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Structure of an Enteric Pathogen Bovine Parvovirus1
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Shweta Kailasan1, Sujata Halder1, Brittney Gurda1#, Heather Bladek1†, Paul R. 3
Chipman1, Robert McKenna1, Kevin Brown2‡, Mavis Agbandje-McKenna1* 4
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1Department of Biochemistry and Molecular Biology and the McKnight Brain Institute, 6
University of Florida, Gainesville, Florida 32607, USA; 2Hematology Branch, National 7
Heart Lung and Blood Institute, NIH, Bethesda, MD 20892. 8
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Running Title: The capsid structure of Bovine Parvovirus 11
Abstract: 250 words, Importance: 150 words, Text: 5,130, Figures: 7, Tables, 3. 12
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*Corresponding author: Mavis Agbandje-McKenna, [email protected] . 15
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Current address: #Department of Pathobiology and Clinical Studies, School of 17
Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA; †633 W. 18
Rittenhouse Street, Philadelphia, PA; ‡Virus Reference Department, Public Health 19
England, London NW9 5HT, United Kingdom. 20
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JVI Accepts, published online ahead of print on 17 December 2014J. Virol. doi:10.1128/JVI.03157-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 25
Bovine Parvovirus (BPV), the causative agent of respiratory and gastrointestinal 26
disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae. 27
Towards efforts to obtain a template for the development of vaccines and small 28
molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from 29
the major capsid viral protein 2 (VP2), was determined using X-ray crystallography and 30
cryo-electron microscopy and three-dimensional image reconstruction to 3.2 and 8.8 Å 31
resolution, respectively. The VP2 region ordered in the crystal structure, residues 39-32
536, conserves the parvoviral eight-stranded jellyroll motif and an A helix. The BPV 33
capsid displays common parvovirus features: a channel at and depressions surrounding 34
the 5-fold axes, and protrusions surrounding the 3-fold axes. However, rather than a 35
depression centered at the 2-fold axes, a raised surface loop divides this feature in 36
BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, 37
compared to the crystal structure, is interpreted as ten additional N-terminal residues, 38
29-38 that radially extends the channel under the 5-fold axis, as observed for Human 39
Bocavirus 1. Surface loops of varying lengths and conformations extend from the core 40
jellyroll motif of VP2. These confer the unique surface topology of the BPV capsid, 41
making it strikingly different from HBoV1 as well as the type members of other 42
Parvovirinae genera for which structures have been determined. For the type members, 43
structurally analogous regions to those decorating the BPV capsid surface serve as 44
determinants of receptor recognition, tissue and host tropism, pathogenicity, and 45
antigenicity. 46
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Importance 47
Bovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is a type 48
member of the Bocaparvovirus genus of the non-enveloped, ssDNA Parvoviridae family. 49
Recent isolation of human bocaparvoviruses from children with severe respiratory and 50
gastrointestinal infections has generated interest in understanding the lifecycle and 51
pathogenesis of these emerging viruses. We have determined high-resolution structure 52
of the BPV capsid assembled from its predominant capsid protein VP2, known to be 53
involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity 54
for other Parvovirinae members. Our results indicate the conservation of the core 55
secondary structural elements and the location of the N-terminal residues for the known 56
bocaparvovirus capsid structures. However, surface loops with high variability in 57
sequence and conformation give BPV a unique capsid surface topology. Similar 58
analogous regions in other Parvovirinae type members are important as determinants of 59
receptor recognition, tissue and host tropism, pathogenicity, and antigenicity. 60
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Introduction 69
Bovine Parvovirus (BPV), the type member of the Bocaparvovirus genus of the 70
Parvoviridae, was discovered in 1961 in the gastrointestinal tract of diarrheal calves (1). 71
In addition to acute gastroenteritis, it is associated with reproductive disorders such as 72
spontaneous abortions and stillbirths (2). The occurrence of BPV infections in herds is 73
high at 83-100% worldwide (2–5). Other known genus members include the human 74
bocaviruses (6–8), canine minute virus (9), porcine bocavirus (10), gorilla bocavirus 75
(11), California sea lion bocavirus (12), feline bocavirus, and canine bocavirus (13) 76
which all have similar disease phenotypes. Currently, no treatment or preventive 77
measures are available for Bocaparvovirus infections. The majority of the data available 78
for these viruses is epidemiological. Hence, there is a need to study them at a cellular, 79
molecular, and structural level. 80
BPV is an autonomously replicating virus with a linear single-stranded (ss) DNA 81
genome of ~5.5 kB flanked by non-identical palindromic terminal hair-pins, similar to 82
other bocaparvoviruses (14). The genome consists of three open-reading frames 83
(ORF1-3). ORF1 encodes a non-structural protein, NS1, important for DNA replication. 84
ORF2 encodes a nuclear phosphoprotein, NP1, unique to the Bocaparvovirus genus, 85
containing nuclear localization signals (NLS), and plays a role in viral RNA processing 86
during gene expression (15, 16). Lastly, ORF3 encodes two capsid viral proteins (VP1, 87
VP2) which are generated as a result of alternative splicing events (1). VP1 (75 kDa) 88
and VP2 (61 kDa) share a common C-terminus end but VP1 contains an additional N-89
terminal region (VP1u). The VP1u has a phospholipase-A2 motif and NLS essential for 90
