• No results found

Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100

N/A
N/A
Protected

Academic year: 2019

Share "Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100"

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

0022-538X/07/$08.00

0

doi:10.1128/JVI.00632-07

Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Proteomics Analysis of

Helicoverpa armigera

Single Nucleocapsid

Nucleopolyhedrovirus Identified Two New Occlusion-Derived

Virus-Associated Proteins, HA44 and HA100

Fei Deng,

1

† Ranran Wang,

1

† Minggang Fang,

1

‡ Yue Jiang,

1

Xushi Xu,

1

Hanzhong Wang,

1

Xinwen Chen,

1

Basil M. Arif,

2

Lin Guo,

3

Hualin Wang,

1

and Zhihong Hu

1

*

State Key Laboratory of Virology and Joint Laboratory of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of

Sciences, Wuhan 430071, People’s Republic of China

1

; Laboratory for Molecular Virology, Great Lakes Forestry Centre,

Sault Ste. Marie, Ontario, Canada

2

; and State Key Laboratory of Virology, Wuhan University and College of

Life Sciences, Wuhan 430072, People’s Republic of China

3

Received 24 March 2007/Accepted 14 June 2007

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the

structural proteins of the occlusion-derived virus (ODV) of

Helicoverpa armigera

single nucleocapsid

nucleo-polyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were

identi-fied, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other

baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66

(AC66), DNA polymerase, GP41, VP39, P33, E25, helicase, P6.9, ODV/BV-C42, VP80, EC43,

ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (

ha44

) and ORF100 (

ha100

) were discovered as

ODV-associated proteins for the first time.

ha44

encodes a protein of 378 aa with a predicted mass of 42.8 kDa.

ha100

encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for

poly(ADP-ribose) glycohydrolase (

parg

). Western blot analysis and immunoelectron microscopy confirmed that

HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope

of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs,

while HA100 is conserved only in group II NPVs.

The

Baculoviridae

, a diverse family of more than 600 viruses,

encompasses two genera, the nucleopolyhedroviruses (NPVs)

and the granuloviruses (GVs) (5). Baculoviruses are generally

host specific, infecting mainly insects of the orders

Lepidoptera

,

Hymenoptera

, and

Diptera

. Two progeny phenotypes are

pro-duced in the replication cycle, the budded virus (BV) and the

occlusion-derived virus (ODV). In larvae, ODVs initiate

pri-mary infections in midgut epithelial cells of susceptible hosts

and BVs spread the virus from cell to cell in the larvae (5, 30,

62). The two phenotypes are genotypically identical, but each

has characteristic structural components to accommodate their

respective functions (7, 50). Based on phylogeny, lepidopteran

NPVs are divided into group I and group II (23, 24, 70). It is

known now that the BVs of group I and group II NPVs use

different fusion proteins to enter host cells. GP64 is the

mem-brane fusion protein of group I NPVs (4, 40), while the F

protein is that of group II NPVs (27, 36, 44).

Identification of ODV structural proteins and comparisons

in different NPVs are fundamental to the functional

investiga-tion of virulence and host specificity. So far, 30 genome

se-quences of baculoviruses have been reported, including 8

group I NPVs, 12 group II NPVs, 7 GVs, 1 dipteran NPV, and

2 hymenopteran NPVs. The availability of the genome

se-quences has facilitated proteomic analysis of baculoviruses. In

2003, proteomic investigations revealed 44 proteins to be ODV

components of

Autographa californica

multiple

nucleopolyhe-drosis virus (AcMNPV), a group I NPV (11). Recent

investi-gations of a dipteran NPV,

Culex nigripalpus

NPV (CuniNPV),

identified 44 ODV-associated proteins (46). By comparison,

little is known about the structural proteins of ODVs from

group II NPVs.

The

Helicoverpa armigera

single nucleocapsid NPV (HearNPV,

also called HaSNPV) was first isolated in 1975 in the Hubei

Province of the People’s Republic of China and has been used

extensively over 25 years in China to control

H

.

armigera

in cotton

(71). Phylogenetic analysis indicated that HearNPV belongs to

the group II NPVs (12, 29). Its DNA genome is 131 kb and

contains 135 open reading frames (ORFs) that potentially encode

proteins of 50 amino acids (aa) or larger (13). Several HearNPV

genes, such as the polyhedrin gene (

polh

) (14), the ecdysteroid

UDP-glucosyltransferase gene (

egt

) (15), the late expression

fac-tor 2 gene (

lef

-

2

) (12), the basic DNA-binding protein gene (

p6

.

9

)

(61),

ha122

(37),

Ha94

(19),

chitinase

(60),

fp25K

(67),

p10

(18),

and the F-protein gene (

ha133

) (36), have been characterized.

In this report, we describe using sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass

spectrometry-based protein analysis techniques to study

struc-tural proteins of the ODV of HearNPV. HearNPV was chosen

to serve as a representative of the group II NPVs. ODV

proteins were separated by SDS-PAGE and analyzed by

pep-tide mass fingerprinting techniques by using matrix-assisted

laser desorption ionization–time of flight mass spectrometry

* Corresponding author. Mailing address: Wuhan Institute of

Virol-ogy, Chinese Academy of Sciences, Wuhan 430071, People’s Republic

of China. Phone and fax: 86-27-87197180. E-mail: huzh@wh.iov.cn.

† F.D. and R.W. contributed equally to this work.

‡ Present address: Department of Agroecology, University of British

Columbia, Vancouver, British Columbia, Canada V6T 1Z4.

Published ahead of print on 20 June 2007.

9377

on November 8, 2019 by guest

http://jvi.asm.org/

(2)

(MALDI-TOF MS). The resulting mass spectra were searched

against the NCBI database and the theoretical ORF database

of HearNPV. A total of 23 proteins were identified as

ODV-associated proteins. Of these 23 proteins, 21 were previously

reported as ODV-associated proteins in other baculoviruses

but 2 were hitherto unknown as ODV-associated proteins.

These two newly identified proteins, encoded by

ha44

and

ha100

, respectively, were further shown to be structural

com-ponents of the ODV by Western blot analysis and

immuno-electron microscopy (IEM).

MATERIALS AND METHODS

Insects, cells, and virus.A culture ofH.armigerainsects was maintained as described by Sun et al. (55). An in vivo-cloned strain of HearNPV

(HearNPV-G4) (13, 55) was used as the wild-type virus and propagated inH.armigera. Cells

of theHeliothis zeaHzAM1 line (39) were used for producing BV of HearNPV.

Purification of HearNPV BV and ODV.BV was purified from the cell culture supernatant of infected HzAM1 cells (72 h postinfection) as described by Brau-nagel and Summers (7). Larvae were homogenized in 0.1% SDS, followed by a few rounds of differential and rate zonal centrifugation in sucrose gradients. All solutions were supplemented with 0.1% SDS (56). Protease inactivation of the

purified occlusion bodies was performed by HgCl2and hot water treatment (54).

