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Deng et al. Two newly identified ODV associated proteins, HA44 and HA100
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Revised to Journal of Virology May 11, 2007 1
Proteomics Analysis of HearNPV Identified Two New ODV associated 2
Proteins, HA44 and HA100 3
4
Fei Deng1,#
, Ranran Wang1,#
, Minggang Fang1,+
, Yue Jiang1, Xushi Xu
1, Hanzhong 5
Wang1, Xinwen Chen
1, Basil M. Arif
2, Lin Guo
3, Hualin Wang
1, Zhihong Hu
1,* 6
7
1State Key Laboratory of Virology and Joint-lab of Invertebrate Virology, Wuhan 8
Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, P.R. China, 2
9
Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste. Marie, Ont. 10
Canada, and 3State Key Laboratory of Virology, Wuhan University and College of Life 11
Sciences, Wuhan 430072, P.R. China 12
13
14
15
#These authors have contributed equally to the work. 16
+The current address for Minggang Fang is Department of Agroecology, University of 17
British Columbia, Vancouver, BC, Canada V6T 1Z4. 18
* Corresponding author 19
Dr. Zhihong Hu 20
Wuhan Institute of Virology 21
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00632-07 JVI Accepts, published online ahead of print on 20 June 2007
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Chinese Academy of Sciences 22
Wuhan 430071 23
P.R. China 24
Tel/Fax: 86-27-87197180 25
Email: huzh@wh.iov.cn 26
27
Running title: Two newly identified ODV associated proteins, HA44 and HA100 28
Words count for the abstracts: 187 29
Words count for the text: 3447 30
31
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Abstract 32
SDS-PAGE and mass spectrometry were used to analyze the structural proteins of the 33
occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid 34
nucleopolyhedrovirus (HearNPV), a Group II NPV. Twenty-three structural proteins of 35
HearNPV ODV were identified. Twenty-one of which have been reported previously as 36
structural proteins or ODV associated proteins in other baculoviruses. These include 37
Polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), 38
DNA-Polymerase, GP41, VP39, P33, ODV-E25, Helicase, P6.9, ODV/BV-C42, VP80, 39
ODV-EC43, ODV-E66 and PIF-1. Two proteins, encoded by HearNPV ORF44 (ha44) 40
and ORF100 (ha100), were discovered as ODV associated proteins for the first time. 41
Ha44 encodes a protein of 378 amino acids with a predicted mass of 42.8 kDa. Ha100 42
encodes a protein of 510 amino acids with predicted mass of 58.1 kDa, and is a 43
homologue of the poly (ADP-ribose) glycohydrolase (parg). Western blot analysis and 44
immunoelectron microscopy confirmed that HA44 is associated with nucleocapsid and 45
HA100 is associated with both nucleocapsid and envelope of HearNPV ODV. HA44 is 46
conserved in Group II NPVs and GVs but does not exist in Group I NPVs, while HA100 47
is conserved only in Group II NPVs. 48
49
50
51
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INTRODUCTION 52
The Baculoviridae, a diverse family of more than 600 viruses, encompass two 53
genera, the Nucleopolyhedroviruses (NPVs) and the Granuloviruses (GVs) (5). 54
Baculoviruses are generally host-specific infecting mainly insects of the orders 55
Lepidoptera, Hymenoptera and Diptera. Two progeny phenotypes are produced in the 56
replication cycle, the budded virus (BV) and the occlusion-derived virus (ODV). In 57
larvae, ODVs initiate primary infections in midgut epithelial cells of susceptible hosts 58
and BVs spread the virus from cell to cell in the larvae (5,30,62). The two phenotypes are 59
genotypically identical but each has characteristic structural components to accommodate 60
their respective functions (7,50). Based on phylogeny, lepidopteran NPVs are divided 61
into Group I and Group II (23,24,70). It is known now that the BVs of Group I and Group 62
II NPVs use different fusion proteins to enter host cells. GP64 is the membrane fusion 63
protein of Group I NPVs (4,40), while the F-protein is that of Group II NPVs (27,36,44). 64
Identification of ODV structural proteins and comparisons in different NPVs are 65
fundamental to functional investigation of virulence and host specificity. So far 30 66
genome sequences of baculoviruses have been reported, including 8 Group I NPVs, 12 67
Group II NPVs, 7 GVs, 1 dipteran NPV and 2 hymenopteran NPVs. The availability of 68
the genome sequences has facilitated proteomic analysis of baculoviruses. In 2003, 69
proteomic investigations revealed 44 proteins to be ODV components of Autographa 70
californica MNPV (AcMNPV), a Group I NPV (11). Recent investigations on a dipteran 71
NPV, Culex nigripalpus Nucleopolyhedrovirus (CuniNPV), identified 44 ODV associated 72
proteins (46). By comparison, little information is known on the structural proteins of 73
ODVs from Group II NPVs. 74
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The Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV, 75
also called HaSNPV), was first isolated in 1975 in the province Hubei of the People’s 76
Republic of China and has been used extensively over 25 years in China to control H. 77
armigera in cotton (71). Phylogenetic analysis indicated that HearNPV belongs to Group 78
II NPVs (12,29). Its DNA genome is 131 kb containing 135 open reading frames (ORFs) 79
that potentially encode proteins of 50 amino acids (aa) or larger (13). Several HearNPV 80
genes, such as polyhedrin (polh) (14), ecdysteroid UDP-glucosyltransferase (egt) (15), 81
late expression factor 2 gene (lef-2) (12), basic DNA-binding protein gene (p6.9) (61), 82
ha122 (37), Ha94 (20), chitinase (60), fp25K (67), p10 (18) and F-protein (ha133) (36) 83
have been characterized. 84
In this report, we describe using SDS-PAGE and mass spectrometry-based protein 85
analysis techniques to study structural proteins of the ODV of HearNPV. HearNPV was 86
chosen to serve as representative of Group II NPVs. ODV proteins were separated by 87
SDS-PAGE and analyzed by peptide mass fingerprinting (PMF) techniques using matrix-88
assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).