infectivity by facilitating release of the virus from the endocytic pathway during trafficking 91
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and entry into the nucleus for initiation of viral replication, respectively. A total of sixty 92
copies of the VP1 and VP2 assemble a T=1 icosahedral capsid. However, sixty copies 93
of Bocaparvovirus VP2 alone is also able to assemble a capsid (17). 94
The parvoviral capsid is involved in a myriad of functions including cell entry, 95
endosomal trafficking to the nucleus, and cell egress (18, 19). These capsids are also 96
reported to be subjected to the selective pressures of the environment, host cells, and 97
the host immune system (20). Several structural “hotspots” have been identified on the 98
capsid surface, specially near the icosahedral axes of symmetry, which are important 99
for different functions including receptor attachment, antigenicity, and pathogenicity 100
(reviewed in (21)). For the bocaparvoviruses, while there is some information on 101
requirements for cellular infection, nothing is known about the capsid determinants of 102
these interactions. BPV, in particular, has been shown to bind to N- and O-linked sialic 103
acid moieties which may serve as primary receptors for cell entry (22, 23). Cell-based 104
studies with human bocavirus 1 (HBoV1) virions predict that primary and co-receptors 105
on the apical membrane of human airway epithelial cells influence efficient virion 106
infection and cellular transduction (24). Information on Bocaparvovirus capsid structure 107
will provide a platform to begin the elucidation of sites on the capsid that serve as 108
important determinants of host-range, tissue-tropism, pathogenicity, and 109
bocaparvovirus-related disease emergence. 110
Here, we report the structure of BPV determined by X-ray crystallography and cryo-111
electron microscopy and three-dimensional (3D) image reconstruction (cryo-112
reconstruction) to 3.2 and 8.8 Å resolution, respectively. The capsid conserves 113
parvovirus features: a channel at and depressions surrounding the 5-fold and 114
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protrusions surrounding the 3-fold axes. Uniquely, the 2-fold axes have a flat surface 115
flanked by a depression. A comparison to the previously published HBoV1 structure, 116
determined to 7.9 Å resolution by cryo-reconstruction (17), revealed conserved density 117
extending the 5-fold channel radially inwards in both viruses while differences were 118
located on the capsid surface. Surface loop differences, clustered at or close to 2-, 3-, 119
and 5-fold axes in addition to the capsid surface region between the 2- and 5-fold axes 120
(the 2/5-fold wall), gave each Bocaparvovirus capsid a distinctive surface topology. 121
These regions may play a role in governing host and tissue specific interactions. A 122
comparison to other type members of the Parvovirinae subfamily, with known crystal 123
structures (adeno-associated virus (AAV2), minute virus mice (MVMp), and human 124
parvovirus B19) or a pseudo-atomic model built into a cryo-reconstructed density 125
(Aleutian mink disease virus (AMDV)), identified similarities and differences in VP2 and 126
the capsid. While maintaining the VP2 secondary structural elements that are likely 127
important for capsid assembly, unique surface features are conferred on BPV due to 128
variations in VP2 surface loop lengths and conformations. These surface variations 129
occur in analogous regions known to control infectious functions, including host range, 130
receptor attachment, antigenicity, and pathogenicity, for other Parvovirinae type 131
members. This structure serves as a template to begin structure-function annotations of 132
bocaparvoviruses. 133
134
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Materials and Methods 135
VP2 virus-like particle (VLP) expression and purification. The VP2 gene of BPV 136
(HADEN strain, ABC69731.1) was cloned into a pFastBac vector to produce a 137
recombinant baculovirus using standard Bac-to-Bac technology (Invitrogen). 138
Spodoptera frugiperda (Sf9) cells, maintained in Grace’s medium with 10% fetal calf 139
serum and antibiotics, were infected with the recombinant baculovirus and harvested 4-140
7 d post infection. The purification of BPV VLPs was carried out using sucrose cushion 141
and sucrose density gradients as previously described for HBoV1 (17). The final sample 142
was isolated from the 25% fraction of the sucrose gradient and concentrated to 10 143
mg/mL using Amicon concentrators (EMD Millipore). The purity and integrity of the 144
VLPs were determined using SDS-PAGE (data not shown) and negative-stain electron 145
microscopy (EM), respectively. A polyclonal primary antibody to the BPV capsid 146
generated in guinea pigs (generously provided by Dr. Brent F. Johnson, BYU) and a 147
secondary anti-guinea pig horseradish peroxidase-conjugated Protein A antibody 148
(Invitrogen) were used for denatured western-blots and native dot blots to confirm the 149
presence of protein and VLPs, respectively (data not shown). 150
151
X-ray diffraction data collection and processing. The purified BPV VLP sample was 152
used to screen for suitable crystallization conditions using the hanging-drop vapor-153
diffusion method (25). Each drop contained 2 l of VLPs and 2 l of the reservoir 154
solution. Crystallization screens included conditions with varying PEG 8000 (0.5-4.0%), 155
NaCl (75-500 mM), MgCl2 (0-8 mM), Li2SO4 (0-50 mM), and pH ranging from 6.0-8.5 156
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(Bis-Tris, Tris-HCl). The crystals used for data collection were flashed cooled in the 157
precipitant solution with 10% PEG 8000 and 30% glycerol added as cryo-protectant. 158
Diffraction images were collected at the Advanced Photon Source (Argonne 159
National Laboratory) using the SER-CAT 22-ID-D beamline on a MAR300 CCD detector 160
with =0.97240 Å. The crystal-to-detector distance, oscillation width, and exposure time 161
were 400 mm, 0.3º, and 4 s, respectively. The measured reflections, with maximum 162