ODVs were released by alkaline treatment (pH 10.9) (7) and purified on con-tinuous sucrose gradients. Purified BV and ODV were further fractionated into envelope and nucleocapsid components (28).

Protein separation, reduction, alkylation, and digestion.Proteins from puri-fied HearNPV ODV were separated by 12% SDS-PAGE and stained with a colloidal blue staining kit (Invitrogen). Protein bands were excised from the one-dimensional polyacrylamide electrophoresis gel and destained by washing

with a mixture of 200 mM NH4HCO3–acetonitrile (1:1). Proteins were reduced

with dithiothreitol, alkylated with iodoacetamide, and digested in gel with trypsin (Promega, Madison, WI) as previously described (53). The peptide mixtures obtained were further desalted by ZipTipC18 (Millipore) and eluted in 50% acetonitrile–0.1% trifluoroacetic acid buffer before MS analysis.

MALDI-TOF MS.A saturated solution of␣-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile was used as the matrix. The sample and the matrix (1:1, vol/vol) were spotted onto a target plate. MALDI-TOF spectra of the peptides were obtained with a Voyager DE STR MALDI-TOF work station mass spectrometer (Applied Biosystems Inc.). The analysis was performed in posi-tive-ion reflector mode with an accelerating voltage of 20 kV and a delayed extrac-tion of 150 ns. Typically, 200 scans were averaged. Data mining was performed with MS-Fit software (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) and Mascot soft-ware (http://www.matrixscience.com/search_form_select.html) against the NCBI da-tabase and the theoretical ORF dada-tabase of HearNPV.

Sequence analysis ofha44andha100.The sequence data were compiled and analyzed with DNASTAR software. Homologues in the GenBank and EMBL databanks were explored with the PSI-BLAST search tool (1). Amino acid sequence alignment was performed with Clustal X and T coffee software (42, 58). GeneDoc software (version 1.1.1004) was used for similarity shading and scoring of alignment. MEGA3.1 (33) was used for generating the phylogenetic trees by the neighbor-joining method, with bootstrap replications. A phylogenetic tree was visualized with the Treeview program.

Preparation of antibodies against HA44 and HA100.The entireha44coding

region and a truncated fragment of theha100gene were amplified with

synthe-sized primers Ha44a/Ha44b (Ha44a, 5⬘-GAATTCATGAGCAATCCCAGCAA

ACAATC-3⬘; Ha44b, 5⬘-GAATTCTCAATAGCGCAAACGAGTTTCG-3⬘) and

Ha100f/Ha100r (Ha100f, 5⬘GCCGGATCCATGACTTTGTCGCGTTTAGATT

GCG-3⬘; Ha100r, 5⬘-GGCTCTAGATTAATAAACCATATTGTAATCGGCAA

C-3⬘), respectively (the sequences in italics are restriction enzyme digestion sites.

The PCR product ofha44was first cloned into pGEM-T-Easy (Promega) and

then into the expression vector pET28a (Novagen) in whichha44was fused in

frame with a six-His tag at the C terminus. The PCR product ofha100was first

cloned into pGEM-T-Easy (Promega) and then into the expression vector

pGEX-KG (22) in whichha100was fused in frame with the gene for glutathione

S-transferase at the C terminus. HA44 expressed inEscherichia coliwas purified

with Ni-nitrilotriacetic acid agarose (QIAGEN), and HA100 was purified by glutathione-agarose beads (Sigma). The purified proteins were used to generate specific antibodies against HA44 and HA100.

Purified HA44 and HA100 (200␮g) were used to immunize rabbits.

Preim-mune sera were withdrawn prior to inoculation. After 3 weeks, the rabbits

received a booster with the same amount of the antigens. Two weeks later, the

antisera were collected and stored at⫺80°C until use. The specificities of the

antisera were tested by Western blot analysis.

Western blot analysis.Purified BVs and ODVs, as well as their nucleocapsid and envelope fractions, were separated by 12% SDS-PAGE and transferred onto Hybond-N membranes (Amersham) by semidry electrophoresis transfer (2). HA44- and HA100-specific antisera and alkaline phosphatase-conjugated immu-noglobulin G (SABC, China) were used as the primary and secondary antibodies,

respectively. The signal was detected with a 5-bromo-4-chloro-3-indolyl-␤-D

-galactopyranoside (BCIP)–nitroblue tetrazolium kit (SABC, China). Polyclonal anti-VP80, anti-ODV-E56, and anti-HaF1 antibodies were used as controls for nucleocapsid-, ODV envelope-, and BV envelope-specific proteins, respectively.

IEM.Purified ODVs were added to carbon-coated nickel grids (150 mesh) and

[image:2.585.310.530.71.527.2]

blocked with 5% bovine serum albumin. The primary antibodies were 1:100 dilutions of anti-HA44 and anti-HA100 antisera. Preimmune sera were used as

FIG. 1. SDS-PAGE profile and MS results of purified HearNPV

ODV. ODV proteins were separated by 12% SDS-PAGE and stained

with colloidal blue. The ODV bands (numbered in the middle) were

subjected to MALDI-TOF MS, and their identities are listed on the right.

9378

DENG ET AL.

J. V

IROL

.

on November 8, 2019 by guest

http://jvi.asm.org/

(3)

Band Size (kDa) from SDS-PAGE

HearNPV ORF

AcMNPV

ORF Protein

Predicted size (kDa)

Sequence

coverage (%) Function Reference(s)