89
The resulting mass spectra were searched against the NCBI database and the theoretical 90
ORF database of HearNPV. A total of 23 proteins were identified as ODV associated 91
proteins. Among the 23 proteins, 21 were previously reported as ODV associated proteins 92
in other baculoviruses, but 2 were hitherto unknown as ODV associated proteins. These 93
two newly identified proteins, encoded by ha44 and ha100, respectively, were further 94
shown to be structural components of the ODV by Western blot analysis and 95
immunoelectron microscopy. 96
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MATERIALS AND METHODS 97
Insects, cells and virus. A culture of H. armigera insects was maintained according 98
to Sun et al. (55). An in vivo cloned strain of HearNPV (HearNPV-G4) (55,13) was used 99
as wild-type virus and propagated in H. armigera. The H. zea cell line HzAM1 cells (39) 100
were used for producing BV of HearNPV. 101
Purification of HearNPV BV and ODV. BV was purified from the cell culture 102
supernatant of infected HzAM1 cells (72hr p.i.) as described by Braunagel and Summers 103
(7). Larvae were homogenized in 0.1% SDS followed by few rounds of differential and 104
rate zonal centrifugation in sucrose gradients. All solutions were supplemented with 0.1% 105
SDS (56). Protease inactivation of the purified OBs was performed by HgCl2 and hot 106
water treatment (54). ODVs were released by alkaline treatment (pH=10.9) (7) and 107
purified on continuous sucrose gradients. Purified BV and ODV were further fractionated 108
into envelope and nucleocapsid components (28). 109
Protein Separation, Reduction, Alkylation, and Digestion. Proteins from purified 110
HearNPV ODV were separated on 12% SDS-PAGE and stained with Colloidal Blue 111
Staining kit (Invitrogen). Protein bands were excised from the 1D-PAGE gel, destained 112
by washing with a mixture of 200 mM NH4HCO3/acetonitrile (1:1). Proteins were 113
reduced with DTT, alkylated with iodoacetamide, and digested in-gel with trypsin 114
(Promega, Madison, WI) as described (53). The peptide mixtures obtained were further 115
desalted by ZipTipC18 (Millipore) and eluted in 50% acetonitrile/0.1% trifluoroacetic 116
acid buffer before MS analysis. 117
MALDI-TOF MS. A saturated solution of α-cyano-4-hydroxycinnamic acid in 0.1% 118
trifluoroacetic acid and 50% acetonitrile was used as the matrix. The sample and the 119
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matrix (1:1, v/v) were spotted on a target plate. MALDI-TOF spectra of the peptides were 120
obtained using a Voyager DE STR MALDI-TOF work station mass spectrometer 121
(Applied Biosystems Inc. USA). The analysis was performed in positive ion reflector 122
mode with an accelerating voltage of 20 kV and a delayed extraction of 150 ns. Typically, 123
200 scans
were averaged. Data mining was performed using MS-Fit
software 124
(http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) and Mascot software 125
(http://www.matrixscience.com/search_form_select.html) against the NCBI database and 126
the theoretical ORF database of HearNPV. 127
Sequence analysis of ha44 and ha100. The sequence data were compiled and 128
analyzed using DNASTAR software. Homologues in GenBank/EMBL databanks were 129
explored by using PSI-BLAST searching tool (1). The amino acid sequence alignment 130
was performed using Clustal X and T coffee software (42,58). GeneDoc software 131
(version 1.1.1004) was used for similarity shading and scoring of alignment. MEGA3.1 132
(33) was used for generating the phylogenetic trees using the neighbor-joining method 133
(NJ), with bootstrap replications. Phylogenetic tree was visualized using the Treeview 134
program. 135
Preparation of antibodies against HA44 and HA100. The entire ha44 coding 136
region and a truncated fragment of ha100 gene were amplified using synthesized primers 137
Ha44a / Ha44b (Ha44a: 5’-GAATTCATGAGCAATCCCAGCAAACAATC-3’; Ha44b: 138
5’-GAATTCTCAATAGCGCAAACGAGTTTCG-3’) and Ha100f / Ha100r (Ha100f: 5’ 139
GCCGGATCCATGACTTTGTCGCGTTTAGATTGCG-3’; Ha100r: 5’-140
GGCTCTAGATTAATAAACCATATTGTAATCGGCAAC-3’), respectively. The PCR 141
product of ha44 was first cloned into pGEM-T-Easy (Promega) and then into the 142
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expression vector pET28a (Novagen) in which ha44 was fused in-frame with 6-His-tag at 143
