radially outward on each of the trimeric prM-E spikes of the 174
virus (Fig. 2a to c). Thus, a total of 180 copies of 2H2 Fab were bound to the 180 copies of pr on 175
the viral surface. Each of the Fab densities consisted of two lobes corresponding to the constant 176
and variable domain dimers, which were connected by two thin stalks or “elbows” (Fig. 3a and 177
b). The cryoEM density of the virus, underneath the bound Fabs, maintained the features of 178
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immature DENV and this was further confirmed by fitting the prM-E trimer crystal structure 179
(Table 2) into the cryoEM map. 180
The structure of virus Fab complexes with three 2H2 Fabs present on each spike did not 181
change either when the pH was lowered to 6 (Fig. 2g to i) or when subsequently changed back to 182
neutral. Previous results had shown that, in the absence of Fab, lowering the pH from 7.0 to 6.0 183
changed the structure from having 60 spiky trimers to a smooth surfaced virus containing 90 184
dimers. In contrast, it is shown here that the presence of three 2H2 Fab molecules bound to each 185
trimeric spike stopped the conformational change that would have occurred in the absence of 186
bound Fab molecules. 187
However, in the structure of the immature DENV virus complexed with low 188
concentration of Fab at neutral pH, only two Fab densities (green and blue positions) were 189
resolved on each of the trimeric prM-E spikes (Fig. 2d to f and Fig. 3c and d). Thus, 2H2 Fab 190
molecules preferentially bound to two of the three prM molecules per trimeric spike on the viral 191
surface resulting in a total of 120 copies of 2H2 Fab molecules presented. Furthermore, when pH 192
was lowered, these complexes disintegrated into heterogonous particle populations. Therefore 193
the presence of only two Fab molecules on a spike is insufficient to stop the conformational 194
change that normally occurs when immature virus encounters an acidic pH. 195
The crystal structure of the 2H2 Fab fragment was fitted into the cryoEM difference map 196
(Materials and Methods). The binding site of the Fab molecule was on the top of the pr peptide 197
and consisted primarily of the highly exposed a and c strands that belong to two adjacent β-198
sheets (12) of the pr peptide (Fig. 4a and b; Table 3). Both hydrophobic and charged 199
interactions participate in these interactions. Assuming the crystal structure of the isolated prM-E 200
heterodimer (12) to interpret the cryoEM results reported here would place this glycan within 6Å 201
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of the Fab binding interface. However the heterodimer was produced in drosophila cells whereas 202
the virus was propagated in mosquito cells. Thus it is not clear whether the glycan moiety at this 203
site is involved in the binding of the 2H2 antibody. Superposition of the three independent pr 204
peptides in the icosahedral asymmetric unit showed that the Fab fragments bound to the pr 205
peptides are in roughly similar orientation with respect to each prM-E heterodimer (Fig. 4a and 206
b). 207
Even though there are slight differences in the positions and orientations of the three 208
independent Fab molecules relative to each prM-E heterodimer (Fig. 4a and b), there are more 209
significant differences in the Fab occupancies. The central blue Fab is the best ordered as 210
measured by the high density at the atomic positions (sumf) and the low percentage of residues 211
in negative density (-den). In contrast, the magenta Fab is the least ordered and almost 212
completely missing at low Fab concentration (Table 4). The contact area between the blue and 213
green Fab molecules was 525Å2 whereas the contact area between the magenta and blue Fab 214
molecules was only 32Å2 (Fig. 5). Thus the interaction between blue and green Fab molecules 215
may have stabilized their binding to the prM-E molecules even when the Fab concentration was 216
low. 217
218
DISCUSSION 219
When the 2H2 Fab molecules were at high concentration, the immature virus did not change its 220
conformation in acid pH (Fig. 2 and Fig. 3), possibly because the association of the three Fab 221