1 110 ha73 ac80 GP41 36.6 43 Tegument main protein 63, 64

2 103 ha73 ac80 GP41 36.6 28 Tegument main protein 63, 64

3 100 ha66 ac66 HA66 88.9 37 ODV-associated protein 11

4 83 ha92 ac104 VP80 69.7 37 Nucleocapsid 38, 41

5 78 ha20 ac138 P74 78.4 25 Oral infectivity 21, 34, 68

6 73 ha73 ac80 GP41 36.6 58 Tegument main protein 63, 64

7 65 ha96 ac46 ODV-E66 76.1 35 ODV envelope 26

8 60 ha100 HA100 58.1 26 Nucleocapsid- and ODV

envelope-associated protein

This study

9 58 ha1 ac8 Polyhedrin 28.8 40 Polyhedron main protein 49

ha96 ac46 ODV-E66 76.1 12 ODV envelope 26

10 56 ha111 ac119 PIF-1 60.3 19 Oral infectivity 31

ha1 ac8 Polyhedrin 28.8 40 Polyhedron main protein 49

11 54 ha2 ac9 P78/83 45.9 49 Nucleocapsid 47, 52

12 50 ha96 ac46 ODV-E66 76.1 25 ODV envelope 26

13 48 ha9 ac142 P49 55.3 36 ODV-associated protein 11

14 45 ha44 Ha44 42.8 29 Nucleocapsid-associated protein This study

15 44 ha44 Ha44 42.8 32 Nucleocapsid-associated protein This study

ha89 ac101 C42 42.6 20 Nucleocapsid 10

16 42 ha96 ac46 ODV-E66 76.1 15 ODV envelope 26

ha89 ac101 C42 42.6 18 Nucleocapsid 10

17 39 ha94 ac109 ODV-EC43 41.5 45 ODV envelope and nucleocapsid 19

18 38 ha94 ac109 ODV-EC43 41.5 51 ODV envelope and nucleocapsid 19

ha84 ac95 Helicase 146 10 DNA replication essential 11, 32

19 36 ha15 ac148 ODV-E56 38.9 24 ODV envelope 8

20 35 ha9 ac142 p49 55.3 29 ODV-associated protein 11

ha78 ac89 VP39 33.4 35 Nucleocapsid 45

21 34 ha73 ac80 GP41 36.6 58 Tegument main protein 63, 64

22 33 ha1 ac8 Polyhedrin 28.8 38 Polyhedron main protein 49

23 32 ha78 ac89 VP39 33.4 50 Nucleocapsid 45

ha11 ac144 ODV-EC27 33.3 32 ODV envelope and nucleocapsid 3, 9

24 30 ha78 ac89 VP39 33.4 42 Nucleocapsid 45

ha1 ac8 Polyhedrin 28.8 34 Polyhedron main protein 49

25 29 ha78 ac89 VP39 33.4 57 Nucleocapsid 45

26 28 ha82 ac94 ODV-E25 25.9 41 ODV envelope 51

27 27 ha78 ac89 VP39 33.4 56 Nucleocapsid 45

ha80 ac92 P33 30.8 34 Stimulation of P53-induced apoptosis 48

28 26 ha80 ac92 P33 30.8 44 Stimulation of P53-induced apoptosis 48

29 25 ha78 ac89 VP39 33.4 45 Nucleocapsid 45

ha15 ac148 ODV-E56 38.9 24 ODV envelope 8

31 23.5 ha80 ac92 P33 30.8 32 Stimulation of P53-induced apoptosis 48

32 22 ha1 ac8 Polyhedrin 28.8 28 Polyhedron main protein 49

ha67 ac65 DNA polymerase 119.3 11 DNA replication essential 11, 32

34 20 ha1 ac8 Polyhedrin 28.8 28 Polyhedron main protein 49

35 19 ha82 ac94 ODV-E25 25.9 45 ODV envelope 51

36 18.5 ha78 ac89 VP39 33.4 40 Nucleocapsid 45

ha9 ac142 P49 55.3 18 ODV-associated protein 11

37 18 ha88 ac100 P6.9 11.5 35 DNA binding protein 65, 66

38 15 ha10 ac143 ODV-E18 8.8 45 ODV envelope 9

39 14 ha10 ac143 ODV-E18 8.8 45 ODV envelope 9

41 12 ha82 ac94 ODV-E25 25.9 40 ODV envelope 51

ha65 ac67 LEF-3 44 20 DNA replication essential 11, 32

a

The order of the bands was the same as that in Fig. 1. MALDI-TOF MS was repeated once.

9379

on November 8, 2019 by guest

http://jvi.asm.org/

[image:3.585.45.537.63.712.2]
(4)

the negative controls. Twelve-nanometer Colloidal Gold-AffiniPure goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch) was used as the secondary antibody for hybridization. The grids were then negatively stained with 2% sodium phosphotungstate and examined with a transmission electron microscope (H-7000 FA; Hitachi).

RESULTS

MS identification of HearNPV ODV proteins.

HearNPV

ODVs were purified, and the proteins were separated by 12%

SDS-PAGE. More than 40 bands ranging from 11 to 110 kDa

were made visible by colloidal blue staining (Fig. 1). Forty-one

bands were excised from the gel, reduced, alkylated, and

di-gested with trypsin, and the peptides were analyzed by

[image:4.585.42.546.66.489.2]

MALDI-TOF MS. Peak lists of tryptic peptide masses were

generated and subjected to an NCBI database and HearNPV

ORF database search with MS-Fit and the Mascot search

en-gine. The SDS-PAGE and MALDI-TOF MS analyses were

performed twice. Reliable gene DNA matches from the

theo-retical HearNPV ORFs were obtained and are summarized in

Table 1. Twenty-three ORFs were identified, including

ha1

(

polh

),

ha2

(

p78

/

83

),

ha9

(

p49

),

ha10

(

odv

-

e18

),

ha11

(

odv

-ec27

),

ha15

(

odv

-

e56

),

ha20

(

p74

),

ha44

,

ha65

(

lef

-

3

),

ha66

(

Ac66

),

ha67

(

dna

-

pol

),

ha73

(

gp41

),

ha78

(

vp39

),

ha80

(

p33

),

ha82

(

odv

-

e25

),

ha84

(

helicase

),

ha88

(

p6

.

9

),

ha89

(

odv

/

bv

-C42

),

ha92

(

vp80

),

ha94

(

odv

-

EC43

),

ha96

(

odv

-

e66

),

ha100

,

and

ha111

(

pif

-

1

).

FIG. 2. Alignment of the amino acid sequences of HA44 and its homologues among the group II NPVs. Three shading levels were set, black

for 100% identity, dark gray for 80% identity, and light gray for 60% identity. The NCBI accession numbers are NP_818692 for AdhoNPV45,

YP_529786 for ORF116 of

Agrotis segetum

NPV (AgseNPV116), YP_249646 for ORF42 of

Chrysodeixis chalcites

NPV (ChchNPV42), NP_075113

for HA44, NP_542668 for ORF45 of HzSNPV45, NP_047691 for ORF55 of

Lymantria dispar

NPV (LdMNPV55), NP_613219 for ORF136 of

Mamestra configurata

NPV A (MacoA136), NP_689309 for ORF135 of

Mamestra configurata

NPV B (MacoB135), NP_037867 for ORF107 of

Spodoptera exigua

NPV (SeMNPV107), NP_258314 for ORF44 of

Spodoptera litura

NPV (SpliNPV44), and YP_308929 for ORF39 of

Trichoplusia

ni

NPV (TnSNPV39).

9380

DENG ET AL.

J. V

IROL

.

on November 8, 2019 by guest

http://jvi.asm.org/

(5)

Of these 23 proteins, VP39 (57), P78/83 (59), VP80 (38, 41),

and ODV/BV-C42 (10) have been reported previously as

nu-cleocapsid proteins of both BV and ODV. P6.9 is the main

basic DNA-binding protein located in the nucleocapsid (65,

66). GP41 is defined as the tegument protein of ODVs (63, 64).