the C-terminus. The PCR product of ha100 was first cloned into pGEM-T-Easy 144
(Promega) and then into the expression vector pGEX-KG (22) in which ha100 was fused 145
in-frame with GST at the C-terminus. HA44 expressed in E. coli was purified with Ni-146
NTA agarose (Qiagen) and HA100 was purified by glutathione-agarose beads (Sigma). 147
The purified proteins were used for generating specific antibodies against HA44 and 148
HA100. 149
Purified HA44 and HA100 (200 µg) were used to immunize rabbits. Pre-immune 150
sera were withdrawn prior to inoculation. After three weeks, the rabbits received a 151
booster with the same amount of the antigens. Two weeks later the anti-sera were 152
collected and stored at –80℃ until use. The specificities of the antisera were tested by 153
Western blot analysis. 154
Western blot analysis. Purified BVs and ODVs, as well as their nucleocapsid and 155
envelope fractionations were separated on 12% SDS-PAGE and transferred onto 156
Hybond-N membranes (Amersham) by a semi-dry electrophoresis transfer (2). HA44 and 157
HA100 specific antisera and an alkaline phosphatase-conjugated IgG (SABC, China) 158
were used as the primary and secondary antibodies, respectively. The signal was detected 159
using a BCIP/NBT kit (SABC, China). Polyclonal anti-VP80, anti-ODV-E56 and anti-160
HaF1 were used as controls for nucleocapid, ODV envelope and BV envelope specific 161
proteins, respectively. 162
Immunoelectron microsopy. Purified ODVs were added to carbon-coated nickel 163
grids (150 mesh) and blocked with 5% bovine serum albumin. The primary antibodies 164
were 1:100 dilutions of anti-HA44 and anti-HA100 antisera. Pre-immune sera were used 165
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as the negative controls. The 12-nm Colloidal Gold-AffiniPure goat anti-rabbit IgG 166
(Jackson ImmunoResearch) was used as the secondary antibody for hybridization. The 167
grids were then negatively stained with 2% sodium phosphotungstate (PTA) and 168
examined with a transmission electron microscope (H-7000 FA, Hitachi). 169
170
RESULTS 171
MS Identification of HearNPV ODV Proteins. HearNPV ODVs were purified and 172
the proteins were separated by 12% SDS-PAGE. More than forty bands ranging from 11 173
to 110 kDa were visible with Colloidal Blue Staining (Fig. 1). Forty-one bands were 174
excised from the gel, reduced, alkylated, digested with trypsin and the peptides were 175
analyzed by MALDI-TOF MS. Peak lists of tryptic peptide masses were generated and 176
subjected to the NCBI database and HearNPV ORF database search using the MS-Fit and 177
Mascot search engine. The SDS-PAGE and MALDI-TOF MS analyses were performed 178
twice. Reliable DNA gene matches from the theoretical HearNPV ORFs were obtained 179
and summarized in Table 1. Twenty-three ORFs were identified, including ha1(polh), 180
ha2(p78/83), ha9(p49), ha10(odv-e18), ha11(odv-ec27), ha15(odv-e56), ha20(p74), 181
ha44, ha65(lef-3), ha66(Ac66), ha67(dna-pol), ha73(gp41), ha78(vp39), ha80(p33), 182
ha82(odv-e25), ha84(helicase), ha88(p6.9), ha89(odv/bv-C42), ha92(vp80), ha94(odv-183
EC43), ha96(odv-e66), ha100 and ha111(pif-1). 184
Among the 23 proteins, VP39 (57), P78/83 (59), VP80 (38,41) and ODV/BV-C42 185
(10) have been reported previously as nucleocapsid proteins of both BV and ODV. P6.9 186
is the main basic DNA-binding protein located in the nucleocapsid (65,66). GP41 is 187
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defined as the tegument protein of ODVs (63,64). ODV-E18 (9), ODV-E25 (51), ODV-188
E56 (8), ODV-E66 (26), as well as oral infectivity related proteins P74 (19,34,68) and 189
PIF-1 (31) were reported to be the ODV envelope proteins. ODV-EC27 (5,9) and ODV-190
EC43 (20) were reported as structural proteins of ODV nucleocapsid and envelope. P33 191
(48), P49, AC66, Helicase, LEF-3, DNA-polymerase and polyhedrin were reported as 192
ODV associated proteins (11). Two proteins HA44 and HA100 have not been reported 193
before and therefore are being described in more detail here. 194
PMF data interpretation using MS-Fit program revealed that 11 experimentally 195
derived tryptic peptide masses were found to match the predicted peptide masses of the 196
HA44 protein (error <100 ppm), covering 29% of its amino acid sequence. For HA100, 197