molecules on each prM-E inhibits the conformational change that is required to form the mature 222
virus. In contrast, at low Fab concentration only two Fab molecules (blue and green positions) 223
were bound to each spike. Presumably the association of these two Fab molecules was unable to 224
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hold the spikes together, when the pH was lowered. As a result, the particles degenerated into a 225
heterogeneous collection of conformations that could not be used for a successful image 226
reconstruction. Possibly, at low concentrations, the prM-E heterodimers lacking a Fab molecule 227
would be free to make the first movement on being exposed to acidic pH, but then would not be 228
able to find another unbound partner to make a dimer as required for the formation of a mature 229
particle. These attempts at re-assortment of trimers into dimers would create a large variety of 230
heterogeneous particles as observed. The difference between the low and high concentration 231
structures of the 2H2 Fab complex with partially mature virus may be related to the increase of 232
infectivity caused by low concentration prM binding antibodies (2, 5, 16) and, hence, would also 233
be associated with the occurrence of ADE. Based on the present results, low concentration of 234
2H2 can bind to partially immature virus that would permit the virus to infect other cells by 235
means of their Fc receptor molecules, thus enhancing the infectivity of the virus. 236
Although the spikes in immature flaviviruses are “trimers”, they do not have an exact 3-237
fold axis, showing that the three prM-E hetrodimers in a spike could have slightly different 238
structures and properties. This asymmetry has been amplified in that the Fab bound to the blue 239
site has greater order at all pH and all Fab concentrations (Table 4). The lack of equivalence 240
between the three heterodimers in a spike must originate in the assembly process, suggesting a 241
sequential pathway for the assembly of the immature virus. This lack of equivalence between 242
heterodimers might also be required in the subsequent conformational processes ending up in a 243
mature structure in which the three heterodimers do not have equivalent T=3 environments. 244
245
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ACKNOWLEDGEMENTS 246
This work was supported by NIH grant AI76331 to MGR. Use of the Advanced Photon Source 247
was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy 248
Sciences, under Contract No. DE-AC02-06CH11357. We thank the National Institutes of Health 249
(S10RR023011A) and Purdue University for their support of the EM facility. 250
We appreciate TJ Battisti’s support in training on cryoEM data collection procedure. We 251
are grateful to Sheryl Kelly for her administrative support. We thank Richard Kuhn for helpful 252
discussions, and Valorie Bowman and Agustin Avila-Sakar for technical support. 253
254
255
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REFERENCES 256
1. Adams, P. D., P. V. Afonine, G. Bunkoczi, V. B. Chen, I. W. Davis, N. Echols, J. J. 257
Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. 258
Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. 259
Terwilliger, and P. H. Zwart. 2010. PHENIX: a comprehensive Python-based system for 260
macromolecular structure solution. Acta Crystallogr. Sect D Biol. Crystallogr. 66:213-261
221. 262
2. Beltramello, M., K. L. Williams, C. P. Simmons, A. Macagno, L. Simonelli, N. T. 263
Quyen, S. Sukupolvi-Petty, E. Navarro-Sanchez, P. R. Young, A. M. de Silva, F. A. 264
Rey, L. Varani, S. S. Whitehead, M. S. Diamond, E. Harris, A. Lanzavecchia, and F. 265
Sallusto. 2010. The human immune response to dengue virus is dominated by highly 266
cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host 267
Microbe 8:271-283. 268
3. Boonnak, K., B. M. Slike, T. H. Burgess, R. M. Mason, S. J. Wu, P. Sun, K. Porter, I. 269
F. Rudiman, D. Yuwono, P. Puthavathana, and M. A. Marovich. 2008. Role of 270
dendritic cells in antibody-dependent enhancement of dengue virus infection. J Virol 271
82:3939-3951. 272
4. Centers for Disease Control and Prevention Dengue Branch 2013, posting date. 273
