ARTICLE pubs.acs.org/jpr
ODV-Associated Proteins of the Pieris rapae Granulovirus Xiao-Feng Wang,†,‡,§ Bao-Qin Zhang,†,‡ Hai-Jun Xu,‡ Ying-Jun Cui,‡ Yi-Peng Xu,‡ Min-Juan Zhang,‡ Yeon Soo Han,|| Yong Seok Lee,^ Yan-Yuan Bao,‡ and Chuan-Xi Zhang*,‡ ‡
)
Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Insect Science, Zhejiang-California International Nanosystem Institute, Zhejiang University, Hangzhou 310058, China § College of Life and Environmental Science, Wenzhou University, Wenzhou 325027, China Department of Agricultural Biology, Chonnam National University, Gwangju, 500-757, Korea ^ Department of Parasitology, College of Medicine and UHRC, Inje University, Busan, 614-735, Korea ABSTRACT: Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/ BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirushost interaction studies. KEYWORDS: baculovirus, Betabaculovirus, Pieris rapae granulovirus (PrGV), ODV proteome
’ INTRODUCTION The family Baculoviridae is a diverse group of rod-shaped, enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 to 180 kb. Baculoviruses have been extensively used for biological control of insect pests and some are widely used as vectors in gene expression systems. Traditionally, the family was classified into two genera, Nucleopolyhedrovirus (NPV) and Granulovirus (GV), based on the morphology of the occlusion bodies (OBs). Recently, it has been classified into four genera based on multiple characteristics: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific GV), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).29 Baculoviruses have been reported worldwide from over 600 host species, mostly from the order Lepidoptera. Both Alphabaculovirus and Betabaculovirus produce two phenotypes in their infection cycle, budded virus (BV) and occlusion-derived virion (ODV). BV, the primary phenotype, can mediate the cell-to-cell spread from the tissue of the infected host while ODV can mediate the transmission of infection from animal to animal.49 Therefore, the two types of virion are critical for the life cycle of the baculoviruses. r 2011 American Chemical Society
The Betabaculovirus largely differ from the Alphabaculovirus in virion structure, that is, Betabaculovirus OBs (ovoid shape: 500 nm by 200 nm)5 are much smaller than Alphabaculovirus OBs (polyhedra, average diameter of 8002000 nm).4 Additionally, Betabaculovirus usually contains a single ODV per OB whereas Alphabaculovirus OBs contain many ODV which may also contain multiple nucleocapsids per virion.28 More recently, mass spectrometry (MS) has been used for exploring the compositions of baculovirus ODV and BV with the intent to reveal infectious mechanisms and virus-host interaction. These studies include the ODV of Autographa californica nucleopolyhedrovirus (AcMNPV) (Alphabaculovirus),9 Culex nigripalpus nucleopolyhedrovirus (CuniNPV) (Deltabaculovirus),55 and Helicoverpa armigera nucleopolyhedrovirus (HearNPV) (Alphabaculovirus)11 as well as the BV of AcMNPV.70 However, only limited virion proteins were identified from betabaculoviruses, such as Granulin,63 Alkaline protease,64 P6.965 Enhancin,36 ODVP-6E/ODV-E56,58 and IAP.67 No proteome from any betabaculovirus has been determined, and the similarities and differences between Received: January 27, 2011 Published: April 26, 2011 2817
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Figure 1. TEM photographs (100 000 ) Of PrGV. Occlusion bodies (upper) and ODV (lower), respectively.
betabaculovirus and alphabaculovirus ODV-associated protein compositions remain unclear. The small cabbage white butterfly (Pieris rapae) is widespread around the world and is an important pest of cultivated cabbages and other mustard family crops. P. rapae granulovirus (PrGV) infects and kills larvae of P. rapae. Presently, PrGV has been widely used for biological control of the small cabbage white butterfly and has become a commercially important biological insecticide.27 Given the importance of PrGV for P. rapae management, it is necessary to explore the infectious molecular mechanism of PrGV. Recently, we sequenced the complete genome of P. rapae GV. The PrGV genome consists of 108 592 bp and 120 open reading frames(ORFs) were predicted (GenBank, GQ884143). In this study, we performed analyses of PrGV ODV proteome by using MS approaches. The resulting data help provide a better understanding of virion structure and gene function(s) as well as reveal similarities and differences between Betabaculovirus and Alphabaculovirus at the molecular level.