ten experimentally derived peptide masses were found to match the predicted peptide
198
masses of the HA100 protein (error <100 ppm), covering 26% of its amino acid sequence. 199
By using Mascot software for database searches (25), high Mascot score were revealed 200
when matched with HA44 and HA100 respectively (>67). 201
Sequence and phylogeny analysis of ha44 and ha100. Sequence analysis indicated 202
that ha44 contains 1134 nucleotides and potentially encodes a protein of 378 amino acids 203
(aa) with a predicted molecular mass of 42.8 kDa. A baculovirus late transcription motif 204
TAAG was found at 76 nt upstream of the initial ATG of ha44, suggesting that it is a late 205
gene. No polyadenylation signal was found within 500 nucleotides downstream of the 206
stop codon. 207
Searches of databases with all available genomes of baculoviruses showed that 208
homologues of HA44 were found in all Group II NPVs and GVs, but not in Group I 209
NPVs or in the dipteran and hymennopteran NPVs. The size of the HA44 homologue 210
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varies, ranging from the 255 aa of ORF45 of Adoxophyes honmai nucleopolyhedrovirus 211
(AdhoNPV45) to the 422 aa of ORF46 of Spodoptera litura nucleopolyhedrovirus 212
(SpliNPV46), although most of the homologues have a size of 311-378 aa. Pairwise 213
comparisons revealed that three proteins were very similar to their counterparts. Amino 214
acid identity was 98%, 83% and 75% for HA44/HzSNPV45, ChchNPV42/TnSNPV39 215
and MacoA136/MacoB135 respectively (for abbreviations please see the legend of Fig. 216
2). In contrast, the amino acid identity for the rest of the pairwise results was lower than 217
50%. The alignment of HA44 homologues from Group II NPVs is presented in Fig. 2. 218
The protein is mostly conserved at the C-terminus (Fig. 2). The N-terminal sequence of 219
HA44 is rich in basic residues (K/R) and serine, and this is a common feature for most of 220
the HA44 homologues. The isoelectric point (pI) of the N-terminal 64 amino acids of 221
HA44 is 10.79. Only 12 amino acids were absolutely conserved in the alignment, which 222
included N236, N264, V265, Y267, F281, N283, L322, N327, L333, K340, T342 and 223
V369 (Fig. 2). These amino acids might be important in the function of HA44. 224
Phylogenetic analysis indicated that the HA44 homologues have a common ancestor and 225
then diverged into the cluster of Group II NPVs and that of GVs (Fig. 3). 226
The Ha100 ORF is 1530 nt and encodes a protein of 510 aa with a predicted 227
molecular mass of 58 kDa. No consensus early transcription initiation motifs were found 228
upstream of the initial ATG, but a TAAG motif was found at -34 nucleotides suggesting 229
ha100 may also be a late gene. A polyadenylation signal (AATAAA) was found at 22-27 230
nucleotides downstream the stop codon. 231
HA100 has homology to poly (ADP-ribose) glycohydrolase (PARG), a ubiquitously 232
expressed exo- and endoglycohydrolase in eukaryotic cells. PARG mediates oxidative 233
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and excitotoxic neuronal death, and is involved in the breakdown and recruitment of 234
polyribose for nuclear functions such as DNA replication and repair (17,69). The 235
vertebrate PARGs contain 4 domains, A, B, C and D (43). Domain A is a putative 236
regulatory domain while B, C and D form catalytic fragments. Homologues of HA100 are 237
conserved in all Group II NPVs sequenced so far. Comparison of PARGs from a range of 238
organisms and from Group II NPVs is shown in Fig. 4. Similar to the PARG of 239
Drosophila melanogaster, the PARG-like proteins of Group II NPVs contain a catalytic 240
fragment, but lack of the putative regulatory A domain (Fig. 4). Alignment of HA100 241
homologues from baculoviruses and PARGs from selected eukaryotes reveals that 242
although the sequence similarity is not high, there were seven amino acids absolutely 243
conserved including F662, K676, Y683, G745, E756, P764 and E765 with respect to 244
bovine PARG sequence (data not shown). All the conserved amino acids are located in 245
conserved catalytic domain, which spans residues 610–795 in bovine PARG (43). 246
Localization of HA44 and HA100 in viral structures. Anti-HA44 and Anti-HA100 247
