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Virology 282, 348–356 (2001)
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Comparative Expressed-Sequence-Tag Analysis of Differential Gene Expression Profiles
in BmNPV-Infected BmN Cells
Kazuhiro Okano,* ,† ,1 Toru Shimada,‡ Kazuei Mita,§ and Susumu Maeda* ,† ,2
*Laboratory of Molecular Entomology and Baculovirology, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan;
†Core Research for Evolutional Science and Technology (CREST) Project, Japan Science and Technology Corporation (JST),
Kawaguchi, Saitama, Japan; ‡Department of Agricultural and Environmental Biology, University of Tokyo, Tokyo, Japan;
and §Genome Research Group, National Institute of Radiological Science, Chiba, Japan
Received September 11, 2000; returned to author for revision October 17, 2000; accepted January 3, 2001
To compare the gene expression profiles of uninfected and Bombyx mori nucleopolyhedrovirus (BmNPV)-infected BmN
cells, we constructed four cDNA libraries for mock-infected cells, and cells at 2, 6, and 12 h postinfection (h.p.i.). A total of
2645 partial sequences obtained for expressed-sequence-tags (ESTs) from the libraries were categorized using BLAST
searches of the public database and the BmNPV genome sequence. The following proportions of BmNPV-derived ESTs were
observed: 0.4, 4.5, and 57% at 2, 6, and 12 h.p.i, respectively. Moreover, 31 BmNPV open reading frames (ORFs) were newly
identified for transcripts and the baculovirus-repeated ORFs (bro) showed the highest levels of expression in the 12 h.p.i.
library. Most of the host genes decreased in number as the infection progressed. However, several, including cytochrome c
oxidase 1, increased in the late stages of infection. Two apoptosis-related host genes were also identified. © 2001 Academic
Press
Key Words: EST; baculovirus; expression profile.
The silkworm, Bombyx mori, was domesticated from
the wild silkmoth, Bombyx mandarina, several thousand years ago and has been widely cultured in many
parts of the world. It is a large insect characterized by
a rapid, almost 10,000-fold, increase in mass from the
first to the fifth instar. The adults of B. mori are about
1000 times larger than the adults of D. melanogaster.
The consequent ease of collecting different organs
and tissues from B. mori at different stages of development offers a significant advantage in investigating
localized gene expression. We have started B. mori
EST database project as a first step in its genome
analysis. To date, over 15,000 pieces of EST data have
been obtained from cDNA libraries of various tissues
and stages.
B. mori nucleopolyhedrovirus (BmNPV) is restricted
to growth in B. mori tissues and cells. Its genome is
about 128 kb and contains 136 putative open reading
frames (ORFs; Gomi et al.,1999), the functions of many
of which are undefined. To gain a better understanding
of the various cellular events and transcriptional
changes occurring in BmN cells during BmNPV infection, we constructed a B. mori EST database consisting of 2645 partial cDNA sequences from different
stages of infection. In this report, we describe the
analysis in terms of the relative expression levels
of host and virus-derived genes during the infection
cycle.
INTRODUCTION
Comparisons of gene expression profiles can provide
useful information on the molecular basis of cellular
events. The use of expressed-sequence-tag (EST) singlepass, partial sequences of randomly selected cDNA
clones offers a rapid and efficient means of obtaining
steady-state mRNA profiles. This potentially permits the
identification of differentially regulated mRNAs (Adams
et al., 1992, 1993). EST projects have greatly progressed
in the construction of various databases, such as the
human genome project (The Institute for Genomic
Research (TIGR), http://www.tigr.org/; WashU-Merck
Human EST Project, http://genome.wustl.edu/est/
esthmpg.html; Adams et al., 1995), Arabidopsis (Delseny
et al., 1997), rice (Yamamoto and Sasaki, 1997), mice
(WashU-HHMI Mouse EST Project, http://genome.wustl.edu/est/mouse._esthmpg.html), Caenorhabditis elegans (http://www.ddbj.nig.ac.jp/htmls/c-elegans/html/
CE_INDEX.html). In insects, the only EST project is that of
Drosophila melanogaster, which is underway as part of
the Berkeley Drosophila Genome Project (BDGP, http://
www.fruitfly.org/).
1
To whom reprint requests should be addressed at Laboratory of
Molecular Entomology and Baculovirology, RIKEN (The Institute of
Physical and Chemical Research), Hirosawa 2-1, Wako 351-0198,
Saitama, Japan. Fax: ⫹81-48-462-4678; E-mail: kokano@postman.
riken.go.jp.
2
Deceased.
0042-6822/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
348
EST DATABASE OF BmNPV-INFECTED BmN CELLS
349
FIG. 1. Distribution of BmN cell and BmNPV ESTs in infected cells. The data for the BmN, 2, 6, and 12 h.p.i. libraries are derived from 757, 558, 684,
and 646 ESTs, respectively. Hatched areas indicate the percentages of host ESTs in each library and solid areas indicate the percentages of viral
ESTs in each library.
RESULTS
Comparison of ESTs in different infection stage cDNA
libraries
To identify differentially regulated host genes and viral
genes during BmNPV infection, we constructed a cDNA
library of mock-infected BmN cells and three cDNA libraries of BmNPV-infected BmN cells at 2, 6, and 12 h
postinfection (h.p.i.). About 65% of the samples yielded
readable 500- to 700-bp (on average) sequences. In all,
2645 ESTs were analyzed from the four libraries. Figure
1 shows proportional changes in the ESTs derived from
the host or the four different infection stage libraries. As
expected, no virus-derived sequence was found in 757
ESTs from the mock-infected BmN library. Only 2 of 558
(0.4%) ESTs found in the 2 h.p.i. library were virus-derived. The EST derived from BmNPV dramatically increased in number in later stages. Thirty viral ESTs of
654 (4.5%) were found from the 6 h.p.i. library. At 12 h.p.i.,
369 out of 646 (57%) ESTs were derived from BmNPV and
they constituted 90 distinct viral ORFs.