’ MATERIALS AND METHODS Preparation of PrGV-ODV
Third-instar P. rapae larvae were infected with PrGV occlusion bodies. Preparation of PrGV was performed according to a modified approach.49 The purified occlusion bodies were observed to preliminarily evaluate both their purity and quantity by transmission electron microscopy (TEM) with negative staining (2% sodium phosphotungstate) (Figure 1). PrGV-ODVs were released
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Figure 2. Twelve percent SDS-PAGE of PrGV-ODVs. Protein markers are to the left, and identified proteins are to the right.
from occlusion bodies by using an alkaline treatment (0.1 M Na2CO3, 166 mM NaCl, 10 mM EDTA) and were purified by using a step sucrose density gradient centrifugation (405060%, w/w) with modifications as described previously.10 Separation and Identification of PrGV-ODV Composition
PrGV-ODV proteins were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Figure 2). Either 23 bands or 3 band regions (F1/F2/F3) were excised from a 1-D electrophoresis gel, and the gels were then placed in destaining solution (2 M NH4HCO3 in 40 mL acetonitrileddH2O (1:1)) at 37 C for about 30 min. Proteins were further reduced with dithiothreitol, alkylated with iodoacetamide, and subsequently digested in-gel with trypsin (Promega Sequencing grade trypsin, V5111) for 20 h. Sample concentration was determined by SpeedVac (Labconco CentriVap concentrator) at room temperature for 20 min. Peptide mixtures were resuspended in 20 μL 0.1% formic acid and were analyzed by PMF (peptide mass fingerprinting) using Bruker-Daltonics AutoFlex TOF/TOF LIFT MS (matrix-assisted laser desorption/ionization time-offlight, MALDI TOF/TOF MS) (Bruker Daltonics, Bremen, Germany), LCLTQ (liquid chromatographytandem mass spectrometry) (LTQ VELOS, Thermo Finnigan) (for sample F1/F2/F3), and MDLC-LTQ-Orbitrap (Ettan MDLC system, GE Healthcare; LTQ-Orbitrap, Thermo Finnigan) (for complete samples). Data mining of PMFs by MALDI-TOF/TOF was conducted by Mascot search56 with filtered parameters: 2818
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Table 1. Primers Used in This Studya primer name
sequence
PrGV02F(411) 50 -AGGATCCATGAAAGAATTTAATTTTA-30 PrGV02R
50 -ACTCGAGTTAAACGTCAAAATCG-30
PrGV21F(1023) 50 -AGGATCCATGTCCAGGCTGATATTTTCG-30 PrGV21R
50 -ACTCGAGTTATTTAGCTTTTATTTGAACG-30
PrGV22F(438) 50 -AGGATCCATGTCCTACTCGTGTC-30 PrGV22R
50 -ACTCGAGTTAAACTGTTGTCAATGGCG-30
PrGV29F(570) 50 -AGGATCCATGTATTATTTGATACACG-30 PrGV29R 50 -ACTCGAGTTATGAGATGTTAAAAAAAGG-30 PrGV35F (345) 50 -AGGATCCATGATACATTATCAGGTG-30 PrGV35R
50 -ACTCGAGTTAGAGATTAGTTGTGG-30
PrGV42F(2412) 50 -AGGATCCATGAAGAGGTCTGTAGCG-30 PrGV42R
50 - ACTCGAGTTAATCGCTGGTGTCGCTTTC-30
PrGV46F(1065) 50 -AGGATCCATGACTTGTTCCGCC-30 PrGV46R
50 -ACTCGAGTTACAGAAAATAATTGTATACCGG-30
PrGV54F(600) 50 -AGGATCCATGGAATCGCTCG-30 PrGV54R 50 -ACTCGAGTTATACACCATTATTGTGC-30 PrGV76F(645) 50 - AGGATCCATGTGGGGAACTATTGTGC-30 PrGV76R
50 - ACTCGAGTTATTGTAATCTGAAAC-30
PrGV78F(816) 50 -AGGATCCTTGGTAAAAATTATTGG-30 PrGV78R
50 -ACTCGAGTCAAATAATACCTAATAAAG-30
PrGV84F(294) 50 -AGGATCCATGAACAACCAATC-30 PrGV84R
50 -ACTCGAGCTACAATTTCTGATTA-30
PrGV86F(516) 50 -AGGATCCGTGGTGTATTGTGAGATTAAGC-30 PrGV86R 50 -ACTCGAGTTACTGTTTTCTATTTGACTCG-30 PrGV114F(618) 50 -AGGATCCGATTCGGACGATGG-30 PrGV114R
50 -ACTCGAGCTATTTGATTATGGTAA-30
a
The numbers in parentheses represented the amplified sizes of corresponding ORFs by PCR reaction and underlined sequences indicated restriction enzyme digestion sites (BamHI/XhoI).