antisera were generated as descripted in Materials and Methods, and were used in 248
Western blot analysis and immunoelectron microscopy. The specificities of the antisera 249
were shown in Fig. S1 of the Supplemental Material. Western blot analyses were 250
performed to identify the localization of HA44 and HA100 in BV and ODV (Fig. 5). The 251
results showed that HA44 is located in the nucleocapsid but not in the envelope of ODV 252
and BV. HA100 was detected in the nucleocapsid and envelope of ODV, as well as in the 253
nucleocapsid of BV (Fig. 5). The sizes of HA44 and HA100 were 44 kDa and 60 kDa 254
respectively, which are in agreement with the predicted sizes deduced from their 255
nucleotide sequences. 256
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The immunoelectron microscopy results showed that HA44 was located in the 257
nucleocapsid of ODV, but not detected in the envelopes of the intact ODV (Fig. 6A). 258
HA100 was detected in intact ODVs, as well as in the nuclocapsids of ODV (Fig. 6B). 259
The IEM results confirmed that HA44 is a nucleocapsid protein of HearNPV ODV, while 260
HA100 is a structural protein of both nucleocapsid and envelope of the ODV. 261
262
DISCUSSION 263
In this study, we identified 23 HearNPV genes that encode ODV structural proteins 264
using SDS-PAGE and MS methods. This is the first such report for a Group II NPV. 265
Bruanagel et al. (2003) were able to identify 44 ODV associated proteins of 266
AcMNPV by using multiple techniques including MALDI-TOF, MUDPIT-MS/MS, 267
library exploring and Western blot. Perera et al. (2007) identified 44 polypeptides in 268
CuniNPV ODV by MALDI-TOF and GeLC-MS/MS. Comparison of the ODV associated 269
proteins of AcMNPV, CuniNPV and HearNPV, 9 proteins are shared by these viruses, 270
and are also conserved in baculoviruses sequenced so far. AcMNPV and CuniNPV ODVs 271
shared another five baculoviral conserved proteins, that of PIF2, F-protein, VP1054, 272
VLF-1 and VP91, which were not detected in HearNPV by MS. However, PIF2 was 273
identified as a HearNPV ODV structural protein by Western Blot analysis (21). Therefore, 274
at least 10 conserved baculoviral proteins are shared by ODVs of AcMNPV, CuniNPV 275
and HearNPV, including P49, ODV-EC27, ODV-E56, P74, GP41, VP39, P33, P6.9, 276
ODV-EC43, and PIF-2. Another baculoviral conserved protein, PIF1, was identified as an 277
ODV component in HearNPV in our study and was also reported as ODV associated 278
protein in CuniNPV (46), but was not identified by multiple approaches in AcMNPV (13). 279
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DNA polymerase and Helicase are conserved baculoviral proteins shared by the ODVs of 280
both AcMNPV and HearNPV, but they were not detected in the CuniNPV ODV (46). 281
The identification of common structural proteins is essential to elucidate the core 282
structure of baculoviruses. Among the 10 conserved proteins, ODV-EC27, ODV-E56, 283
GP41, VP39, P6.9 and ODV-EC43 are known to be structural proteins. It is interesting to 284
see that P74 and PIF-2, which are essential for oral infection, are also associated with the 285
ODV. With more data derived from different viruses becoming available, the importance 286
and the functions of these proteins can be further revealed. 287
In this study, approximately 41 ODV protein bands separated by SDS-PAGE were 288
subjected to MALDI-TOF MS analysis, 38 bands had matches to viral ORFs while 3 289
bands did not produce significant matches and were not identified by this technique (Fig. 290
1, table 1). The data of unmatched bands suggested that additional host proteins may 291
present. In vaccinia virus, MS techniques revealed 23 virion-associated host proteins in 292
addition to the 75 viral proteins (16). Our HearNPV ODV data have not matched any host 293
proteins which may be due to the lack of the genetic information and database on H. 294
armigera. Some proteins of HearNPV ODV were not identified possibly due to their low 295
molar content in ODVs, and/or resistance to staining, or some proteins may not be 296
amenable to MALDI-TOF MS. For example, HA122 and PIF-2 have already been 297
identified and located in the HearNPV ODV (37,21) but we were not able to detect them 298
in this study. 299
