2 proteins, ha44 and ha100 accepted - journal of...

Deng et al. Two newly identified ODV associated proteins, HA44 and HA100

1

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: [email protected] 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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analysis of FP25K of Helicoverpa armigera single nucleocapsid 551

nucleopolyhedrovirus. J. Gen. Virol. 86: 2439-2444. 552

68. Yao, L., W. Zhou, H. Xu, Y Zheng, and Y. Qi. 2004. The Heliothis armigera single 553

nucleocapsid nucleopolyhedrovirus envelope protein P74 is required for infection of 554

the host midgut. Virus Res. 104: 111-121. 555

69. Ying, W., M. B. Sevigny, Y. Chen, and R. A. Swanson. 2001. Poly(ADP-ribose) 556

glycohydrolase mediates oxidative and excitotoxic neuronal death. Proc. Natl. Acad. 557

Sci. U S A. 98: 12227-12232. 558

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70. Zanotto, P. M., B. D. Kessing, and J. E. Maruniak. 1993. Phylogenetic 559

interrelationships among baculoviruses: evolutionary rates and host associations. J. 560

Invertebr. Pathol. 62:147-164. 561

71. Zhang, G. 1994. Research, development and application of Heliothis viral pesticide 562

in China. Resources and Environment in the Yangtze Valley. 3: 1-6. 563

72. Zooq, S. J., J. J. Schiller, J. A. Wetter, N. Chejanovsky, and P. D. Friesen. 2002. 564

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resistant to P35 in vivo. EMBO J. 21: 5130-5140. 566

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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


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


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


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


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9 142 p49 55.3 29% Apoptotic suppressor 72 20 35

78 89 VP39 33.4 35% Nucleocapsid 45


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


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


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



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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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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Figure 2

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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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Figure 4.

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Figure 5.

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Figure 6.

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2 proteins, ha44 and ha100 accepted - journal of...

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