ODV-E18 (9), ODV-E25 (51), ODV-E56 (8), and ODV-E66

(26), as well as oral infectivity-related proteins P74 (21, 34, 68)

and PIF-1 (31), were reported to be the ODV envelope

pro-teins. ODV-EC27 (5, 9) and ODV-EC43 (19) were reported as

structural proteins of the ODV nucleocapsid and envelope.

P33 (48), P49, AC66, helicase, LEF-3, DNA polymerase, and

polyhedrin were reported as ODV-associated proteins (11).

Two proteins, HA44 and HA100, have not been reported

be-fore and therebe-fore are being described in more detail here.

Peptide mass fingerprinting data interpretation by the

MS-Fit program revealed that 11 experimentally derived tryptic

peptide masses were found to match the predicted peptide

masses of the HA44 protein (error,

100 ppm), covering 29%

of its amino acid sequence. For HA100, 10 experimentally

derived peptide masses were found to match the predicted

peptide masses of the HA100 protein (error,

100 ppm),

cov-ering 26% of its amino acid sequence. By using Mascot

soft-ware for database searches (25), high Mascot score were

re-vealed when matched with HA44 and HA100, respectively

(

67).

Sequence and phylogeny analysis of

ha44

and

ha100

.

Se-quence analysis indicated that

ha44

contains 1,134 nucleotides

(nt) and potentially encodes a protein of 378 aa with a

pre-dicted molecular mass of 42.8 kDa. A baculovirus late

tran-scription motif, TAAG, was found 76 nt upstream of the initial

ATG of

ha44

, suggesting that it is a late gene. No

polyadenyl-ation signal was found within 500 nt downstream of the stop

codon.

Searches of databases with all of the available genomes of

baculoviruses showed that homologues of HA44 were found in all

group II NPVs and GVs but not in group I NPVs or in the

dipteran and hymenopteran NPVs. The size of the HA44

homo-logue varies, ranging from the 255 aa of ORF45 of

Adoxophyes

honmai

NPV (AdhoNPV45) to the 422 aa of ORF46 of

Spodopt-era litura

NPV (SpliNPV46), although most of the homologues

have a size of 311 to 378 aa. Pairwise comparisons revealed that

three proteins were very similar to their counterparts. The amino

acid identities were 98%, 83%, and 75% for HA44/HzSNPV45,

ChchNPV42/TnSNPV39, and MacoA136/MacoB135,

respec-tively (for definitions of abbreviations, see the legend to Fig. 2). In

contrast, the amino acid identity for the rest of the pairwise results

was lower than 50%. The alignment of HA44 homologues from

group II NPVs is presented in Fig. 2. The protein is mostly

conserved at the C terminus (Fig. 2). The N-terminal sequence of

HA44 is rich in basic residues (K/R) and serine, and this is a

common feature of most of the HA44 homologues. The

isoelec-tric point (pI) of the N-terminal 64 aa of HA44 is 10.79. Only 12

aa were absolutely conserved in the alignment, which included

N236, N264, V265, Y267, F281, N283, L322, N327, L333, K340,

T342, and V369 (Fig. 2). These amino acids might be important

in the function of HA44. Phylogenetic analysis indicated that the

HA44 homologues have a common ancestor and then diverged

into the cluster of group II NPVs and that of GVs (Fig. 3).

[image:5.585.132.461.72.300.2]

The

Ha100

ORF is 1,530 nt and encodes a protein of 510 aa

with a predicted molecular mass of 58 kDa. No consensus early

transcription initiation motifs were found upstream of the

ini-tial ATG, but a TAAG motif was found at

34 nt, suggesting

FIG. 3. Neighbor-joining tree derived from HA44 and its homologues among NPVs and GVs. Bootstrap values (1,000 replicates, nodes

supported with more than 50%) are on the branch lines. The accession numbers for NPVs are as described in the legend to Fig. 3. The additional

ones are NP_872567 for ORF113 of

Adoxophyes orana

GV (AdorGV113), YP_006220 for ORF124 of

Agrotis segetum

GV (AgseGV124),

NP_891969 for ORF122 of

Cryptophlebia leucotreta

GV (CrleGV122), NP_148919 for ORF135 of

Cydia pomonella

GV (CpGV135), NP_663288

for ORF123 of

Phthorimaea operculella

GV (PhopGV123), NP_068332 for ORF113 of

Plutella xylostella

GV (PlxyGV113), and NP_059320 for

ORF172 of

Xestia c

-

nigrum

GV (XecnGV172).

on November 8, 2019 by guest

http://jvi.asm.org/

(6)

that

ha100

may also be a late gene. A polyadenylation signal

(AATAAA) was found at 22 to 27 nt downstream of the stop

codon.

HA100 has homology to poly(ADP-ribose) glycohydrolase

(PARG), a ubiquitously expressed exo- and

endoglycohydro-lase in eukaryotic cells. PARG mediates oxidative and

excito-toxic neuronal death and is involved in the breakdown and

recruitment of polyribose for nuclear functions such as DNA

replication and repair (17, 69). The vertebrate PARGs contain

four domains, A, B, C, and D (43). Domain A is a putative

regulatory domain, while B, C, and D form catalytic fragments.

Homologues of HA100 are conserved in all of the group II

NPVs sequenced so far. A comparison of PARGs from a range

of organisms and from group II NPVs is shown in Fig. 4.

Similar to the PARG of

Drosophila melanogaster

, the

PARG-like proteins of group II NPVs contain a catalytic fragment but

lack the putative regulatory A domain (Fig. 4). Alignment of

HA100 homologues from baculoviruses and PARGs from

se-lected eukaryotes reveals that although the sequence similarity

is not high, there were 7 aa absolutely conserved, including

F662, K676, Y683, G745, E756, P764, and E765, with respect

to the bovine PARG sequence (data not shown). All of the

conserved amino acids are located in the conserved catalytic

domain, which spans residues 610 to 795 in bovine PARG (43).

Localization of HA44 and HA100 in viral structures.

Anti-HA44 and anti-HA100 antisera were generated as described in

Materials and Methods and were used in Western blot analysis

and IEM. Western blot analyses were performed to identify

the localization of HA44 and HA100 in BV and ODV (Fig. 5).

The results showed that HA44 is located in the nucleocapsid

but not in the envelope of ODV and BV. HA100 was detected

in the nucleocapsid and envelope of ODV, as well as in the

nucleocapsid of BV (Fig. 5). The sizes of HA44 and HA100

were 44 kDa and 60 kDa, respectively, which are in agreement

with the predicted sizes deduced from their nucleotide

se-quences.

The IEM results showed that HA44 was located in the

nu-cleocapsid of ODV but not detected in the envelopes of the

intact ODV (Fig. 6A). HA100 was detected in intact ODVs, as

well as in the nucleocapsids of ODV (Fig. 6B). The IEM

results confirmed that HA44 is a nucleocapsid protein of

HearNPV ODV, while HA100 is a structural protein of both

the nucleocapsid and the envelope of the ODV.