The degradation or losses of proteins during virus purification could also affect 300
protein detection. Although HgCl2 treatment, heat inactivation of proteases and a protease 301
inhibitor cocktail were used during the purification of virions, multiple bands of a single 302
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protein were still present in the gel including those of Polyhedrin, VP39, GP41, P49, P33, 303
ODV-E66, ODV-E56 and ODV-E25 (Fig. 1 and Table 1). Similar results were observed 304
from a single protein during an investigation of the CuniNPV ODV (46). Various reasons 305
can be attributed to the fragile nature of some proteins including the refractory profile of 306
alkaline proteases and the different methods used in virus purification (11,54), However, 307
we cannot exclude the possibility of some protein degradation during the experimental 308
procedures. On the other hand, there may be polymorphisms, oligomerization and post-309
translational modification of the gene products in the matrix of ODVs. 310
During data mining by MS-Fit, some peptides footprints were matched to HearNPV 311
ORFs but with a low MOWSE Score, such as ha26, ie1, me53, bro-b, bro-c, alk-exo, pk1, 312
ha133 (f protein gene), etc. Therefore, they were not included as ODV structural proteins 313
in our results. Some of them, such as IE1, Alk-exo and F-protein were identified to be 314
located to the ODVs of AcMNPV (11), while F-protein and Bro were identified in the 315
ODVs of CuniNPV (46). The importance of employing multiple techniques to identify 316
ODV structural proteins has been elucidated by Braunagel et al. (11). We are using 317
antibodies against HearNPV ORFs to verify the protein localization in the ODV by 318
Western blot analysis and immunoelectron microscopy. Location of the above proteins in 319
the ODV awaits confirmation pending preparation of specific antibodies. 320
Two structural proteins of HearNPV ODV, HA44 and HA100, were newly identified 321
here. ORF ha44 contains a TAAG late gene promoter motif, which is in agreement with 322
its function as a structural protein. Western blot analysis has confirmed that HA44 was a 323
nucleocapsid component in both BV and ODV with a molecular mass of 44 kDa. 324
Homologues of HA44 were found in all the Group II NPVs and GVs whose complete 325
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sequences have been determined, but not in Group I NPVs, dipteran or hymennopteran 326
baculoviruses. It is generally believed that GVs separated from the ancestor of NPVs and 327
GVs before the radiation of Group I and Group II NPVs (35). It is, therefore, likely that 328
the ancestor of HA44 existed in both NPV and GV but was lost during the emergence of 329
Group I NPVs. 330
Western blot analysis and immunoelctron microscopy revealed that HA100 is a 331
component of the nucleocapsid and the envelope of ODV. HA100 is conserved in all 332
Group II NPVs and it is a homologue of poly(ADP-ribose) glycohydrolase (PARG). 333
PARG is critical for the maintenance of a steady-state poly(ADP-ribose) levels and plays 334
important roles in modulating chromatin structure, transcription, DNA repair and 335
apoptosis (6). It is interesting that the Group II members of the NPVs encode a PARG-336
like protein as a structural protein. It remains to be determined whether the PARG-like 337
proteins in Group II NPVs are enzymatically functional. 338
With the knowledge of baculovirus ODV composition, it is possible to study the 339
functions of the relevant proteins and their potential role during virus primary infection 340
(11). In this study, we identified two new ODV structural proteins: HA44 and HA100. 341
Currently we are investigating the biological functions of these two proteins. 342
343
ACKNOWLEDGMENTS 344
The work is supported by a 973 project (2003CB114202) and a NSFC key project 345
from China, and a joint PSA project from China and the Netherlands (2004CB720404). 346
We acknowledge the State Key Laboratory of Virology Proteomics/MS Center (Wuhan 347
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University) and Shanghai GeneCore BioTechnologies Co. Ltd. for technical support. We 348
thank Jian-Lan Yu and Fang-Ke Huang for experimental assistance. 349
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FIGURE LEGENDS 567