Peptide Mass Tolerance: ( 100 ppm; Fragment Mass Tolerance: ( 0.4 Da; Peptide Charge State: 1þ; Max Missed Cleavages: 1. MS data by LC-LTQ was analyzed by BuildSummary software14 with parameters (DelCN g 0.1): when Charge þ1, Xcorr g1.9; when Charge þ2, Xcorr g2.2; and when Charge þ3, Xcorr g3.75. MS data by LTQ-Orbitrap was determined by BioWorks/SEQUEST software52 with parameters: Peptide tolerance 10.00 PPM, Fragment ions tolerance 1.00; number results scored 250; B lons; PTMs per peptide 3 (m/z 3002000; Resolution 60 000). The above three approaches were performed against the virus protein database and the theoretical protein database of PrGV, respectively. Expression of PrGV-ORFs in Escherichia coli (E. coli)
Eleven ODV-encoding genes were randomly selected and expressed for preparation of primary antibody, including Pr2, Pr21, Pr22, Pr29, Pr35, Pr42, Pr46, Pr54, Pr78, Pr84, Pr114. Protein expression was conducted as described by Wang et al.69 The complete coding region of each ORF was amplified with its corresponding primers designed based on our sequenced genome sequence (GenBank, GQ884143) (Table 1). The target protein expressed in E. coli was separated and excised from a 12% SDS-PAGE gel. The proteins were electroeluted and dialyzed to remove ion and other impurities, freeze-dried, and finally dissolved in sterilized ddH2O. Purified products were used for the preparation of primary antibodies.
Preparation of Antibodies and Western Blotting Analysis
The preparation of antibodies and immunoassays were performed as described by Xu et al.81 Briefly, preimmune sera were withdrawn for a negative control of immunoassay before inoculation. Purified target protein (200 μg) in complete Freund’s adjuvant was injected multipoint-subcutaneously to immunize New Zealand white rabbits. Three weeks later, three booster injections were executed in complete Freund’s adjuvant per two weeks. Exsanguinations were performed after 8 days of the last booster injection. The acquired polyclonal rabbit antibodies were used for Western blotting analysis on purified PrGV ODV proteins as described previously.81 Purified protein samples were separated by 12% SDS-PAGE gel and immunoreactive proteins were detected using goat antirabbit IgG-HPR (1:4000, Southern Biotech, AL) with 0.1% H2O2 and diaminobenzidine (DAB) as a chromogenic substrate. Protein Bioinformatics Analysis
Proteinprotein similarity was analyzed by Protein BLAST program with default settings (Scoring Parameters: Matrix, BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: conditional compositional score matrix adjustment)43 (http://blast.ncbi.nlm.nih.gov). Signal peptide was predicted by online software SignalP 3.0 Server3 (http://www.expasy.org/ tools). Pfam families were predicted by Pham HMM search with default threshold: E-value (1.0)18 (http://pfam.sanger.ac.uk/ search?tab=searchSequenceBlock).
’ RESULTS MS Identification of PrGV-ODV Proteins
Forty-seven ODV-associated proteins were identified by different MS methods used in this study (Table 2). A MALDITOF/TOF approach identified 16 proteins from 23 bands separated by SDS-PAGE. The LCLTQ approach identified 37 proteins from 3 fragments (F1/F2/F3) while the LTQOrbitrap approach identified 31 proteins from the protein complex. Notably, some proteins, such as granulin, ODV-E66b, and Pr114, were identified in several bands or from different fragments (Figure 2 and Table 2). Similar instances have also been observed in other NPVs by proteomic analyses.11,55,70 Among the 47 ODV-associated proteins, eleven proteins, Pr1 (Granulin), Pr15 (p49), Pr16 (ODV-E56), Pr20 (PEP1), Pr39 (ODV-E66a), Pr42, Pr44 (ODV-E66b), Pr46 (ODV-EC43), Pr70(BV/ODV-C42), Pr88 (GP41) and Pr114, were identified by all three MS methods. These were also highly abundant proteins (Figure 2 and Table 2). Fifteen proteins, Pr2 (P78/83), Pr21(PEP/P10), Pr22 (PEP2), Pr40 (PIF-2), Pr51 (P74), Pr61 (PIF-1), Pr68 (P45/P48), Pr78 (P33), Pr81 (VP39), Pr83, Pr84, Pr89, Pr90 (VIF-1), Pr92, and Pr120 (ME-53), were identified using two of the three MS methods, whereas other 21 proteins were only found using one of the three MS methods, including ODV-E18, P10, Pr26 (F-protein), Pr29, Pr30 (PIF-3), Pr35, Pr45 (ubiquitin), Pr54, Pr69, Pr71 (P6.9), Pr73 (38K), Pr74 (ODV-E28), Pr75 (Helicase-1), Pr82 (ODV-EC27), Pr85 (VP91), Pr93 (DNA pol), Pr95 (LEF-3), Pr96, Pr111, Pr116 (Vp1054), and Pr118 (FGF-3). Comparison of ODV Proteins Identified in This Study and Proteins in Other Baculoviruses
Because the ODV/BV compositions of AcMNPV, and ODV compositions of CuniNPV and HearNPV have been successfully resolved by MS approaches, we comprehensively analyzed our 2819
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PIF-2
Pr42
ODV-E66b
Ubquitin
ODV-EC43
P74 Pr54
PIF-1
P45/P48
Pr69 (Ac102)
BV/ODV-C42
P6.9
38K ODV-E28
Helicase-1
P33
VP39
BV/ODV-EC27
42
44
45
46
51 54
61
68
69
70
71
73 74
75
78
81
82
F-protein
26
40
PEP2
ODV-E66a
PEP/P10
21
22
39
PEP1
20
PIF-3 Pr35
P10
17
Pr29
ODV-E56
16
29
P49
15
30 35
F1,F2,F3 F1,F2,F3
ODV-E18
14
2820
16,17,18,21
20
12
14
1,5,6,9
2,3,4
7
23
12,13,21,23
10,11
38
32
16
28
47
84
51
70
46
66
F1,F3
F1,F2,F3
F3
F3 F3
F3
F1,F2,F3
F3
F2
F1
F3
F1,F2,F3
F1,F2
F1,F2,F3
F1,F2
F2
F1,F2,F3
F3
F3
F1,F2,F3
F3
F1,F2,F3
F1,F2,F3
F3
F3
F1,F2,F3
P78/83
89
2
19,11,19,22
Granulin
fragment
1
(score) pept.