DISCUSSION

In this study, we identified 23 HearNPV genes that encode

ODV structural proteins by SDS-PAGE and MS methods.

This is the first such report for a group II NPV.

[image:6.585.136.444.67.244.2]

Braunagel et al. (11) were able to identify 44

ODV-associ-ated proteins of AcMNPV by using multiple techniques,

in-cluding MALDI-TOF, multidimensional protein identification

technology-tandem MS, library exploring, and Western

blot-ting. Perera et al. (46) identified 44 polypeptides in CuniNPV

ODV by MALDI-TOF and gel electrophoresis-liquid

chroma-tography-tandem MS. Comparison of the ODV-associated

proteins of AcMNPV, CuniNPV, and HearNPV shows that

nine proteins are shared by these viruses and are also

con-served in the baculoviruses sequenced so far. AcMNPV and

CuniNPV ODVs shared another five conserved baculovirus

proteins, i.e., PIF2, F protein, VP1054, VLF-1, and VP91,

which were not detected in HearNPV by MS. However, PIF2

was identified as a HearNPV ODV structural protein by

West-ern blot analysis (20). Therefore, at least 10 conserved

bacu-lovirus proteins are shared by ODVs of AcMNPV, CuniNPV,

and HearNPV, including P49, ODV-EC27, ODV-E56, P74,

GP41, VP39, P33, P6.9, ODV-EC43, and PIF-2. Another

pro-tein conserved in baculoviruses, PIF1, was identified as an

FIG. 4. Comparison of PARGs from a wide range of organisms and from group II NPVs. A, putative regulatory domain; B, C, and D, catalytic

fragments; C, PARG catalytic domain. Percent conservation is indicated in each block with respect to the bovine PARG. The amino acid positions

of bovine PARG domains and the lengths of PARGs are indicated. The accession numbers of PARGs are NP_776563 for

Bos taurus

, AAH52966

for

Homo sapiens

, NP_036090 for

Mus musculus

, NP_112629 for

Rattus norvegicus

, NP_477321 for

Drosophila melanogaster

, NP_501508 for

Caenorhabditis elegans

, NP_075169 for HA100, NP_818756 for AdhoNPV, YP_529728 for AgseNPV, YP_249712 for ChchNPV, NP_542726 for

HzSNPV, NP_047778 for LdMNPV, NP_613153 for MacoNPV A, NP_689244 for MacoNPV B, NP_037812 for SeMNPV, NP_258370

for SpliNPV, and YP_308992 for TnSNPV.

9382

DENG ET AL.

J. V

IROL

.

on November 8, 2019 by guest

http://jvi.asm.org/

(7)

ODV component in HearNPV in our study and was also

re-ported as an ODV-associated protein in CuniNPV (46) but

was not identified by multiple approaches in AcMNPV (13).

DNA polymerase and helicase are conserved baculovirus

pro-teins shared by the ODVs of both AcMNPV and HearNPV,

but they were not detected in the CuniNPV ODV (46). The

identification of common structural proteins is essential to

elucidate the core structure of baculoviruses. Of the 10

con-served proteins, ODV-EC27, ODV-E56, GP41, VP39, P6.9,

and ODV-EC43 are known to be structural proteins. It is

interesting that P74 and PIF-2, which are essential for oral

infection, are also associated with the ODV. With more data

derived from different viruses becoming available, the

impor-tance and functions of these proteins can be further revealed.

In this study, approximately 41 ODV protein bands

sepa-rated by SDS-PAGE were subjected to MALDI-TOF MS

anal-ysis; 38 bands had matches to viral ORFs, while 3 bands did not

produce significant matches and were not identified by this

technique (Fig. 1 and Table 1). The finding of unmatched

bands suggested that additional host proteins may be present.

In vaccinia virus, MS techniques revealed 23 virion-associated

host proteins in addition to the 75 viral proteins (16). Our

HearNPV ODV data have not matched any host proteins; this

may be due to the lack of genetic information about or a

database for

H

.

armigera

. Some proteins of HearNPV ODV

were not identified, possibly because of their low molar content

in ODVs and/or resistance to staining or because some

pro-teins may not be amenable to MALDI-TOF MS. For example,

HA122 and PIF-2 have already been identified and located in

the HearNPV ODV (37, 20) but we were not able to detect

them in this study.

The degradation or losses of proteins during virus

purifica-tion could also affect protein detecpurifica-tion. Although HgCl

2

[image:7.585.122.462.68.275.2]

treat-ment, heat inactivation of proteases, and a protease inhibitor

cocktail were used during the purification of virions, multiple

[image:7.585.44.286.354.679.2]

FIG. 6. IEM of HA44 and HA100 and localization in HearNPV

ODVs and nucleocapsids (NC) of ODVs. A 1:100 dilution of

anti-HA44 or anti-HA100 antiserum was used as the primary antibody.

Preimmune serum was used as a negative control. A, IEM of HA44; B,

IEM of HA100. Bars, 100 nm.

FIG. 5. Western blot analysis of the HearNPV ODV/BV nucleocapsid (NC) and envelope (E) fractions with anti-HA44 and anti-HA100

antibodies. Healthy HzAM1 cells (H) and virus-infected cells (I) were loaded as negative and positive controls. VP80, ODV-E56, and the F protein

were detected by their specific antibodies for illustrating the NC-specific protein, ODV E-specific protein, and BV E-specific protein, respectively.

M, molecular size markers.

on November 8, 2019 by guest

http://jvi.asm.org/

(8)

bands of a single protein were still present in the gel, including

those of polyhedrin, VP39, GP41, P49, P33, E66,

ODV-E56, and ODV-E25 (Fig. 1 and Table 1). Similar results were

observed for a single protein during an investigation of the

CuniNPV ODV (46). Various factors can be responsible for

the fragility of some proteins, including the refractory profile

of alkaline proteases and the different methods used in virus

purification (11, 54); however, we cannot exclude the

possibil-ity of some protein degradation during the experimental

pro-cedures. On the other hand, there may be polymorphisms,

oligomerization, and posttranslational modification of the gene

products in the matrix of ODVs.

During data mining by MS-Fit, some peptide footprints were

matched to HearNPV ORFs but with a low MOWSE score,

such as

ha26

,

ie1

,

me53

,

bro

-

b

,

bro

-

c

,

alk

-

exo

,

pk1

,

ha133

(F-protein gene), etc. Therefore, they were not included as ODV

structural proteins in our results. Some of them, such as IE1,

Alk-exo, and the F protein, were found to be located in the

ODVs of AcMNPV (11), while the F protein and Bro were

identified in the ODVs of CuniNPV (46). The importance of

using multiple techniques to identify ODV structural proteins

has been elucidated by Braunagel et al. (11). We are using

antibodies against HearNPV ORFs to verify the protein

local-ization in the ODV by Western blot analysis and IEM.