resolution of 3.2 Å, were indexed, integrated, and scaled using the HKL2000 suite of 163
programs (26). The space group was assigned as C2221 with unit cell parameters 164
a=323.4, b=381.2, and c=376.6 Å. The Mathews coefficient was calculated using the 165
MATTHEWS_COEF subroutine in the CCP4 suite of programs to be 3.23 Å/Da3 and 166
corresponding solvent content of 61.9%, assuming a VLP molar mass of ~3.66 x 106 Da 167
(27, 28). A self-rotation function was calculated using the GLRF program with 10% of 168
the data between 10 to 5 Å resolution, 120 Å radius of integration, and = 72°, 120°, 169
and 180° to search the 5-fold, 3-fold, and 2-fold non-crystallographic symmetry (NCS) 170
elements, respectively, of the icosahedral virus capsid (29). The rotation function was 171
consistent with four VLPs in the unit cell with the NCS 2-fold and crystallographic 2-fold 172
axes coincident resulting in 30 VP2 monomers per asymmetric unit. 173
174
Molecular replacement and structure refinement. Molecular replacement methods 175
were used to obtain the initial phases, using the crystal structure of B19 VP2 (PDB ID: 176
1S58) as the phasing model to 7 Å resolution using the AutoMR subroutine within the 177
PHENIX program (30–32). A polyalanine model of B19 VP2 was generated in the 178
MAPMAN program and an oligomer of 30 VP2 monomers was built in VIPERdb2 179
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(calculated by icosahedral matrix multiplication) (33, 34). Phases were extended to the 180
final high resolution of 3.2 Å using the density modification subroutine in the CNS 181
program suite, which includes solvent flattening and NCS averaging (35). Subsequent 182
model coordinate refinement was conducted in the CNS program using simulated 183
annealing, energy minimization, position and individual temperature factor refinement 184
(Bfactor) while applying 30-fold NCS. The refinement process was monitored using 5% of 185
the total data set for the calculation of Rfree (defined in Table 1). This step was followed 186
by real space electron density averaging using a VP2 molecular mask generated in the 187
CNS program. The electron density was interpretable for residues 39 to 536 (C-terminal 188
end according to VP2 numbering) of BPV VP2 in the averaged sigma-weighted 2Fo-Fc 189
electron density map (with Fo and Fc as defined in Table 1). The VP2 model was built 190
into this density by substitution of the alanine residues, in the initial B19 model, with the 191
specific BPV residues using the COOT program (36). This was followed by alternating 192
cycles of refinement and model building guided by the calculated averaged density map 193
until no further improvement was observed in the model as monitored by the Rfactor (as 194
defined in Table 1). Density below a threshold of 0.5σ extending from residue 39 below 195
the icosahedral 5-fold axis was not modeled because the backbone direction could not 196
be unambiguously determined. The quality of the BPV VP2 model was assessed in the 197
COOT and MOLPROBITY programs (36, 37). The CNS program was used to calculate 198
the root mean square deviations (R.M.S.D.) from ideal bond lengths and angles while 199
the average Bfactor values for the VP2 model was determined by the MOLEMAN 200
program (35, 38). Data collection, processing and refinement statistics are summarized 201
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in Table 1. Figures were generated in the UCSF-Chimera and PyMOL programs (39, 202
40). 203
Cryo-electron microscopy and image reconstruction. Three microliter aliquots of 204
purified BPV VLPs (~0.2 mg/mL) were applied to C-flat holey carbon grids (Protochips, 205
Inc.) and vitrified using a Vitrobot™ Mark IV (FEI Co.). The sample was examined 206
using a 16-megapixel CCD camera (Gatan, Inc.) in a Tecnai (FEI Co.) G2 F20-TWIN 207
Transmission Electron Microscope operated at a voltage of 200 kV using low dose 208
conditions (~20 e/Å2). Images were recorded with a defocus range of ~1.2 to 4 µm at a 209
magnification of ~69,000X resulting in a pixel size of 2.24 Å. The RobEM subroutine 210
within the AUTO3DEM software suite (41) was used to extract 1414 particles from 63 211
cryo-micrographs. Estimations for the defocus values of the micrographs were made 212
using the ctffind3 subroutine in AUTO3DEM, and corrections for the microscope related 213
contrast transfer functions were applied during the search and refine modes in 214
AUTO3DEM (42). An initial low resolution (30 Å) cryo-reconstructed model map was 215
generated using an ab initio random model method and imposing icosahedral symmetry 216
with 150 particle images (43). This map was used for the initial determination of the 217
orientations and origins followed by refinement of these parameters for the entire set of 218
particle images using AUTO3DEM (41). The final 3D map was reconstructed from 1131 219
particle images to an estimated resolution of ~8.8 Å based on a Fourier shell correlation 220
(FSC) threshold criterion of 0.5. An inverse temperature factor of 1/100 Å2 was used to 221
improve the high-resolution features at 8.8 Å resolution. To avoid amplification of noise 222
in addition to signal, the structure factors were multiplied by a noise suppression factor 223
post refinement in AUTO3DEM (44). 224
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Difference map calculations. A 60mer (generated in VIPERdb2 (33)) from the BPV 226
VP2 crystal structure was docked into the cryo-reconstructed density map using the 227
UCSF-Chimera program with a standard cross-correlation as a criterion to measure the 228
fit (39). The docked crystal structure coordinates were back-filtered and used to 229
calculate a map to the resolution of the reconstructed map using the ‘pdb2mrc’ 230
subroutine in the EMAN2 program (45) for a difference map calculation. The two maps 231
(model and cryo-reconstructed) were normalized in the MAPMAN program prior to use 232
(46). A difference map was calculated subtracting the crystal structure map from the 233