product name
(s)
48.25
76.92
28.29
7.97 11.18
15.52
63.37
66.06
3.34
2.22
7.53
62.43
40
45.54
44.33
12
32.38
8.42
27.51
64.83
23.82
58.82
76.92
30.51
58.48
19.7
24.82
49.8
coverage(%)
LCLTQ
orf
band
MALDI-TOF/TOF
6(9)
2(2)
4(4)
4(5)
6(11) 3(6)
5(7)
5(5)
3(3)
4(4)
7(7)
3(3)
6(6)
1(1)
4(7)
5(10)
3(3)
4(6)
1(1)
9(43)
uni./tot.
no.
26
13.4
30.3
17.6
18.6 32.2
37.3
24.5
8.6
29.1
32.8
33.3
35.3
16.6
37.6
29.9
25.7
20.9
35.8
57.5
(%)
coverage
LTQ-Orbitrap
32
24
14
89
87 90
23
29
74
69
38
46
104
102
30
CuniNPV (ODV)
144 (ODV/BV)
89 (ODV/BV)
92 (ODV)
95 (ODV)
98 (BV) 96
100 (ODV/BV)
101 (ODV/BV)
102 (ODV)
103
119
138 (ODV)
109 (ODV/BV)
35 (BV)
46 (ODV)
22 (ODV)
46 (ODV)
115
23 (ODV/BV)
131 (BV)
131 (BV)
137
148 (ODV/BV)
142 (ODV/BV)
143 (ODV/BV)
9 (ODV/BV)
8
AcMNPV (ODV/BV)
11
78
80
84
86 85
88
89
90
91
111
20
94
28
96
132
96
98
141
120
120
57
15
9
10
2
1
HearNPV (ODV)
and references
function description
ODV envelope and nucleocapsid2
major capsid protein51
BV production and ODV formation45,77
necessary for DNA replication33
nucleocapsid formation78 ODV envelope protein81
DNA binding protein65
mediate the nuclear entry of P78/837,72
ODV-associated protein9
and ODV envelopment84
virus replication;38 BV production
structure protein for oral infectivity61
to be involved in oral infectivity85 not be characterized in Betabaculovirus15
ODV envelope and nucleocapsid protein17
to direct protein degradation73
ODV envelope26
this study
Structure protein for oral infectivity61
ODV envelope26
required for oral infectivity47 this study
this study
protein40 viral pathogenicity factor41
baculovirus envelope fusion
OB envelope protein15
this study
OB envelope protein15
polyhedrin morphogenesis68 or cell-infected lysis75
function as Pif23,62,79
ODV-envelope protein6,58,79 and
unknown
mediate budded virus production42
essential for virus replication19
main structure protein63
Table 2. Proteins Identified in This Study and Their Homologues Previously Identified (boldface) in CuniNPV, AcMNPV, or HearNPVa
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a
GP41 Pr89
VLF-1
Pr92
DNA pol.
LEF-3
Pr96(Ac68)
88 89
90
92
93
95
96
2821
FGF-3
ME-53
114 116
118
120
33
16
31 F1,F2,F3 F2
57.89 17.27
13.6
15.33
20.27
60.84 23.71
20.62
21.41
3(3)
6(6)
3(3)
5(5)
3(3)
2(2)
4(6)
5(7)
3(3)
1(1)
2(4)
uni./tot.
no.
25.1
58.5
19.2
22.7
16.2
7.3
15.5
22.7
13.7
28.9
20.9
(%)
coverage
LTQ-Orbitrap
8
58
91
18
33
35
CuniNPV (ODV)
139 (BV)
32
54 (ODV/BV)
68
67 (ODV)
65 (ODV)
75
77 (ODV/BV)
80 (ODV) 78
83 (ODV)
AcMNPV (ODV/BV)
16
47
64
67
69
71
73 72
76
HearNPV (ODV)
Underlined numbers in the second column represented that these ORFs (proteins) were randomly chosen to make antiserum for immunoassays.