Loca-tion of the above proteins in the ODV awaits confirmaLoca-tion

pending the preparation of specific antibodies.

Two structural proteins of HearNPV ODV, HA44 and HA100,

were newly identified here. ORF

ha44

contains a TAAG late

gene promoter motif, which is in agreement with its function as a

structural protein. Western blot analysis has confirmed that HA44

was a nucleocapsid component in both BV and ODV with a

molecular mass of 44 kDa. Homologues of HA44 were found in

all of the group II NPVs and GVs whose complete sequences

have been determined but not in group I NPVs or dipteran or

hymenopteran baculoviruses. It is generally believed that GVs

separated from the ancestor of NPVs and GVs before the

radi-ation of group I and group II NPVs (35). It is therefore likely that

the ancestor of HA44 existed in both NPV and GV but was lost

during the emergence of group I NPVs.

Western blot analysis and IEM revealed that HA100 is a

component of the nucleocapsid and the envelope of ODV.

HA100 is conserved in all group II NPVs, and it is a

homo-logue of PARG. PARG is critical for the maintenance of

steady-state poly(ADP-ribose) levels and plays important roles

in modulating chromatin structure, transcription, DNA repair,

and apoptosis (6). It is interesting that the members of NPV

group II encode a PARG-like protein as a structural protein. It

remains to be determined whether the PARG-like proteins in

group II NPVs are enzymatically functional.

With the knowledge of baculovirus ODV composition, it is

possible to study the functions of the relevant proteins and

their potential role during virus primary infection (11). In this

study, we identified two new ODV structural proteins, HA44

and HA100. Currently we are investigating the biological

func-tions of these two proteins.

ACKNOWLEDGMENTS

This work was supported by a 973 project (2003CB114202), an

NSFC key project from China (30630002), and a joint PSA project

from China and The Netherlands (2004CB720404).

We acknowledge the State Key Laboratory of Virology

Proteom-ics/MS Center (Wuhan University) and Shanghai GeneCore

Bio-Technologies Co. Ltd. for technical support. We thank Jian-Lan Yu

and Fang-Ke Huang for assistance with the experiments.

REFERENCES

1.Altschul, S. F., W. Gish, W. Miller, E. W. Meyers, and D. J. Lipman.1992.

Basic local alignment search tool. J. Mol. Biol.215:403–410.

2.Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl.1994. Current protocols in molecular biology. Greene and Wiley InterScience, New York, NY.

3.Belyavskyi, M., S. C. Braunagel, and M. D. Summers.1998. The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a

multifunctional viral cyclin. Proc. Natl. Acad. Sci. USA95:11205–11210.

4.Blissard, G., and J. R. Wenz.1992. Baculovirus gp64 envelope glycoprotein

is sufficient to mediate pH-dependent membrane fusion. J. Virol.66:6829–

6835.

5.Blissard, G., B. Black, N. Crook, B. A. Keddie, R. Possee, G. Rohrmann, D. Theilmann, and L. E. Volkman.2000.Baculoviridae: taxonomic structure

and properties of the family, p. 195–202.InM. H. V. van Regenmortel, C. M.

Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and E. B. Wickner (ed.), Virus taxonomy: classification and nomenclature of viruses. Seventh report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, CA.

6.Bonicalzi, M. E., J. F. Haince, A. Droit, and G. G. Poirier.2005. Regulation of poly(ADP-ribose) metabolism by poly(ADP-ribose) glycohydrolase:

where and when? Cell. Mol. Life Sci.62:739–750.

7.Braunagel, S. C., and M. D. Summers.1994.Autographa californicanuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids:

struc-tural proteins, antigens, lipid and fatty acid profiles. Virology202:315–328.

8.Braunagel, S. C., D. M. Elton, H. Ma, and M. D. Summers.1996.

Identifi-cation and analysis of anAutographa californicanuclear polyhedrosis virus

structural protein of the occlusion-derived virus envelope: ODV-E56.

Virol-ogy217:97–110.

9.Braunagel, S. C., H. He, P. Ramamurthy, and M. D. Summers.1996. Tran-scription, translation, and cellular localization of three Autographa califor-nica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35,

and ODV-EC27. Virology222:100–114.

10.Braunagel, S. C., P. A. Guidry, G. Rosas-Acosta, L. Engelking, and M. D. Summers.2001. Identification of BV/ODV-C42, an Autographa californica

nucleopolyhedrovirusorf101-encoded structural protein detected in

infected-cell complexes with ODV-EC27 and p78/83. J. Virol.75:12331–12338.

11.Braunagel, S. C., W. K. Russell, G. Rosas-Acosta, D. H. Russell, and M. D. Summers.2003. Determination of the protein composition of the

occlusion-derived virus of Autographa californicanucleopolyhedrovirus. Proc. Natl.

Acad. Sci. USA100:9797–9802.

12.Chen, X. W., W. F. J. IJkel, C. Dominy, P. M. de Andrade Zanotto, Y. Hashimoto, O. Faktor, T. Hayakawa, H. Wang, A. Premkumar, S. Matha-van, P. J. Krell, Z. Hu, and J. M. Vlak.1999. Identification, sequence analysis and phylogeny of the lef-2 gene of Helicoverpa armigera single-nucleocapsid

baculovirus. Virus Res.65:21–32.

13.Chen, X. W., W. F. J. IJkel, R. Tarchini, X. L. Sun, H. Sandbrink, H. L. Wang, S. Peters, D. Zuidema, R. K. Lankhorst, J. M. Vlak, and Z. H. Hu.

2001. The sequence of theHelicoverpa armigerasingle nucleocapsid

nucleo-polyhedrovirus genome. J. Gen. Virol.82:241–257.

14.Chen, X. W., Z. H. Hu, and J. M. Vlak.1997. Nucleotide sequence analysis

of the polyhedrin gene of Heliothis armigerasingle nucleocapsid nuclear

polyhedrosis virus. Virol. Sin.12:346–353.

15.Chen, X. W., Z. H. Hu, J. A. Jehle, and J. M. Vlak.1997. Characterization of

the ecdysteroid UDP-glucosyltransferase gene ofHeliothis armigera

single-nucleocapsid nucleopolyhedrovirus. Virus Genes15:219–225.

16.Chung, C. S., C. H. Chen, M. Y. Ho, C. Y. Huang, C. L. Liao, and W. Chang.

2006. Vaccinia virus proteome: identification of proteins in vaccinia virus

intracellular mature virion particles. J. Virol.80:2127–2140.

17.D’Amours, D., S. Desnoyers, I. D’Silva, and G. G. Poirier.1999. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem. J.

342:249–268.

18.Dong, C., D. Li, G. Long, F. Deng, H. Wang, and Z. Hu.2005. Identification of functional domains of HearNPV P10 for filament formation. Virology

338:112–120.

19.Fang, M. G., H. Wang, L. Yuan, X. W. Chen, J. M. Vlak, and Z. H. Hu.2003.