cryo-reconstructed map using the “vop (volume of operation)” subroutine in the UCSF-234
Chimera program (39). Positive density observed below the 5-fold axis was modeled as 235
ten additional N-terminal residues 29-38 (VP2 numbering). 236
237
Revision of the pseudo-atomic model built into the 7.9 cryo-reconstructed HBoV1 238
structure. To improve the original pseudo-atomic model built for HBoV1, the BPV 239
crystal structure coordinates were used as the template to build a new VP2 homology 240
model in the UCSF-CHIMERA program (39). This exercise was carried out because the 241
original HBoV1 pseudo-atomic model utilized B19, with ~23% sequence identity to 242
HBoV1, while BPV shares ~46% identity. In lieu of an HBoV1 crystal structure, the 243
revised model served as a better representative of the structure. Docking of a 60mer of 244
the revised HBoV1 homology model into the previously reported HBoV1 cryo-245
reconstructed map was carried out using the ‘fit in map’ subroutine in the UCSF-246
CHIMERA program (39). Further interactive model adjustments to improve the model 247
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fitting guided by the density map envelope, followed by real-space refinement, were 248
carried out using the COOT program (36). Readjustments were mainly made in the 249
following surface variable regions (VRs) (defined in Table 3): VR-II (DE loop), VR-III, 250
VR-IV, VR-V, and VR-VIIIB (the HI loop). 251
252
Sequence and structural comparison to other Parvovirinae members with 253
available 3D structures. BPV, AMDV, AAV2, MVMp, and B19 VP2 sequences 254
(Accession IDs: ABC69731.1, AAA96023.1, YP_680427.1, CAB46508.1, and 255
AAV35058.1, respectively) were aligned in the CLUSTALW-2 online server (47). The 256
C atoms of the VP2 atomic coordinates for MVM, B19, AAV2 (PDB IDs: 1Z14, 1S58, 257
and 1LP3, respectively), and pseudo-atomic coordinates modeled into the cryo-258
reconstructed density maps of AMDV (48) and HBoV1 (EMDB accession number 1739) 259
were superposed onto the BPV atomic coordinates using the secondary structure 260
matching (SSM) tool found in the PDBefold online server (49). The server generates 261
R.M.S.D for the superposed structures and the distances between the aligned C 262
positions were used to identify VRs. The definition of VRs for the autonomously 263
replicating viruses, regions with two or more consecutive residues with a R.M.S.D >2.0 264
Å between the superposed structures (50), was used. Cartoon and surface 265
representations were generated in the PyMOL and UCSF-Chimera programs, 266
respectively (39, 40). 267
268
PDB and EMDB accession numbers. The coordinates and structure factors for the 269
crystal structure of BPV VP2 have been deposited to the PDB database 270
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(http://www.rcsb.org) with the accession number 4QC8. The cryo-reconstructed map 271
was deposited into the EMDB database with the accession number EMD-6168. 272
. 273
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Results and Discussion 274
The crystal structure of the BPV VP2. Purified and homogenous BPV VLPs, which 275
formed two-dimensional crystalline arrays on EM-micrographs, produced flat crystals in 276
10 mM Tris-HCl (pH 7.5), 4% PEG 8000, 8 mM MgCl2, and +/- 25-50 mM Li2SO4 (Fig. 277
1A and B). The crystals diffracted X-rays to 3.2 Å resolution (Table 1). Residues 39-278
536 of the VP2 were ordered in the structure determined using molecular replacement 279
and assigned to the averaged density map (Fig. 2A and B). Atomic detail comparison of 280
the BPV map and refined model with the coordinates of the B19 phasing model showed 281
no phase bias at both the residue-level and on a secondary structure-level wherein the 282
density was consistent with the BPV sequence (Fig. 2A and B). The first 38 residues at 283
the N-terminus are not present in the refined model due to the lack of interpretable 284
density. This lack of density is consistent with observations for most other parvoviruses, 285
except B19. This could be a result of multiple conformations of the VP2 N terminus or 286
inherent disorder in this region which is incompatible with the 30-fold NCS applied 287
during model refinement and density averaging (51, 52). The refined parameters for the 288
crystal structure are provided in Table 1 and are within the range reported for other virus 289
structures (including parvoviruses) determined to similar resolution. The similarity of 290
Rfactor and Rfree for the virus structures stems from the non-crystallographic icosahedral 291
symmetry of the capsid that does not permit random selection of unique reflections 292
required for the Rfree calculation. 293
The BPV VP2 (39-536) structure displays the conserved features of the 294
parvovirus VP topology. It maintains the core eight-stranded anti-parallel jellyroll motif, 295
organized in two -sheets, BIDG and CHEF, seen commonly in most virus structures 296
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(Fig. 2C and 3). This -barrel motif constitutes 17% of the VP2 sequence and forms the 297
contiguous capsid shell. Additional anti-parallel -strand stretches are present in the 298
loops between the core -strands as reported for some of the other parvoviruses (53) 299
(Fig. 2C and 3). A small helix (A) spanning residues 109-121 (VP2 numbering), also 300
conserved in all other Parvovirinae structures solved thus far, was observed in BPV. 301
The BPV VP2 uniquely contains an additional 2-turn -helix at residues 211-218 not 302
reported in other parvovirus structures (Fig. 2C and 3). Loops of varying lengths and 303
conformations, named for their flanking β-strands, for example, the DE loop for the loop 304
between βD and βE, were inserted between the -strands. These loop regions often 305
clustered at the capsid surface as a consequence of the icosahedral symmetry, 306
contribute to the BPV capsid surface topology (Fig. 2D). 307
The characteristic dimple-like depression centered directly at the 2-fold axis of 308
symmetry, common in other parvoviruses (53), is divided by a raised flat surface in BPV 309