23
23
15,19,21
Pr111
Pr114 VP1054
111
F3
F3
F2
F1,F2,F3 F3
VP91 14 93
F3
Pr84
84
85
3,18,21 23
F2
coverage(%)
LCLTQ
fragment
Pr83
(score) pept.
83
(s)
product name
band
MALDI-TOF/TOF
orf
Table 2. Continued
and references
metabolism regulation80
accelerate host mortality13
this study structural protein for virion assembly48
this study
involved in transcription37 or virus production82
single-stranded DNA-binding protein21
key enzyme for DNA replification33
this study
Involvement in late gene expression44
Tegument main protein74 this study
capsid structure protein59
this study
this study
function description
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Figure 3. Western blotting analyses on purified ODV proteins of PrGV. The result disclosed molecular weighs of 4 proteins imcompatible with their corresponding predicted sizes: Pr21* (36 kDa), Pr22* (16 kDa), Pr2* (15.9 kDa) and Pr84* (11.4 kDa). The remain 7 proteins showed bands located in their predicted sizes positions, including Pr29 (21.6 kDa), Pr54 (26.4 kDa), Pr78 (30.0 kDa), Pr46 (40.3 kDa), Pr114 (36.9 kDa), Pr42 (93.5 kDa) and Pr35 (13.4 kDa). Four proteins (Pr78, Pr46, Pr42 and Pr2) had more than two immunoblot bands, 3 (Pr78, Pr46, Pr42) of which had another band below parallel predicted size, respectively, whereas Pr2 has 3 bands over the parallel predicted size. MW: molecular weight marker, vindicates the molecular weigh bigger than the predicted size, V denotes the molecular weigh smaller than predicted sizes. r denotes protein size compatible with its predicted one.
MS data by comparing among these three baculoviruses (Table 2). (i) Comparison of the results of this study and the other ODV proteomic analyses identified 9 proteins common to all baculovirus. These included VP39, GP41, P49, ODV-EC27, ODV-EC43, ODV-E56, P33, P74, and P6.9. Of these proteins, three proteins (VP39, GP41, and P49) were highly abundant structural proteins in baculovirus. VP39 is located in the nucleocapsid,51 and GP41 has been reported as the tegument protein of ODVs.50,74 P49 has been shown to be a baculovirus apoptotic suppressor34,86 but the exact function needs to be identified in Betabaculovirus. Two proteins (ODV-EC27 and ODV-EC43) are located in both the ODV nucleocapsid and envelope; ODV-EC27 participates in cell cycle regulation when a virus infects host midgut cells,2 whereas ODV-EC43 has been regarded as a structural component of ODV.17 ODV-E56 and P74, two core ODV envelope proteins, function as per os infectivity factors (See Discussion). P33 is needed for ODV and BV production45,77 and multiply enveloped ODV formation.77 P6.9 is a DNA-binding protein located in the nucleocapsid.76 (ii) Twenty-four homologues of PrGV ODV proteins were identified either in AcMNPV, CuniNPV, or HearNPV. These included granulin (homologue of polyhedrin), P78/83, ODV-E18, PEP1, PEP2, PIF-3, F-protein, ODV-E66a/b, viral ubiquitin, PIF2, PIF1,
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Pr69, Pr70 (BV/ODV-C42), 38K, ODV-E28, Helicase-1, VP91, VLF-1, DNA pol, LEF3, Pr96, VP1054 and ME-53. Of these proteins, four proteins (PIF1, PIF2, and ODV-E66a/b) were regarded as core ODV envelope proteins. PIF1 and PIF2 are essential for oral infectivity (see Discussion) and the exact function of ODV-E66 remains unclear though a highly hydrophobic region is identified in the N-terminus of the protein.26 Two proteins (helicase and DNA polymerase) are involved in DNA replication and located within the nucleocapsid of ODV.28,33,39 VLF-1 typically regulates very late gene expression and is also located within the nucleocapsid.44 VP1054, a type of capsid-associated protein, could promote capsid assembly and arrangement of VP39 monomers.48 VP91 is one of the main capsid structural proteins59 while Granulin is a main OB structural protein.63 Pr70 (BV/ODV-C42) has been proven to mediate the nuclear entry of P78/83.7,72 As a conserved ODV envelope protein, ODV-E18 has been shown to mediate budded virus production,42 whereas ODV-E28 has been characterized as a structural ODV-associated envelope protein.81 LEF3 also participates in DNA replication and (or) the very late expression of genes.24,28,39 PEP1 and PEP2 are a type of polyhedrin envelope proteins that are located on the surface of OBs and play an important role in polyhedrin formation.15,22 ME-53 is an early expression