Open reading frame 94 ofHelicoverpa armigerasingle nucleocapsid

nucle-opolyhedrovirus encodes a novel occlusion-derived virion protein,

ODV-EC43. J. Gen. Virol.84:3021–3027.

20.Fang, M. G., Y. Nie, Q. Wang, F. Deng, R. Wang, H. Wang, H. Wang, J. M. Vlak, X. Chen, and Z. Hu.2006. Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor

(PIF-2). J. Gen. Virol.87:2563–2569.

21.Faulkner, P., J. Kuzio, G. V. Williams, and J. A. Wilson.1997. Analysis of p74, a PDV envelope protein of Autographa californica

nucleopolyhedrovi-9384

DENG ET AL.

J. V

IROL

.

on November 8, 2019 by guest

http://jvi.asm.org/

(9)

rus required for occlusion body infectivity in vivo. J. Gen. Virol.78:3091– 3100.

22.Guan, K. L., and J. E. Dixon.1991. Eukaryotic proteins expressed in Esch-erichia coli: an improved thrombin cleavage and purification procedure of

fusion proteins with glutathioneS-transferase. Anal. Biochem.192:262–267.

23.Herniou, E. A., J. A. Olszewski, D. R. O’Reilly, and J. S. Cory.2004. Ancient

coevolution of baculoviruses and their insect hosts. J. Virol.78:3244–3251.

24.Herniou, E. A., J. A. Olszewski, J. S. Cory, and D. R. O’Reilly.2003. The genome sequence and evolution of baculoviruses. Annu. Rev. Entomol.

48:211–234.

25.Hirosawa, M., M. Hoshida, M. Ishikawa, and T. Toya.1993. MASCOT: multiple alignment system for protein sequences based on three-way

dy-namic programming. Comput. Appl. Biosci.9:161–167.

26.Hong, T., S. C. Braunagel, and M. D. Summers.1994. Transcription, trans-lation, and cellular localization of PDV-E66: a structural protein of the PDV

envelope ofAutographa californicanuclear polyhedrosis virus. Virology204:

210–222.

27.IJkel, W. F., M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema.2000. A novel baculovirus envelope fusion protein with a

proprotein convertase cleavage site. Virology275:30–41.

28.IJkel, W. F., R. J. Lebbink, M. L. Op den Brouw, R. W. Goldbach, J. M. Vlak, and D. Zuidema.2001. Identification of a novel occlusion derived

virus-specific protein inSpodoptera exiguamulticapsid nucleopolyhedrovirus.

Vi-rology284:170–181.

29.Jehle, J. A., M. Lange, H. Wang, Z. Hu, Y. Wang, and R. Hauschild.2006. Molecular identification and phylogenetic analysis of baculoviruses from

Lepidoptera. Virology346:180–193.

30.Keddie, B. A., G. W. Aponte, and L. E. Volkman.1989. The pathway of

infection ofAutographa californicanuclear polyhedrosis virus in an insect

host. Science243:1728–1730.

31.Kikhno, I., S. Gutierrez, L. Croizier, G. Croizier, and M. L. Ferber.2002. Characterization of pif, a gene required for the per os infectivity of

Spo-doptera littoralis nucleopolyhedrovirus. J. Gen. Virol.83:3013–3022.

32.Kool, M., C. H. Ahrens, R. W. Goldbach, G. F. Rohrmann, and J. M. Vlak.

1994. Identification of genes involved in DNA replication of the Autographa

californica baculovirus. Proc. Natl. Acad. Sci. USA91:11212–11216.

33.Kumar, S., K. Tamura., and M. Nei.2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief.

Bioinform.5:150–163.

34.Kuzio, J., R. Jaques, and P. Faulkner.1989. Identification of p74, a gene

essential for virulence of baculovirus occlusion bodies. Virology173:759–

763.

35.Lange, M., H. Wang, Z. Hu, and J. A. Jehle.2004. Towards a molecular identification and classification system of lepidopteran-specific

baculovi-ruses. Virology325:36–47.

36.Long, G., M. Westenberg, H. L. Wang, J. M. Vlak, and Z. H. Hu.2006.

Function, oligomerization and N-linked glycosylation of theHelicoverpa

ar-migeraSNPV envelope fusion protein. J. Gen. Virol.87:839–846. 37.Long, G., X. W. Chen, D. Peters, J. M. Vlak, and Z. H. Hu.2003. Open

reading frame 122 ofHelicoverpa armigerasingle nucleocapsid

nucleopoly-hedrovirus encodes a novel structural protein of occlusion-derived virions.

J. Gen. Virol.84:115–121.

38.Lu, A., and E. B. Carstens.1992. Nucleotide sequence and transcriptional

analysis of the p80 gene ofAutographa californicanuclear polyhedrosis virus:

a homologue of the Orgyia pseudotsugata nuclear polyhedrosis virus

capsid-associated gene. Virology190:201–209.

39.McIntosh, A. H., and C. M. Ignoffo.1983. Characterization of 5 cell lines

established from species ofHeliothis. Appl. Entomol. Zool.18:262–269.

40.Monsma, S. A., A. G. Oomens, and G. W. Blissard.1996. The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell

transmission of infection. J. Virol.70:4607–4616.

41.Muller, R., M. N. Pearson, R. L. Russell, and G. F. Rohrmann.1990. A capsid-associated protein of the multicapsid nuclear polyhedrosis virus of

Orgyia pseudotsugata: genetic location, sequence, transcriptional mapping,

and immunocytochemical characterization. Virology176:133–144.

42.Notredame, C., D. Higgins, and J. Heringa.2000. T-Coffee: a novel method

for multiple sequence alignments. J. Mol. Biol.302:205–217.

43.Patel, C. N., D. W. Koh, M. K. Jacobson, and M. A. Oliveira.2005. Identi-fication of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain. Biochem. J.

388:493–500.

44.Pearson, M. N., C. Groten, and G. F. Rohrmann.2000. Identification of the

Lymantria disparnucleopolyhedrovirus envelope fusion protein provides

ev-idence for a phylogenetic division of theBaculoviridae. J. Virol.74:6126–

6131.

45.Pearson, M. N., R. L. Russell, G. F. Rohrmann, and G. S. Beaudreau.1988. p39, a major baculovirus structural protein: immunocytochemical

character-ization and genetic location. Virology167:407–413.

46.Perera, O., T. B. Green, S. M. Stevens, Jr., S. White, and J. J. Becnel.2007.

Proteins associated withCulex nigripalpusnucleopolyhedrovirus occluded

virions. J. Virol.81:4585–4590.

47.Pham, D. Q., R. H. Hice, N. Sivasubramanian, and B. A. Federici.1993. The 1629-bp open reading frame of the Autographa californica multinucleocap-sid nuclear polyhedrosis virus encodes a virion structural protein. Gene

137:275–280.