(Fig. 2D). Residues between 502-506 give rise to this raised flat surface appearance 310
while residues 109-121 (of the conserved A helix) and 499-519 (of the loop between I 311
and C terminus) form the floor/wall of the 2-fold depression. A depression is also 312
centered at the 3-fold axis surrounded by three distinct protrusions composed of three 313
large loops within the GH loop (comprising of 199 residues), contributed from two 3-fold 314
symmetry-related monomers, residues 246 to 297 and 385 to 405 from one VP2 and 315
residues 304 to 320 from the other (Fig. 2D). A second outer ring of slightly smaller 316
protrusions, formed by residues 81 to 90, 200 to 220, and 321 to 358 of the same VP2 317
monomer, is located on the 2/5-fold wall surrounding the 3-fold protrusions (Fig. 2D). 318
The DE loop, comprised of 22 residues, cluster to form a cylindrical channel at the 5-319
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fold axis (Fig. 2C and D). This feature is maintained in all other known Parvovirinae 320
structures (53). Surrounding this 5-fold channel is a shallow depression lined by HI 321
loops (residues 455-469) from 5-fold neighboring symmetry-related monomers (Fig. 2C-322
D). Overall, the BPV capsid diameter is ~220 Å at the 2-fold axis, ~275 Å at the peak of 323
the 3-fold protrusions, and ~240 Å at the 5-fold (Fig. 2D). 324
325
Density extending the 5-fold channel is interpreted as the N terminus of VP2. As 326
already mentioned, N-terminal residues 1-38 of the BPV VP2 structure determined by 327
X-ray crystallography were disordered (Fig. 2C). This observation is similar to all 328
structures determined to date for other parvoviruses by X-ray crystallography. This has 329
been postulated to be due to either low copy number, flexibility in conformation or 330
intrinsic disorder in this VP region. These properties are incompatible with the NCS 331
applied during structure determination and refinement, and the finer spatial frequency of 332
data sampling as resolution is increased in crystallographic structure determination. 333
Cryo-reconstruction, at low or median resolution, is thus often a more applicable method 334
for overcoming this limitation. Consistently, this method was used to visualize the N-335
terminus of VP2 in B19 (52). Thus, VLPs extracted from cryo-micrographs were used to 336
generate an 8.8 Å resolution cryo-reconstructed map for BPV (Fig. 1C and 4A). The 337
map is similar in size and surface features to the crystal structure (Fig. 2D and 4A). The 338
conserved secondary structural elements of the BPV VP2 crystal structure, A, BIDG-339
CHEF β-barrel, and A helix (Fig. 4B), were readily fitted into the cryo-reconstructed 340
density map. In addition, although the apexes of most of the surface loops displayed 341
high flexibility and correspondingly large thermal-temperature factors in the VP2 crystal 342
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structure, some of these loops, for example the DE (Fig. 4D) and HI loops (data not 343
shown), were precisely located in the cryo-reconstructed map. The correlation 344
coefficient for the fit between the cryo-reconstructed map and a model map generated 345
from a 60mer of BPV VP2 crystal (residues 39-536) was 0.91. 346
Densities, not satisfied by the fitted VP2 crystal structure (residues 39-536), were 347
ordered inside the capsid of the cryo-reconstructed map, at the base of each 5-fold 348
vertex radially extending the channel inwards (Fig. 4C). A difference map (crystal 349
structure subtracted from the cryo-reconstructed map) showed that the largest positive 350
difference was consistent with these densities, which were immediately adjacent to the 351
first residue, 39, in the VP2 crystal structure model (Fig. 4D). For each VP2, this 352
difference density was modeled as a stretch of ten additional N-terminal residues, 29-353
SVGGGGRGGS-38 (Fig. 4D). The model extends the channel to a total length of ~60 Å, 354
with a capsid exterior width of ~14 Å and a capsid interior width of ~10 Å and ~18 Å at 355
the neck and innermost edge of the channel, respectively (Fig. 4D). Any additional 356
density radially extending inwards from the innermost end of this channel beyond 357
residue 29 was not modeled due to ambiguity and low signal to noise ratio. The 358
correlation coefficient for the new 60mer capsid model (with each monomer comprising 359
of residues 29-536) fitted into the cryo-reconstructed map increased to 0.93 from 0.91. 360
This is consistent with satisfaction of the previously unoccupied interior 5-fold density. 361
Similar VP1/2 N terminus localization under the 5-fold axis have been reported for other 362
autonomous parvovirus members, for example, MVMp and CPV (54, 55). 363
364
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Bocaparvovirus capsids conserve the extended 5-fold channel but show 365
variability in capsid surface features. The cryo-reconstructed BPV capsid structure 366
was compared to a median-resolution, 7.9 Å, structure of the HBoV1 VP2 capsid, also 367
determined using cryo-reconstruction (17) (Fig. 5A and B). Similar to the BPV cryo-368
reconstructed map ordered density was observed below the 5-fold axes in the HBoV1 369
cryo-reconstructed map (Fig. 5B) and 9 residues were built into this density (17). 370
However, the interior end of the channel appears to be morphologically different in 371
HBoV1 compared to BPV (Fig. 5B). Due to conservation of this density feature in both 372
the Bocaparvovirus capsids, the current hypothesis is that the N-termini of their VP2 are 373
bundled under the 5-fold axis and extend the 5-fold channel into the capsid interior. 374
Given the overlap between VP1 and VP2, the anticipation is that the VP1 N-termini are 375
similarly localized. The VP1u is postulated to become externalized through this channel 376
for other parvoviruses (56–60). How the VP1u is extruded through the channel for its 377
PLA2 function during infection requires further studies for members of this genus. 378
The BPV and HBoV1 capsids have dissimilar surface topologies. The VP2 for 379