FIG. 1. SDS-PAGE profile and MS results of purified HearNPV ODV. ODV proteins 568
were separated on 12% SDS-PAGE and stained with Colloidal Blue. The ODV bands 569
(numbered in the middle) were subjected to MALDI-TOF MS and their determined 570
identity is listed on the right. 571
572
FIG. 2. Alignment of HA44 amino acid sequence and its homologues from Group II 573
NPVs. Three shading levels were set: black for 100% identity, dark gray for 80% identity 574
and light gray for 60%. The NCBI accession numbers are: NP_818692 for AdhoNPV45, 575
YP_529786 for ORF116 of Agrotis segetum NPV (AgseNPV116), YP_249646 for 576
ORF42 of Chrysodeixis chalcites NPV (ChchNPV42), NP_075113 for HA44, 577
NP_542668 for ORF45 of (HzSNPV45), NP_047691 for ORF55 of Lymantria dispar 578
NPV (LdMNPV55), NP_613219 for ORF136 of Mamestra configurata NPV A 579
(MacoA136), NP_689309 for ORF135 of Mamestra configurata NPV B (MacoB135), 580
NP_037867 for ORF107 of Spodoptera exigua NPV (SeMNPV107), NP_258314 for 581
ORF44 of Spodoptera litura NPV (SpliNPV44), and YP_308929 for ORF39 of 582
Trichoplusia ni NPV (TnSNPV39). 583
FIG. 3. A neighbor-joining tree derived from HA44 and its homologues from NPVs and 584
GVs. Bootstrap values (1000 replicates, nodes supported with more than 50%) are given 585
on the branch lines. The accession numbers for NPVs are as described in Fig. 3. The 586
additionals are: NP_872567 for ORF113 of Adoxophyes orana GV (AdorGV113), 587
YP_006220 for ORF124 of Agrotis segetum GV (AgseGV124), NP_891969 for ORF122 588
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of Cryptophlebia leucotreta GV (CrleGV122), NP_148919 for ORF135 of Cydia 589
pomonella GV (CpGV135), NP_663288 for ORF123 of Phthorimaea operculella GV 590
(PhopGV123), NP_068332 for ORF113 of Plutella xylostella GV (PlxyGV113), and 591
NP_059320 for ORF172 of Xestia c-nigrum GV (XecnGV172). 592
593
FIG. 4. Comparison of PARGs from a wide range of organisms and from Group II NPVs. 594
A: Putative regulatory domain; B-C-D: Catalytic fragment; C: PARG catalytic domain. 595
Percentage conservation is indicated in each block with respect to the bovine PARG. The 596
amino acid position of bovine PARG domains and the length of PARGs were indicated. 597
The accession numbers of PARGs are: NP_776563 for Bos Taurus (B. Taurus), 598
AAH52966 for Homo sapiens (H. sapiens), NP_036090 for Mus musculus (M. musculus), 599
NP_112629 for Rattus norvegicus (R. norvegicus), NP_477321 for Drosophila 600
melanogaster (D. melanogaster), NP_501508 for Caenorhabditis elegans (C. elegans), 601
NP_075169 for HA100, NP_818756 for AdhoNPV, YP_529728 for AgseNPV, 602
YP_249712 for ChchNPV, NP_542726 for HzSNPV, NP_047778 for LdMNPV, 603
NP_613153 for MacoNPV A, NP_689244 for MacoNPV B, NP_037812 for SeMNPV, 604
NP_258370 for SpliNPV, and YP_308992 for TnSNPV. 605
606
Figure 5. Western blot analysis of the HearNPV ODV/BV nucleocapsid (NC) and 607
envelope (E) fractions using anti-HA44 and anti-HA100 antibodies. Healthy HzAM1 608
cells (H) and virus infected cells (I) were loaded as negative and positive controls. VP80, 609
ODV-E56 and F protein were detected by their specific antibodies for illustrating the NC-610
specific protein, ODV E-specific protein, and BV E-specific protein, respectively. 611
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Figure 6. Immunoeclectron microscopy (IEM) of HA44 and HA100 and localization in 612
HearNPV ODVs and nucleocapsids (NC) of ODVs. A 1:100 dilution of anti-HA44 and 613
anti-HA100 antisera were used as primary antibodies. Pre-immune sera were used for 614
negative controls. A: IEM of HA44; B: IEM of HA100. Bar, 100nm.615
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Table 1. HearNPV ODV Proteins identified by MALDI-TOF MS*
Band
No.
Size
(kDa)
from
SDS-
PAGE
HearNPV
ORF
AcMNPV
ORF Protein
Predicted
size
(kDa)
Sequence
coverage Function References
1 110 73 80 GP41 36.6 43% Tegument main protein 63,64
2 103 73 80 GP41 36.6 28% Tegument main protein 63,64
3 100 66 92 HA66 88.9 37% ODV associated protein 11
4 83 92 104 VP80 69.7 37% Nucleocapsid 38,41
5 78 20 138 P74 78.4 25% Oral infectivity 19,34,68
6 73 73 80 GP41 36.6 58% Tegument main protein 63,64
7 65 96 46 ODV-E66 76.1 35% ODV envelope 26
8 60 100 \ HA100 58.1 26% Nucleocapsid and ODV envelope associated
protein This study