Dengue Homepage - Clinical Guidance. Centers for Disease Control. [Online.] 274
5. Dejnirattisai, W., A. Jumnainsong, N. Onsirisakul, P. Fitton, S. Vasanawathana, W. 275
Limpitikul, C. Puttikhunt, C. Edwards, T. Duangchinda, S. Supasa, K. 276
Chawansuntati, P. Malasit, J. Mongkolsapaya, and G. Screaton. 2010. Cross-reacting 277
antibodies enhance dengue virus infection in humans. Science 328:745-748. 278
on June 22, 2018 by guesthttp://jvi.asm
.org/D
ownloaded from
14
6. Guo, F., and W. Jiang. In press. Single particle cryo-electron microscopy and 3-D 279
reconstruction of viruses. In J. Kuo (ed.), Methods in Molecular Biology: Electron 280
Microscopy. Humana Press. 281
7. Halstead, S. B., and E. J. O'Rourke. 1977. Antibody-enhanced dengue virus infection 282
in primate leukocytes. Nature 265:739-741. 283
8. Henchal, E. A., J. M. McCown, D. S. Burke, M. C. Seguin, and W. E. Brandt. 1985. 284
Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using 285
monoclonal antibodies. Am. J. Trop. Med. Hyg. 34:162-169. 286
9. Junjhon, J., T. J. Edwards, U. Utaipat, V. D. Bowman, H. A. Holdaway, W. Zhang, P. 287
Keelapang, C. Puttikhunt, R. Perera, P. R. Chipman, W. Kasinrerk, P. Malasit, R. J. 288
Kuhn, and N. Sittisombut. 2010. Influence of pr-M cleavage on the heterogeneity of 289
extracellular dengue virus particles. J Virol 84:8353-8358. 290
10. Krissinel, E., and K. Henrick. 2007. Inference of macromolecular assemblies from 291
crystalline state. J. Mol. Biol. 372:774-797. 292
11. Kuhn, R. J., W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C. T. 293
Jones, S. Mukhopadhyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H. 294
Strauss. 2002. Structure of dengue virus: implications for flavivirus organization, 295
maturation, and fusion. Cell 108:717-725. 296
12. Li, L., S. M. Lok, I. M. Yu, Y. Zhang, R. J. Kuhn, J. Chen, and M. G. Rossmann. 297
2008. The flavivirus precursor membrane-envelope protein complex: structure and 298
maturation. Science 319:1830-1834. 299
13. Ludtke, S. J., P. R. Baldwin, and W. Chiu. 1999. EMAN: semiautomated software for 300
high-resolution single-particle reconstructions. J. Struct. Biol. 128:82-97. 301
on June 22, 2018 by guesthttp://jvi.asm
.org/D
ownloaded from
15
14. Otwinowski, Z., and W. Minor. 1997. Processing of X-ray diffraction data collected in 302
oscillation mode, p. 307-326. In Charles W. Carter, Jr. (ed.), Methods in Enzymology, 303
vol. 276. Academic Press. 304
15. Plevka, P., A. J. Battisti, J. Junjhon, D. C. Winkler, H. A. Holdaway, P. Keelapang, 305
N. Sittisombut, R. J. Kuhn, A. C. Steven, and M. G. Rossmann. 2011. Maturation of 306
flaviviruses starts from one or more icosahedrally independent nucleation centres. EMBO 307
Rep. 12:602-606. 308
16. Rodenhuis-Zybert, I. A., H. M. van der Schaar, J. M. da Silva Voorham, H. van der 309
Ende-Metselaar, H. Y. Lei, J. Wilschut, and J. M. Smit. 2010. Immature dengue virus: 310
a veiled pathogen? PLoS Pathog. 6:e1000718. 311
17. Roehrig, J. T., R. A. Bolin, and R. G. Kelly. 1998. Monoclonal antibody mapping of the 312
envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 246:317-328. 313
18. Rossmann, M. G., R. Bernal, and S. V. Pletnev. 2001. Combining electron microscopic 314
with X-ray crystallographic structures. J. Struct. Biol. 136:190-200. 315
19. Tang, G., L. Peng, P. R. Baldwin, D. S. Mann, W. Jiang, I. Rees, and S. J. Ludtke. 316
2007. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. 317
Biol. 157:38-46. 318
20. Vagin, A., and A. Teplyakov. 1997. MOLREP: an automated program for molecular 319
replacement. J. Appl. Crystallogr. 30:1022-1025. 320
21. Vagin, A. A., and M. N. Isupov. 2001. Spherically averaged phased translation function 321
and its application to the search for molecules and fragments in electron-density maps. 322
Acta Crystallogr D Biol Crystallogr 57:1451-1456. 323
22. Winn, M. D., C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. 324
on June 22, 2018 by guesthttp://jvi.asm
.org/D
ownloaded from
16
M. Keegan, E. B. Krissinel, A. G. W. Leslie, A. McCoy, S. J. McNicholas, G. N. 325
Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin, and K. 326
S. Wilson. 2011. Overview of the CCP4 suite and current developments. Acta 327