these viruses share a sequence identity of 46%. The revised pseudo-atomic model 380
(described in Methods) of HBoV1 VP2 docked into the HBoV1 cryo-reconstructed map 381
with a correlation coefficient of 0.92. Comparison of HBoV1 and BPV VP2 shows 382
conservation of the BIDG-CHEF core and -helix regions with variability localized to 383
residues in the loop regions characterized by insertions and deletions within a number 384
of the VRs defined for the Parvovirinae (Fig. 3 and 5C, Table 2). BPV has four regions 385
with deletions of two or more residues compared to HBoV1: VR-III (between E and 386
F), VR-VIII (between G and H), VR-VIIIB (the HI loop), and VR-IX (betweenI and 387
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C-terminus). A combination of a 7 amino acid deletion in VR-III located on the 2/5-fold 388
wall and a 6 amino acid deletion in VR-IX spanning the 2-fold results in a shallow and 389
wider 2-fold depression in BPV compared to HBoV1 in which the depression is more 390
delineated (Fig. 3, 5A, and 5C). In contrast, BPV has an insertion of 3 amino acids 391
(residues 218 to 220) downstream in VR-III, which gives rise to a small raised 392
morphology seen near the 2/5-fold wall (Fig. 3, 5A, and 5C). 393
BPV also has an insertion of 3 amino acids in VR-VIII, which along with the 394
alternate conformations of VR-IV and VR-V around the 3-fold axis give BPV larger 395
protrusions compared to HBoV1 (Fig. 3, 5A, and 5C). This is consistent with the smaller 396
overall diameter seen at the 3-fold axis for HBoV1 (~260 Å). A conformational difference 397
at the apex of the DE loop (VR-II) gives the exterior surface of the 5-fold channel a 398
wider appearance in HBoV1 (Fig. 5A and 5C). Additionally, a conformational difference 399
in the HI loops (VR-VIIIB) lining the floor of the depression around the 5-fold channels in 400
BPV and HBoV1 creates a difference in their surface topology (Fig. 5A). How these VRs 401
correspond to tissue- and/or host-specific functions need to be further studied. 402
403
Commonalities in subfamily and genus-level VRs localize to functional regions. 404
The Parvovirinae members show high sequence divergence with percentage identities 405
in the range of 22-26% in VP2 (Table 3). Shared sequence identities are localized to the 406
residues within the conserved -barrel core and A helix (data not shown). Consistently, 407
superposition of the BPV VP2 crystal structure onto the VP2/3 (depending on virus) 408
structures available for Parvovirinae type members, AAV2, AMDV, B19, and MVMp, 409
showed conservation of secondary structure elements (Fig. 6A). The overall R.M.S.D.s 410
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for the BPV VP2 Cpositions superposed onto the C positions for the other structures 411
were in the range of 1.6-3.2 Å (Table 3). 412
To date, parvovirus VP VRs have only been defined for the members within the 413
same genus, namely Protoparvovirus (VR0-8) and Dependoparvovirus (VRI-IX), which 414
have sequence identities in the range of 50-60 and 60-99%, respectively (50, 61, 62). 415
The BPV structure increased the number of type member structures available for the 416
Parvovirinae to five, enabling a subfamily level definition of VRs. The type member 417
structural comparison identified twelve VRs, nine (VR-I to VR-IX) of which coincide with 418
those defined at the genus level for the dependoparvoviruses, plus three additional VRs 419
(VR-IIA, VR-VIIIA, and VR-VIIIB), named for their preceding VRs and for consistency 420
with the Dependoparvovirus nomenclature (Fig. 6B and C, Table 2). Of these, VR-VIIIA 421
and VR-VIIIB are equivalent to VR6 and VR7, respectively, defined for the 422
protoparvoviruses (50), while VR-VIIIB, or the difference in the HI loop, was recently 423
observed for the Dependoparvovirus member AAV5, following its structure 424
determination (63). VR-IIA is the only novel VR identified but is located in the capsid 425
interior under the 2-fold. Similarly, VR-VIIIA is located inside the capsid between the 2- 426
and 3-fold. The functional relevance of these buried VRs needs to be further 427
investigated. Thus, the Parvovirinae subfamily level VR definition combines the 428
information previously reported with the addition of VR-IIA. These VRs are localized to 429
loops, which display high structural variability (>2 Å R.M.S.D.) and mostly do not 430
superpose, consistent with large insertions and/or deletions, which cluster at or around 431
the symmetry axes (Table 2, Fig. 6B and C). 432
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The 2-fold region is characterized by VR-III and VR-IX (Table 2, Fig. 6C). VR-III 433
has a large deletion in AAV2 compared to the other type members, reducing the height 434
of its 2-fold wall (Fig. 6C and 7). VR-IX is shorter in BPV, AMDV, and MVMp and a 435
conformational difference leads to a raised surface in the middle of the 2-fold axis in 436
BPV (Fig. 7). This 2-fold region has receptor attachment properties in MVMp (reviewed 437
in (64)). VR-I, VR-III, a portion of VR-V, and VR-VII make up the 2/5-fold wall (Fig. 6C 438
and D). VR-I is shorter in BPV and AAV2 (<11 residues) compared to other type 439
members (>20 residues) resulting in a more pronounced 2/5-fold wall in MVMp, B19, 440
and AMDV (Fig. 7). VR-I, VR-III, and a portion of VR-V form the protrusions on the 2/5-441
fold wall (which surround the 3-fold protrusions) in B19 and BPV (Fig. 6B, C, and 7). 442
VR-VII is shorter in BPV, MVMp, and AMDV compared to AAV2 and B19 (Table 2 and 443
Fig. 6A and B). Residues in the 2/5-fold wall are reported to play a role in AMDV tissue 444
tropism and pathogenicity (48, 65) as well as antibody reactivity for AAV2 (66). 445
Variable regions, VR-IV, VR-V, and VR-VIII cluster to form the 3-fold protrusions 446
(Table 2, Fig. 6B and C). A large insertion in AMDV VR-VIII combined with another in 447
VR-III creates protrusions, which are significantly more pronounced than the other 448
viruses (Fig. 6 and 7). Differences in the size and topologies of VR-IV, VR-V, and VR-449