1 8 Polyhedrin 28.8 40% Polyhedra main protein 49 9 58
96 46 ODV-E66 76.1 12% ODV envelope 26
111 119 PIF-1 60.3 19% Oral infectivity 31 10 56
1 8 Polyhedrin 28.8 40% Polyhedra main protein 49
11 54 2 9 P78/83 45.9 49% Nucleocapsid 47,52
12 50 96 46 ODV-E66 76.1 25% ODV envelope 26
13 48 9 142 P49 55.3 36% Apoptotic suppressor 72
14 45 44 \ Ha44 42.8 29% Nucleocapsid associated protein This study
44 \ Ha44 42.8 32% Nucleocapsid associated protein This study 15 44
89 101 C42 42.6 20% Nucleocapsid 10
96 46 ODV-E66 76.1 15% ODV envelope 26 16 42
89 101 C42 42.6 18% Nucleocapsid 10
17 39 94 109 ODV-EC43 41.5 45% ODV envelope & Nucleocapsid 20
94 109 ODV-EC43 41.5 51% ODV envelope & Nucleocapsid 20 18 38
84 95 Helicase 146 10% DNA replication essential 11,32
19 36 15 148 ODV-E56 38.9 24% ODV envelope 8
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9 142 p49 55.3 29% Apoptotic suppressor 72 20 35
78 89 VP39 33.4 35% Nucleocapsid 45
21 34 73 80 GP41 36.6 58% Tegument main protein 63,64
22 33 1 8 Polyhedrin 28.8 38% Polyhedra main protein 49
78 89 VP39 33.4 50% Nucleocapsid 45 23 32
11 144 ODV-EC27 33.3 32% ODV envelope & Nucleocapsid 3,9
78 89 VP39 33.4 42% Nucleocapsid 45 24 30
1 8 Polyhedrin 28.8 34% Polyhedra main protein 49
25 29 78 89 VP39 33.4 57% Nucleocapsid 45
26 28 82 94 ODV-E25 25.9 41% ODV envelope 51
78 89 VP39 33.4 56% Nucleocapsid 45 27 27
80 66 P33 30.8 34% Stimulating P53-induced apoptosis 48
28 26 80 66 P33 30.8 44% Stimulating P53-induced apoptosis 48
78 89 VP39 33.4 45% Nucleocapsid 45 29 25
15 148 ODV-E56 38.9 24% ODV envelope 8
31 23.5 80 66 P33 30.8 32% Stimulating P53-induced apoptosis 48
1 8 Polyhedrin 28.8 28% Polyhedra main protein 49 32 22
67 65 DNA-pol 119.3 11% DNA replication essential 11,32
34 20 1 8 Polyhedrin 28.8 28% Polyhedra main protein 49
35 19 82 94 ODV-E25 25.9 45% ODV envelope 51
78 89 VP39 33.4 40% Nucleocapsid 45 36 18.5
9 142 P49 55.3 18% Apoptotic suppressor 72
37 18 88 100 P6.9 11.5 35% DNA binding protein 65,66
38 15 10 143 ODV-E18 8.8 45% ODV envelope 9
39 14 10 143 ODV-E18 8.8 45% ODV envelope 9
82 94 ODV-E25 25.9 40% ODV envelope 51 41 12
65 67 LEF-3 44 20% DNA replication essential 11,32
*The order of the bands was the same as that of Fig. 1. The MALDI-TOF MS were repeated once.
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 1.
11 kDa
17 kDa
24 kDa
33 kDa
40 kDa
55 kDa
72 kDa
100 kDa
130 kDa
170 kDa
GP41
HA66
VP80P74GP41
ODV-E66HA100ODV-E66 + PolyhedrinPIF-1 + PolyhedrinP78/83
ODV-E66
P49HA44
ODV-EC43
ODV-E56
GP41P49 + VP39
ODV-EC43 + Helicase
ODV-E66 + C42
VP39 + ODV-EC27
VP39 + Polyhedrin
VP39
ODV-E25
VP39 + P33
VP39 + ODV-E56
?
Polyhedrin + DNA-pol
?
ODV-E25
VP39 + P49
P6.9
ODV-E18
ODV-E25 + LEF-3
?
123
456
7
8
910
11
12
13
141516
17
18
19
20
2122
23
24
25
26
272829
3031
32
33
34
35
36
37
38
39
40
41
GP41
HA44 + C42
P33
Polyhedrin
ODV-E18
P33
Polyhedrin
11 kDa
17 kDa
24 kDa
33 kDa
40 kDa
55 kDa
72 kDa
100 kDa
130 kDa
170 kDa
GP41
HA66
VP80P74GP41
ODV-E66HA100ODV-E66 + PolyhedrinPIF-1 + PolyhedrinP78/83
ODV-E66
P49HA44
ODV-EC43
ODV-E56
GP41P49 + VP39
ODV-EC43 + Helicase
ODV-E66 + C42
VP39 + ODV-EC27
VP39 + Polyhedrin
VP39
ODV-E25
VP39 + P33
VP39 + ODV-E56
?
Polyhedrin + DNA-pol
?
ODV-E25
VP39 + P49
P6.9
ODV-E18
ODV-E25 + LEF-3
?
123
456
7
8
910
11
12
13
141516
17
18
19
20
2122
23
24
25
26
272829
3031
32
33
34
35
36
37
38
39
40
41
GP41
HA44 + C42
P33
Polyhedrin
ODV-E18
P33
Polyhedrin
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 2
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 3.
AgseNPV116
SeMNPV107 ChchNPV42
TnSNPV39
MacoA136
MacoB135
HA44 HzSNPV45
LdMNPV55
AdhoNPV45
PlxyGV113
PhopGV123
AdorGV113
AgseGV124
XecnGV172
CrleGV122
CpGV135
100
99
100
100
100
74
50
80
67
100
0.2
SpliNPV46
Group II NPVs
GVs
AgseNPV116
SeMNPV107 ChchNPV42
TnSNPV39
MacoA136
MacoB135
HA44 HzSNPV45
LdMNPV55
AdhoNPV45
PlxyGV113
PhopGV123
AdorGV113
AgseGV124
XecnGV172
CrleGV122
CpGV135
100
99
100
100
100
74
50
80
67
100
0.2
SpliNPV46
Group II NPVs
GVs
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 4.
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 5.
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Deng et al. HA44 and HA100 are two novel structural proteins of HearNPV
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Figure 6.
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