protein with zinc finger-like motifs at its C-terminus.71 An me53 knockout could block viral DNA replication, nucleocapsid formation, and consequently BV and ODV production.80 However, new data suggest that ME-53 does not affect DNA replication but is required for efficient BV production.12 Viral ubiquitin is a low molecular weight peptide (see Discussion).73 Other proteins, including P78/83, F-protein, PIF-3, 38K, Pr69 (Ac102), Pr96 (Ac68), and their functional descriptions are listed in Table 2. (iii) Fourteen ODV proteins (FGF-3, P10, P45/P48, Pr89, Pr92, Pr21(PEP/P10), Pr29, Pr35, Pr42, Pr54, Pr83, Pr84, Pr111, and Pr114) were newly identified in PrGV but their homologues failed to be identified in ODV/BVs of AcMNPV, CuniNPV, or HearNPV by MS studies.9,11,55 Of these 14 proteins, homologous genes of 5 proteins (FGF-3, P10, P45/P48, Pr89, and Pr92) existed, but those of the remaining 9 proteins were absent either in the AcMNPV, CuniNPV, or HearNPV genome by Protein BLAST search with default settings. Viral FGF, a secreted protein,30 may disseminate virus infection,13 enhance systemic virus propagation,31 stimulate cell motility and affect virus attachment.35 P10 features multiple functions in the virus infection process such as polyhedron morphogenesis68 as well as cellinfected lysis.75 P45/P48 is essential for virus replication in cell culture38 as well as BV production and ODV envelopment in the AcMNPV life cycle.84 Though the homologue of Pr21 was regarded as PEP protein,15,22 its precise function remains unclear. The homologue of Pr54 has been found in a number of betabaculoviruses such as Choc48 in Choristoneura occidentalis GV,15 but its function has not been characterized in Betabaculovirus. The other 9 proteins functions (including Pr89, Pr92, Pr29, Pr35, Pr42, Pr83, Pr84, Pr111 and Pr114) also need to be further investigated. Conserved Proteins and Betabaculovirus-specific Proteins Identified in This Study
Comparison of the genes encoding the 47 proteins identified in this study with baculovirus genomes deposited in the database showed homologous ORFs of 21 proteins were present in all baculovirus genomes, including genes encoding VP39, GP41, P49, ODV-EC27, ODV-EC43, ODV-E56, P74, P33, P6.9, PIF-1, 2822
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Journal of Proteome Research PIF-2, PIF-3, Helicase-1, DNA pol., VP1054, VLF-1, VP91, ODVE28, F-protein, 38K and Pr96 (Ac68). Homologous ORFs of 19 additional proteins25 exist in all or partial lepidopteran baculovirus genomes, including genes encoding granulin (homologue of polyhedrin), P78/83, ODV-E18, P10, Pr20 (PEP1), Pr21, Pr22 (PEP2), Pr29, ODV-E66 (a/b), viral ubiquitin, P45/P48 (Pr68), Pr69 (Ac102), Pr70 (BV/ODV-C40), Pr89 (Ac78) Pr92, (Ac75), LEF-3, FGF-3 (Ac32) and ME-53. Noticeably, among the 19 additional proteins, though no homologues of Pr21 and Pr29 were found in the two lepidopteran NPVs (AcMNPV and HearNPV) and the dipteran baculovirus (CuniNPV), their homologues were found in some other lepidopteran NPV genomes. Pr21 has homologues in Chrysodeixis chalcites NPV and Trichoplusia ni SNPV genomes, and Pr29 has homologues in Choristoneura fumiferana DEF MNPV and Epiphyas postvittana NPV genomes. Seven proteins are specific to Betabaculovirus ODV: Pr35, Pr42, Pr54, Pr83, Pr84, Pr111, and Pr114. Of the 7 proteins, homologous genes of Pr83 and Pr84 are found in all Betabaculovirus genomes while the homologue of Pr114 in 11 Betabaculovirus genomes deposited in NCBI up to date. Western Blotting Analysis
To confirm the results from the MS approaches by immunoblotting analysis, we randomly selected 11 ODV-encoding genes and expressed them in E. coli. All the fusion proteins were purified and used for polyclonal antibody preparation. Our results demonstrated that all the prepared polyclonal antibodies were capable of specifically recognizing PrGV-ODV proteins (Figure 3). Of the 11 proteins, immunoblotting signals against 7 proteins Pr29 (21.6 kDa), Pr35 (13.4 kDa), Pr42 (93.5 kDa), Pr46 (ODVEC43) (40 kDa), Pr54 (26.4 kDa), Pr78 (P33) (30 kDa), and Pr114 (37 kDa) occurred in the predicted location (size) deduced from their amino acid sequences. Whereas 4 proteins, Pr22 (PEP2) (16 kDa), Pr2 (P78/83) (15.9 kDa), Pr84 (11.4 kDa), and Pr21 (PEP/P10) (36 kDa), exhibited molecular weight discrepancies in comparison to their calculated sizes.