VIII give rise to two main types of 3-fold morphologies: a mound-like protrusion formed 450
by the clustering of the VRs from three 3-fold related monomers as seen MVMp or three 451
distinct protrusions as seen in AMDV, BPV, B19, and AAV2, with each protrusion 452
created by two 3-fold related monomers (Fig. 7). The BPV capsid, unlike HBoV1 (Fig. 453
5), does not have features similar to B19 in this region, but rather more closely 454
resembles AAV2 (Fig. 5A and 7). The 3-fold region contains residues important for 455
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receptor attachment (in AAV2 and B19) and antibody recognition functions (in AAV2) 456
(53, 67–72). 457
Three VRs characterize the 5-fold: VR-II located at the apex of the channel, and 458
VR-IIA and VR-VIIIB (HI loop), which are part of the canyon floor (Table 2, Fig. 6B, and 459
C). VR-II displays conformational variability in all members and is shorter in BPV, AAV2, 460
and B19 as opposed to MVMp and AMDV (Table 2, Fig. 6A, and 7). B19 has a distinct 461
conformation at the apex of its DE loop resulting in a closed exterior end of the 5-fold 462
channel compared to the other type members (52) (Fig. 6A and 7). The residues in the 463
5-fold channel have been reported to play a role in VP1/VP2 externalization and 464
endosomal escape for AAV2 and MVMp (57, 73). The conformational differences in VR-465
VIIIB give the canyon floor distinctive morphologies in each type member (Fig. 7). The 466
HI loop is reported to be important for genome packaging in AAV2 (74). 467
468
Summary 469
This work represents the first high-resolution structure of BPV, the type member 470
for the relatively new yet continually emerging Bocaparvovirus genus of the 471
Parvoviridae. The structure conserves the core parvovirus VP2 topology consistent with 472
functional importance such as in capsid assembly. Variability in surface loops result in a 473
unique capsid morphology, including the lack of a centered 2-fold depression in BPV. 474
This suggests a host specific role yet to be determined. In contrast, the extended 475
channel at the 5-fold axes was found to be conserved in the two bocaparvovirus 476
members compared suggesting a genus-specific role, which requires further 477
characterization. Significantly, commonalities in location of VRs for the Parvovirinae 478
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type member with those defined at the genus level, which overlap with functional 479
regions, suggest analogous family level functions. These functions are yet to be 480
defined for the bocaparvoviruses. The BPV structure provides a framework for studies 481
aimed at fulfilling this gap as well as understanding parvovirus capsid evolution. It also 482
serves as a design platform for strategies to control pathogenic BPV infection, in the 483
form of structure-guided design of peptide vaccines and/or small molecule inhibitors. 484
485
Acknowledgment: We thank the UF Interdisciplinary Center for Biotechnology 486
Research Electron Microscopy core for cryo-electron microscopy access (funded by the 487
UF College of Medicine and Division of Sponsored Programs). This project was funded 488
by UF College of Medicine and McKnight Brain Institute funds (to MAM). 489
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Table 1. Crystallography data collection, processing and refinement statistics 490
Parameter VLP
Wavelength (Å) 0.9
Crystal System, Space Group Orthorhombic, C2221
Unit cell parameters (Å) a=323.4, b=381.2, c=376.6
Resolutiona (Å) 50-3.2 (3.31-3.2)
Total number of reflections 4271548
No. of unique reflectionsa 340823 (34994)
Completenessa (%) 90.1 (93.2)
Rsym
a,b(%) 12.4 (36.5)
Redundancya 2.4 (2.3)
Average I/(I) 7.6 (1.8)
No. of atoms (protein) 3948
Average Bfactor
(Å2) (protein) 46.7
Rfactor
c/R
free
d(%) 30.97/31.15
RMSD bonds (Å), angles (°) 0.008, 1.72 a Values in parenthesis are for the highest resolution shell. 491
b R
sym = (Σ|I − <I>|/Σ<I>) × 100, where I is the intensity of an individual reflection with 492
indices h, k, and l, and <I> is the average intensity of all symmetry equivalent 493 measurements of that reflection; the summation is over all intensities. 494 c R
factor = (Σ|Fo| − |Fc|/Σ|Fo|) × 100, where Fo and Fc are the observed and calculated 495
structure factor amplitudes, respectively. 496 d R
free is calculated similarly to Rfactor, except it uses 5% of the reflection data partitioned 497
from the refinement process. 498
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Table 2. Variable Regions across the Parvovirinae Subfamily 499
Residues in (*) VP1 and (#) VP2 numbering. 500 501
VRs BPV-1
# MVMp
# AAV2* B19# AmDV
# Capsid Location
Functional Role
I 80-90
85-104 (VR0)
258-276 (VRI)
57-81 82-107 2/5-fold wall AAV2 Antibody
Recognition & Genome
Packaging II 143-
147 157-164 (VR1)
319-333 (VRII)
133-135
160-167 Top of 5-fold channel
MVM VP2 Externalization
IIA 174-179
191-196 355-357 164-171
194-197 Capsid interior MVM Endosomal
Escape/ VP1u Externalization
/AAV2 Genome
packaging III 200-
219 217-238 (VR2)
378-394 (VRIII)
189-208
220-254 2/5-fold wall
IV 256-294
282-289 (VR3. VR4a)
427-439 (VRIV)
246-283
290-351 3f shoulder
V 298-344
336-356 (VR5)
482-511 (VRV)
296-297
357-383 3-fold protrusion/ 2/5-fold wall
AAV2 Genome
Packaging
VI 346-357
358-373 523-541 (VRVI)
299-352
384-401 3-fold protrusion AAV2 Antibody
Recognition & Genome
Packaging VII 367-
370 391-394 544-560
(VRVII) 356-368
408-426 2/5-fold wall AAV2 Antibody
Recognition VIII 385-
405 407-450 (VR4b)
571-604 (VRVIII)
374-387
438-510 3f protrusion AMDV tissue tropism/patho
genicity B19 Globoside
binding VIIIA 433-
441 479-486 (VR6)
628-633 437-445
539-546 Capsid interior VIIIB 458-
465 504-516 (VR7)
655-667 466-475
564-575 Floor of 5-fold canyon
IX 502-506
558-562 (VR8)
701-720 (VRIX)
509-518
617-631 2-fold MVM Receptor
Attachment, AAV2
Transduction Efficiency
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Table 3. Sequence and structure comparison for the Parvovirinae subfamily. 502
Virus Total number of
VP2 residues
Sequence
identitya
Sequence
identityb
R.M.S.D.