Crystallogr. D Biol Crystallogr. 67:235-242. 328
23. Yang, C., W. Jiang, D. H. Chen, U. Adiga, E. G. Ng, and W. Chiu. 2009. Estimating 329
contrast transfer function and associated parameters by constrained non-linear 330
optimization. J Microsc 233:391-403. 331
24. Yu, I.-M., H. A. Holdaway, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. 332
Chen. 2009. Association of the pr peptides with dengue virus at acidic pH blocks 333
membrane fusion. J Virol 83:12101-12107. 334
25. Yu, I.-M., W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. 335
J. Kuhn, M. G. Rossmann, and J. Chen. 2008. Structure of the immature dengue virus 336
at low pH primes proteolytic maturation. Science 319:1834-1837. 337
26. Zhang, Y., J. Corver, P. R. Chipman, W. Zhang, S. V. Pletnev, D. Sedlak, T. S. Baker, 338
J. H. Strauss, R. J. Kuhn, and M. G. Rossmann. 2003. Structures of immature 339
flavivirus particles. EMBO J. 22:2604-2613. 340
341
342
on June 22, 2018 by guesthttp://jvi.asm
.org/D
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FIGURE LEGENDS 343
FIG. 1. SDS-PAGE gel showing the concentration ratio of 2H2 Fab to prM. (a) Immature DENV 344
complexed with high concentration of 2H2 Fab. (b) Immature DENV complexed with low 345
concentration of 2H2 Fab. 346
347
FIG 2. Cryo EM density maps of immature DENV complexed with 2H2 Fab fragments. (a, b, c) 348
High Fab concentration at pH 7.0; (d, e, f) Low Fab concentration at pH 7.0; (g, h, i) High Fab 349
concentration at pH 6.0. (a, d, g) Density map of the whole particle. (b, e, h) Enlarged view 350
showing one asymmetric unit identified by the white triangle. (c, f, i) Side view showing the Fab 351
fragment bound to a trimeric prM-E spike. The maps are colored by radius as indicated. Note 352
that one of the three Fab fragments is missing in d, e and f (see inside the dashed circle) at low 353
concentration of the Fab molecules. 354
355
FIG 3. Structure of the 2H2 Fab crystal structures shown as ribbon drawings fitted into cryoEM 356
density (colored by radius in mesh) of the virus-Fab complex. (a, b) high concentration of the 357
Fab molecules at pH 7.0. (c, d) Low concentration of the Fab molecules at pH 7.0. The three 358
independent positions are colored green, blue and magenta. (a, c) Top view showing one 359
asymmetric unit. (b, d) Side view showing one trimeric spike of the virus-Fab complex. The 360
prM-E trimer is colored baby blue. 361
362
FIG 4. Superposition in pairs of the three 2H2 Fab molecule complexed with the pr peptide of 363
the immature virus, aligned by superimposing the pr peptide. (a) Superposition of the green and 364
blue Fab molecules. (b) Superposition of the blue and magenta Fab molecules. The pr peptide is 365
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shown in baby blue. Note that all 2H2 Fab molecules are in close proximity to the β-sheet 366
structure of the pr peptide. 367
368
FIG 5. Contacts between bound Fab molecules fitted into the map of the immature dengue virus 369
at pH 7.0 complexed with the Fab molecules at high concentration. (a) Side view of the green 370
and blue Fab molecules. (b) Side view of the blue and magenta Fab molecules. The pr peptide is 371
shown in baby blue. 372
373
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TABLES 374
TABLE 1. 2H2 Fab data collection and refinement statistics. 375
Data Collection
Beamline APS 23ID-D
Temperature 100K
Wavelength(Å) 1.03
Resolution(Å) a 2.3
Space group P22121
Unit cell(Å) a = 51.55 b = 87.71 c = 85.31
Unique reflections 16,607
Redundancy 9.5
I/σ 24.7 (3.6)
Completeness(%) 99.9 (100.0)
Rmerge(%)b 10.5
Refinement
Resolution range(Å) 44.1-2.3
Rwork(%)c 17.90
Rfree(%)d 23.35
Average B factor (Å2) 24.42
Rmsd bonds from idealized values (Å) 0.008
Rmsd angels from idealized values (◦) 1.20
Residues in disallowed region of the Ramachandran plot (%)
0.7
a Values in parentheses throughout the table correspond to the outermost resolution shell
b Rmerge =∑ | I- <I> | / ∑ I, where I is measured intensity for reflections with indices hkl. c Rwork= ∑ ||Fobs| - |Fcalc|| ⁄ ∑ |Fobs|
d Rfree has the same formula as Rwork except that calculation was made with the structure factors from the test set.