’ DISCUSSION Proteins Comparison Identified in Different Baculoviruses by Different MS Approaches
Mass spectrometry83 is a high-throughput technique and an extremely sensitive approach to identify proteins, protein composition, or other biomacromolecules in cells, tissue, or organisms. Each MS approach features its own sensitivity and applicability.83 Generally, MALDI-MS is typically used to analyze relatively simple peptide mixtures, whereas integrated liquid chromatography ESIMS systems (LCMS) are preferred for the analyses of complex samples.83 Previous studies have successfully applied these techniques to the analysis of baculovirus ODV-associated proteins. For instance, Braunagel et al.9 identified 44 proteins in AcMNPV ODV by different approaches (MUDPIT, MALDI-TOF, qTOF). Deng et al.11 identified 23 proteins in HearNPV ODV by MALDI-TOF MS, and Perera et al.55 also characterized 44 proteins in CuniNPV ODV through the Edman sequencing approach and two MS approaches (MALDI-TOF and GeLC-MS/MS). Recently, Wang et al.70 identified 34 proteins in the BV proteome of AcMNPV by three methods (LC-Qtrap, LC-Q-TOF, and MALDI-TOF). In the present study, forty seven proteins were identified in PrGV ODV as a result of diverse, high-sensitivity MS approaches as well as different sample determinations (single band, band region, and
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protein complex). However, some conserved ODV proteins in lepidopteran baculoviruses were not identified in this study but present in HearNPV or AcMNPV. For instance, ODV-E25 (Pr76) was identified in HearNPV (Hear25)11 and AcMNPV (Ac94),9 and Hear66 (homologue of Pr94) was also identified in HearNPV11 and Ac66 (homologue of Pr94) in AcMNPV9 by MS approaches, respectively. The failure to detect these proteins may possibly result from relative lower intensities or limited MS sensitivity. As an alternative explanation, relative higher abundances of other structural proteins might interfere with the signals of these proteins during the course of MS analysis.70 Newly-Identified ODV Proteins in This Study
Our MS analyses of PrGV revealed 14 newly identified ODV proteins. Of these proteins, four protein homologues (Pr21, Pr54, P45/48 (Pr68) and FGF3) failed to be previously identified as ODV components, although homologues of these four genes are present, and functionally studied by gene knockout or rescue experiments in some baculoviruses. Three proteins Pr83, Pr84 and Pr114 are highly conserved in betabaculoviruses sequenced to date, implying they may be essential for betabaculovirus ODV. Two proteins Pr42 and Pr114 were identified by all three MS approaches (Figure 2 and Table 2), implying high abundance in PrGV. Pr42, a putative protein composed of 803 amino acids, had one Pfam-A match (TrbI, Ftype; not a significant match) and no Pfam-B matches by Pham HMM search (Sanger Center). Additionally, it has no signal peptide by SignalP 3.0 prediction. Protein BLAST indicated that homologue of Pr42 only occurred in betabaculoviruses, Trichomonas vaginalis G3 (25% identity), Tetrahymena thermophila (31% identity) and Babesia bovis (25% identity). Interestingly, the Pr42 homologue in Trichomonas vaginalis G3 or T. thermophila was characterized as a viral A-type inclusion protein (or viral A-type inclusion protein repeat containing protein). Therefore, Pr42 may be an important structural protein in PrGV. Similarly, the Pr114 also had no signal peptide by SignalP 3.0 prediction and no Pfam-A matches or Pfam-B matches by Pham HMM search (Sanger Center). Protein BLAST indicated that Pr114 homologues shared identities within 11 betabaculoviruses and with the footand-mouth disease virus (FMDV, a kind of RNA virus). Notably, its homologue in FMDV is a polyprotein, which was processed into different sizes of VP proteins by virus-encoded proteases during synthesis.1 Therefore, we reasoned that Pr114 might participate in PrGV ODV structure formation. Additionally, the above information should also provide some clues to track the origin or evolution of Pr42 and Pr114. However, further investigations to explore the functions and locations as well as the protein interactions of the 14 newly- identified proteins in this study will be required to obtain precise and accurate information. Per os Infectivity Factors (PIF)
PIFs are highly conserved genes, nearly present in all baculovirus genomes sequenced to date. So far, at least 6 PIF proteins were characterized to involve in oral infectivity of baculovirus ODV in insect larvae. PIF-1 was first identified in Spodoptera littoralis NPV.32 Subsequently, PIF2,57 PIF3,47 PIF4,16 and P7460 have also been identified. Additionally, ODV-E56, a core ODV envelope protein,6,58 was also regarded as per os infectivity factor and named as PIF5,23,62,79 but is not essential for binding and fusion of ODV to the host midgut62 and for virus replication.79 Previous studies revealed that four PIFs (PIF1, PIF2, PIF3 and P74) are structural components of the envelope of HearNPV ODV, and that deletion of any of the above four pifs results in loss 2823