() to
BPV1c
B19 523 23.0 23.1 1.9
AAV2 598 26.0 29.0 1.6
MVMp 587 22.0 18.5 3.2
AMDV 638 22.3 18.0 2.2
a Sequence identity using full-length VP2 sequences to BPV shown as a percentage. 503
b Sequence identity using only residues ordered in the crystal structures (AAV2, B19 504
and MVMp) or pseudo-atomic model (ADMV) compared to the BPV VP2 crystal 505 structure shown as a percentage. 506 c The crystal structures were superposed based on secondary structures and root mean 507
square deviation (RMSD) values were calculated for the main chain -carbons between 508 the known crystal structures or pseudo-atomic models and the BPV crystal structure. 509 510
511
512
513
514
515
516
517
518
519
520
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Figure Legends 521 Figure 1. Purification, crystallization, and freezing of BPV VLPs. (A) Micrograph of 522
negatively stained BPV VLPs imaged at a magnification of 97,000X. (B) Flat crystals of 523
BPV (indicated by white arrows) obtained in conditions of 10mM Tris pH 7.5, 4 % PEG 524
8000, 8 mM MgCl2 and 25 mM LiSO4. (C) Cryo-micrograph of unstained BPV VLPs. 525
526
Figure 2. The crystal structure of BPV. (A-B) Sections of the 2Fo-Fc electron density 527
map, contoured at a threshold of 1.5 (grey mesh) highlighting that the unbiased BPV 528
structure on a (A) residue and (B) polypeptide chain level. The stick and cartoon 529
representations show the superposed coordinates of BPV (magenta) and B19 (orange). 530
(C) Cartoon representation of the BPV VP2 crystal structure (magenta) with the 531
secondary structure elements highlighted in the following colors: -strands (red), loops 532
(magenta), and helices (cyan). The conserved core consisting of the -strands (A-533
BIDG-CHEF) and -helix (A) are labeled accordingly. The positions of the icosahedral 534
2-, 3-, and 5-fold symmetry axes are indicated as a filled oval, triangle and a pentagon, 535
respectively. The N- and C-termini of the polypeptide chain are also labeled. (D) 536
Radially depth-cued surface representation of BPV capsid shown approximately along 537
the 2-fold axis of symmetry. A color key corresponding to the depth-cue radii (in units of 538
Å) is shown. Panels A-C were generated in PyMOL and panel D was generated in 539
UCSF- CHIMERA (39, 40). 540
541
Figure 3. Structural alignment of BPV and HBoV1. Secondary structures elements, β-542
strands and α-helices are indicated with arrows (blue) and cylinders (red), respectively. 543
The conserved core elements (A-BIDG-CHEF and A) are labeled. Insertions, 544
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deletions, or VRs (as defined in Methods) are colored in red in the HBoV1 sequence 545
and labeled. Symbols below the alignment indicate standard ClustalW2 nomenclature: 546
(*) identity, (:) high conservation, and (.) conservation. 547
548
Figure 4. Cryo-EM structure of BPV. (A) Surface representation of the cryo-549
reconstructed density map, viewed along the 2-fold axis, radially depth-cued as per the 550
color key (in units of Å). An equilateral triangle, depicting the viral asymmetric unit, with 551
the 3-fold axes at the base vertices separated by the 2-fold axis and the 5-fold axis at its 552
vertex (labeled 3f, 2f, and 5f, respectively), is shown. (B) A zoom-in view of the 553
conserved α-helix (αA) from 2-fold related monomers showing the fit into the 554
reconstructed density (grey mesh). (C) A cross-sectional view of the 60mer of the BPV 555
crystal structure docked into the cryo-reconstructed density map (shown as a grey mesh 556
and contoured at 1). The dashed lines indicate the icosahedral symmetry axes. (D) 557
Side-view of a cross-section of the map and fitted crystal structure (shown in rainbow 558
colors) highlighting the extended 5-fold channel. The first N terminal residues observed 559
in the crystal structure and in the pseudo-atomic BPV model built into the cryo-560
reconstructed map are indicated. Approximate dimensions for the width and height of 561
the channel are also shown. This figure was generated using UCSF-CHIMERA (39). 562
563
Figure 5. Comparison of Bocaparvovirus cryo-EM structures. (A) Surface and (B) cross-564
sectional views of BPV and previously reported HBoV1 (17) radially depth-cued by the 565
color key (in units of Å). (C) Superposition of the C of the crystal structure of BPV 566
(magenta) and the revised pseudo-atomic model of HBoV1 (red) depicted as smooth 567
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loops. The positions of the icosahedral 2-, 3-, and 5-fold symmetry axes are indicated 568
as a filled oval, triangle, and pentagon, respectively. VRs are labeled on the VP2 569
monomer (left) and colored on the surface of the BPV capsid (on the right; in grey) in 570
different colors: VR-II (blue), VR-III (yellow), VR-IV (red), VR-VIII (green), VR-VIIIB 571
(wheat), and VR-IX (chocolate). Panels A-B were generated using UCSF- CHIMERA 572
and panel C was generated in PyMOL (39, 40). 573
574
Figure 6. Superposition of Parvovirinae VP2 structures and identification of VRs. (A) 575
Cartoon representation of superposed atomic coordinates of B19 (orange), AAV2 (royal 576
blue), and MVMp (green) as well as pseudo-atomic coordinates of AMDV (cyan) with 577
the BPV crystal structure (magenta). The positions of the icosahedral 2-fold, 3-fold, and 578
5-fold axis of symmetry are labeled as in Fig 2C. (B) A cartoon diagram (grey) of BPV 579
with the VRs depicted in the following colors: VR-I (purple), VR-II (blue), VR-IIA (forest 580
green), VR-III (yellow), VR-IV (red), VR-V (grey), VR-VI (pink), VR-VII (cyan), VR-VIII 581
(green), VR-VIIIA (orange), VR-VIIIB (wheat), and VR-IX (chocolate). (C) Surface 582
representation of BPV capsid (grey) with VRs colored as in Fig. 6B. This figure was 583
generated in PyMOL (40). 584
585
Figure 7. Comparison of Parvovirinae capsids. Radially depth-cued surface 586
representations (in units of Å) of the parvovirus capsids viewed along the icosahedral 2-587
fold axis of symmetry. These images were generated using atomic coordinates of BPV, 588
AAV2, MVMp, and B19 or pseudo-atomic coordinates built into cryo-reconstructed 589
density of AMDV. This figure was generated in UCSF- CHIMERA (39). 590
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50 nm 50 nm
A B C
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EXTERIOR
A B
C
B19 (387-402)
BPV (387-400)
B19 (Y179)
BPV (W189)
Kailasan et al. Fig. 2
C-Term N-Term
(38)
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Kailasan et al. Fig. 4
5-f 3-f
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14
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Kailasan et al. Fig. 5
BPV HBoV-1
B
A
C-Term N-Term
DE loop
HI loop
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VR-IX
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VR-III
INTERIOR
EXTERIOR
C
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VR-I
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VR-II
VR-VIIIB
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Kailasan et al. Fig. 6
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MVMp AMDV
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B19 BPV
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