48.Prikhod’ko, G. G., Y. Wang, E. Freulich, C. Prives, and L. K. Miller.1999. Baculovirus p33 binds human p53 and enhances p53-mediated apoptosis.

J. Virol.73:1227–1234.

49.Rohrmann, G. F.1986. Polyhedrin structure. J. Gen. Virol.67:1499–1513. 50.Rohrmann, G. F.1992. Baculovirus structural proteins. J. Gen. Virol.73:

749–761.

51.Russell, R. L., and G. F. Rohrmann.1993. A 25-kDa protein is associated

with the envelopes of occluded baculovirus virions. Virology195:532–540.

52.Russell, R. L., C. J. Funk, and G. F. Rohrmann.1997. Association of a

baculovirus-encoded protein with the capsid basal region. Virology227:142–

152.

53.Shevchenko, A., M. Wilm, O. Vorm, and M. Mann.1996. Mass spectrometric

sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem.68:

850–858.

54.Summers, M. D., and G. E. Smith.1975.Trichoplusia nigranulosis virus

granulin: a phenol-soluble, phosphorylated protein. J. Virol.16:1108–1116.

55.Sun, X. L., G. Y. Zhang, Z. X. Zhang, Z. H. Hu, J. M. Vlak, and B. M. Arif.

1998. In vivo cloning ofHelicoverpa armigerasingle nucleocapsid nuclear

polyhedrosis virus genotypes. Virol. Sin.13:83–88.

56.Sun, X. L., and G. Y. Zhang.1994. A comparison of four wild isolates of

Heliothisnuclear polyhedrosis virus. Virol. Sin.9:309–318.

57.Thiem, S. M., and L. K. Miller.1989. Identification, sequence, and tran-scriptional mapping of the major capsid protein gene of the baculovirus

Autographa californicanuclear polyhedrosis virus. J. Virol.63:2008–2018. 58.Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.

Higgins.1997. The ClustalX Windows interface: flexible strategies for mul-tiple sequence alignment aided by quality analysis tools. Nucleic Acids Res.

24:4876–4882.

59.Vialard, J. E., and C. D. Richardson.1993. The 1,629-nucleotide open

reading frame located downstream of theAutographa californicanuclear

polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated

phos-phoprotein. J. Virol.67:5859–5866.

60.Wang, H., D. Wu, F. Deng, H. Peng, X. Chen, H. Lauzon, B. M. Arif, J. A. Jehle, and Z. Hu.2004. Characterization and phylogenetic analysis of the

chitinase gene from theHelicoverpa armigerasingle nucleocapsid

nucleopoly-hedrovirus. Virus Res.100:179–189.

61.Wang, H., X. W. Chen, B. M. Arif, J. M. Vlak, and Z. H. Hu.2001. Nucleotide sequence and transcriptional analysis of a putative basic DNA-binding

pro-tein ofHelicoverpa armigeranucleopolyhedrovirus. Virus Genes22:113–120.

62.Washburn, J. O., B. A. Kirkpatrick, and L. E. Volkman.1995. Comparative

pathogenesis ofAutographa californicaM nuclear polyhedrosis virus in larvae

ofTrichoplusia niandHeliothis virescens. Virology209:561–568.

63.Whitford, M., and P. Faulkner.1992. A structural polypeptide of the

bacu-lovirusAutographa californicanuclear polyhedrosis virus contains O-linked

N-acetylglucosamine. J. Virol.66:3324–3329.

64.Whitford, M., and P. Faulkner.1992. Nucleotide sequence and transcrip-tional analysis of a gene encoding gp41, a structural glycoprotein of the

baculovirusAutographa californicanuclear polyhedrosis virus. J. Virol.66:

4763–4768.

65.Wilson, M. E., and K. H. Price.1988. Association of Autographa californica nuclear polyhedrosis virus (AcMNPV) with the nuclear matrix. Virology

167:233–241.

66.Wilson, M. E., T. H. Mainprize, P. D. Friesen, and L. K. Miller.1987. Location, transcription, and sequence of a baculovirus gene encoding a small

arginine-rich polypeptide. J. Virol.61:661–666.

67.Wu, D., F. Deng, X. Sun, H. Wang, L. Yuan, J. M. Vlak, and Z. Hu.2005.

Functional analysis of FP25K ofHelicoverpa armigerasingle nucleocapsid

nucleopolyhedrovirus. J. Gen. Virol.86:2439–2444.

68.Yao, L., W. Zhou, H. Xu, Y. Zheng, and Y. Qi.2004. The Heliothis armigera single nucleocapsid nucleopolyhedrovirus envelope protein P74 is required

for infection of the host midgut. Virus Res.104:111–121.

69.Ying, W., M. B. Sevigny, Y. Chen, and R. A. Swanson.2001. Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death.

Proc. Natl. Acad. Sci. USA98:12227–12232.

70.Zanotto, P. M., B. D. Kessing, and J. E. Maruniak. 1993. Phylogenetic interrelationships among baculoviruses: evolutionary rates and host

associ-ations. J. Invertebr. Pathol.62:147–164.

71.Zhang, G.1994. Research, development and application ofHeliothisviral

pesticide in China. Resour. Environ. Yangtze Valley3:1–6.

on November 8, 2019 by guest

http://jvi.asm.org/

on November 8, 2019 by guest

Figure

FIG. 1. SDS-PAGE profile and MS results of purified HearNPVODV. ODV proteins were separated by 12% SDS-PAGE and stained
TABLE 1. HearNPV ODV proteins identified by MALDI-TOF MSa
FIG. 2. Alignment of the amino acid sequences of HA44 and its homologues among the group II NPVs
FIG. 3. Neighbor-joining tree derived from HA44 and its homologues among NPVs and GVs
+3

References

Related documents

Since E dyn according to Equation 1 is one of the main characteristics of the resonance material, in dendroacoustic studies of tree species with hard wood or young

In our simulation results we consider a secondary net- work with K = 20, 30, 40 relay nodes, and the channel coefficients are generated independently as complex Gaussian

When sensory evoked potentials were measured in relation to blood glucose concentrations in infants and children with episodes of hyperglycemia, abnormalities were noted at

We believe in one Lord, Jesus Christ, the only Son of God, eternally begotten of the Father, God from God, Light from Light, true God from true God, begotten, not made, of one Being

Given the difficulty of modelling the human error in this type of facilities, the objective of the present work is to determine if there is correlation between production and the

In this initial study, a cohort (n = 343) of English-speaking individuals from countries located on four continents (United States, United Kingdom, Australia, India) answered

See the conservation objectives supporting document for lesser horseshoe bat (NPWS, 2018) for further information on all attributes and targets Winter roosts Condition No

In the current study, we sought to assess the expression levels of CDKN1A/p21 and TGFBR2 in a cohort of breast cancer patients from our institute, and evaluate their correlation