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Journal of Proteome Research of oral infectivity of OBs.61 Recently, new evidence further discloses that 3 PIFs proteins (PIF1, PIF2, and PIF3) form a stable complex on the surface of ODV particles of the AcMNPV and P74 is also associated with this complex. Deletion of any genes of PIF1, PIF2, or PIF3 affects the formation of the complex while deletion of the p74 gene fails to affect formation of the PIF1PIF2-PIF3 complex. So the four proteins (PIF1, PIF2, PIF3, and P74) are proposed to form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.53 In present study, all 6 PIF proteins were identified, including PIF1 (Pr61), PIF2 (Pr40), PIF3 (Pr30), PIF4 (ODV-E28, homologue of Ac96), PIF5 (ODV-E56) (Pr16), P74 (Pr51). Interesting, these 6 PIF proteins are also identified in CuniNPV55 and most of them in AcMNPV9 or HearNPV,11 which further confirmed the conservation of these proteins as well as validity and feasibility of MS approaches for identification of protein composition. Two Low-Molecular Weight Proteins Identified in This Study
In this study, we also identified two low-molecular weight proteins or peptides (P6.9 and viral ubiquitin) only by the LCLTQ approach with coverage of 15.5 and 40.0%, respectively. P6.9 (58 amino acid residues), a high-pI protein (theoretical pI 12.56), was rich in basic amino acids (Arg, 39.7%) and can bind DNA to form a condensation state.76 A homologue of P6.9 was also identified in CuniNPV by Gel-MS/MS55 (coverage 12.31%), in AcMNPV by MUDPIT-MS/MS9 (coverage 35.0%), and in HearNPV by MALDI-TOF (coverage 35.0%).11 Additionally, P6.9 was also identified in AcMNPV budded virions by MALDITOF MS analysis.70 Ubiquitin (or ubiquitin-like protein) is widely present in eukaryotes, prokaryote cells, or viruses.20,73 Ubiquitin can covalently attach to cellular proteins to signal their subsequent degradation and, thereby, change the stability, localization, or activity of the target protein. Ultimately, this activity results in the degradation of the target protein.73 Protein BLAST analyses indicated that viral ubiquitin shared high identities with its homologues in 12 betabaculoviruses but only in several alphabaculoviruses genomes deposited in the NCBI database to date, such as Apocheima cinerarium NPV (ORF99), AcMNPV and HearNPV. However, the homologue of viral ubiquitin was not identified in ODV of AcMNPV or HearNPV9,11 but was identified in AcMNPV-BV.70
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granulin, ODV-E66) (Figure 2 and Table 2). Previous studies have also reflected these phenomena.11,55,70 One possible explanation is that these proteins could interact with themselves or other protein(s) to form chimeric fusion proteins such as homo(hetero-) dimers or multimers,8,53,54 or undergo different forms (or degrees) of modification (such as phosphorylation or glycosylation). However, another credible explanation is that these proteins have been subjected to partial degradation or contamination during sample processing and SDS-PAGE electrophoresis.46,70
’ CONCLUSION PrGV is the first Betabaculovirus whose ODV proteins were revealed in the family Baculoviridae. This study identified 47 proteins, including 14 newly identified ODV proteins in PrGV by three MS approaches, and the data derived from these MS approaches were further validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV proteins. Comparison analyses revealed the similarities and differences in the ODV compositions of Betabaculovirus, Alphabaculovirus and Deltabaculovirs. These results expand our understanding of the baculovirus virion structure, ODV proteins complexity as well as their interaction with hosts, which will now be of great interest as a result of this study and their potential impact on related field.
’ AUTHOR INFORMATION Corresponding Author
*C.-X. Zhang, Tel.: þ86 571 88982991. Fax: þ86 571 88982991. E-mail:
[email protected]. Author Contributions †
These authors contributed equally to this work.
’ ACKNOWLEDGMENT The research was supported by the grants from the National Basic Research Program of China (No. 2010CB126205 and 2010CB126102) and the National Natural Science Foundation of China (31070136 and 30570074).
Immunoassays and SDS-PAGE Analyses
Immunoassays confirmed the presence of 11 proteins in PrGV and also confirmed the validity of the MS identifications in this study. Not unexpectedly, four proteins exhibited different degrees of disagreement with their predicted sizes or represented more than two immunoblot bands (Figure 3). These phenomena have also been observed in AcMNPV and HearNPV analyses, that is, antiserum to ODV-E18 recognized bands of 18 and 35 kDa, while antiserum to ODV-EC27 recognized bands of 27 and 35 kDa on Western blots of proteins from purified ODV.8 Additional investigation observed that rabbit polyclonal antiserum against a GST-HA2 fusion protein could react with three proteins of 50, 46, and 35 kDa at HzAM1 cells-infected.46 Notably, it has been shown by others using an HA tagged ORF, that this antiserum was likely defective and was not recognizing the correct ORF band.66 Therefore, further study should be needed to disclose exact blotting band in immunoassays analysis. In this study, some proteins migrated to different positions than their predicted molecular masses and occurred in many bands or in different fragments in SDS-PAGE gels (such as
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