376
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TABLE 2. Fitting of the prM-E trimer into cryoEM density maps. 377
Fab concentration pH sumfa clashb -denc θ1d θ2 θ3 cxe cy cz
0 8.0 45.3 4.5 1.3 329.5 -0.7 300.0 46.9 50.2 212.6
high 7.0 50.9 0.2 2.2 331.0 1.0 300.0 43.7 50.2 208.4
high 6.0 38.1 0.2 2.5 331.0 1.0 300.0 43.9 49.8 208.6
high bk7.0f 50.2 0.1 3.2 331.0 1.0 300.0 44.0 50.4 208.6
low 7.0 41.3 0.2 1.7 331.0 0.0 300.0 44.3 50.5 209.6asumf is the average density for all atom positions normalized by the highest density in the map 378
to 100. 379
bclash (%) describes the percentage of atoms in the map that have steric clashes with symmetry-380
related subunits. 381
c-den (%) represents the percentage of atoms that are positioned outside of the density. 382
dθ1, θ2, and θ3 (°) are the Eulerian angles that rotate the molecules from their initial position to 383
their fitted positions. 384
ecx, cy and cz (Å) are the final center positions of the molecules after fitting. 385
fbk7.0 represents back neutralized to pH 7.0. 386
387
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TABLE 3. Amino acids in the interface between the virus and Fab molecule. 388
pr 2H2 Fab Heavy Chain
1F Y102
3L Y102
21K S30, S31
24L F32, Y102
25F Y102, P103,
26K N101, Y102, P103, H104, Y49
27T Y102, P103
28E H104
389
390
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TABLE 4. Fitting of the 2H2 Fab structure into the cryoEM density maps. 391
Fab
position
Fab
concentration pH sumfa -denb θ1
c θ2 θ3 cxd cy cz
blue high 7.0 34.0 4.2 121.0 49.5 289.8 72.1 66.9 284.0
high 6.0 24.7 6.9 129.8 52.0 290.0 71.7 66.3 284.5
high bk7.0e 33.1 2.8 121.0 51.0 297.0 72.0 67.0 294.1
low 7.0 18.1 4.6 121.0 50.0 299.0 71.6 67.1 284.0
green high 7.0 32.6 4.1 129.0 47.8 221.3 33.5 91.2 284.2
high 6.0 23.5 8.5 139.3 49.8 201.0 29.3 90.3 284.0
high bk7.0 30.8 5.1 135.3 49.0 218.0 33.6 90.9 282.7
low 7.0 17.1 7.2 141.0 40.0 210.3 33.7 91.7 283.3
magenta high 7.0 27.4 9.8 150.0 63.0 92.0 47.2 28.4 295.1
high 6.0 20.3 17.9 150.3 70.0 90.0 47.2 27.9 296.1
high bk7.0 28.1 8.5 149.8 63.0 92.0 47.3 27.8 295.8
low 7.0 8.6 34.8 159.0 62.0 98.5 47.4 28.4 295.8asumf is the average density for all atom positions normalized by the highest density in the map 392
to 100. 393
b-den (%) represents the percentage of atoms that are positioned outside of the density. 394
cθ1, θ2, and θ3 (°) are the Eulerian angles that rotate the molecules from their initial position to 395
their fitted positions. 396
dcx, cy and cz (Å) are the final center positions of the molecules after fitting. 397
ebk7.0 represents back neutralized to pH 7.0. 398
399
400
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