Expression profiles of EST derived from BmNPV in 2,
6, and 12 h.p.i. libraries
To ascertain the differential gene expression profiles of BmNPV ORFs during the progression of viral
infection, the ESTs derived from BmNPV from the 2, 6,
and 12 h.p.i. libraries were compared (Table 1; Gomi et
al.,1999). The two BmNPV ESTs from the 2 h.p.i. library
were ie-0 and ie-2. Other immediate early genes, ie-1
and pe38, however, were not identified. Thirty ESTs,
from 16 distinct viral ORFs, were found in the 6 h.p.i.
library. Among these ORFs, the most prevalent were
ie-2 (3 times), egt (3 times), and 39k (4 times). Several
genes associated with viral DNA replication and late
transcription, including lef-1, dnapol, dnahel, lef-7, p40,
and p35, appeared once each, as did bv/odv-e26, p43,
p47, and gp64/67. Moreover, two of baculovirus-repeated ORFs (bro), bro-a and bro-c (Kuzio et al., 1999;
Gomi et al.,1999; Kang et al., 1999), also appeared
twice and once, respectively. Finally, we found ESTs
for ORF4 (4 times) and ORF21 (2 times); their function
and transcription have never been described. We detected the transcripts at 6 h.p.i., although ORF4 has
an early promoter motif and ORF21 has an early enhancer-like element (Gomi et al.,1999).
Ninety distinct ORFs in 369 viral ESTs, representing
66% of the 136 putative BmNPV ORFs, were found in the
12 h.p.i. library. They represented many of the viral structural protein genes and late expression factor (lef) genes.
ESTs of odv-ec27 appeared 16 times; vp39 and odv-e25
each appeared 14 times, and ESTs of pp34 and gp41
appeared 13 and 11 times in the ESTs of this library,
respectively. Altogether, the transcripts of these structural protein genes comprised 15% of the total viral transcripts at this stage. The EST of a very late gene, polhedrin, was already observed once. ESTs of 31 ORFs that
have not been extensively characterized were also identified.
Although bro a and bro c were the only representatives
of this gene family found among the ESTs from the 6 h.p.i.
library, cDNAs from all five members were highly expressed in the 12 h.p.i. library. The bro c ESTs appeared
25 times and were the most frequently appearing viral
EST from the 12 h.p.i. library. Of the other bro genes, bro
a, bro b, bro d, and bro e ESTs were found 8, 5, 4, and 2
times, respectively. Altogether, the ESTs derived from the
bro family genes comprised 12% of the viral ESTs in the
12 h.p.i. library.
350
OKANO ET AL.
TABLE 1
Expression Profiles of BmNPV ORFs in 2, 6, and 12 h.p.i. Libraries
Library
ORF
1 (8)
2 (9)
3 (10)
4 (11)
5 (13)
6 (14)
7 (15)
8 (16)
9 (17)
10 (18)
11 (19)
12 (20/21)
13 (22)
14 (23)
15 (24)
16 (25)
17 (26)
18 (27)
19 (28)
20 (29)
21 (30)
22
23 (31)
24 (32)
25 (34)
26 (35)
27 (36)
28 (37)
29 (38)
30 (39)
31 (40)
32 (41)
33 (42)
34 (43)
35 (44)
36 (45)
37 (46)
38 (47)
39 (50)
40 (51)
41 (52)
42 (53)
42a (53a)
43 (54)
44 (55)
45 (56)
Gene
Library
2 h.p.i. 6 h.p.i. 12 h.p.i.
polh
orf1629
pk-1
1
3
4
8
lef1
egt
bv/odv-e26
1
3
1
1
1
10
arif-1
1
2
pkip
dbp
9
8
3
1
iap1
lef-6
bro-a
sod
fgf
ubiquitin
39k
lef-11
p43
p47
lef-12
gta
2
2
4
2
1
3
8
4
1
2
13
4
2
2
1
2
4
5
odv-e66
ets
lef-8
lef-10
vp1054
2
1
4
7
1
ORF
46 (57)
47 (58/59)
48 (60)
49 (61)
50 (62)
51 (63)
52 (64)
53 (65)
54 (66)
55 (67)
56 (68)
57 (69)
58 (71)
59 (73)
60 (74)
61 (75)
62 (76)
63 (77)
64 (78)
65 (79)
66 (80)
67 (81)
68 (82)
69 (83)
70 (87)
71 (88)
72 (89)
73 (90)
74 (91)
75 (92)
76 (93)
77 (94)
78 (95)
79 (96)
80
81
82 (98)
83 (99)
84 (100)
85 (101)
86 (102)
87 (103)
88 (104)
89 (105)
90 (106/107)
91 (108)
Gene
2 h.p.i. 6 h.p.i. 12 h.p.i.
1
1
4
1
fp
lef-9
gp37
dnapol
Library
1
1
lef-3
3
3
1
iap2
1
2
vlf-1
2
4
11
3
3
2
gp41
p95
vp15
cg30
vp39
lef-4
odv-e25
dnahel
bro-b
bro-c
38k
lef-5
dna binding
p40
p12
p45
vp80
he65
7
14
2
1
1
1
5
2
14
5
5
25
4
5
3
7
10
2
1
ORF
92 (109)
93 (111)
94 (114)
95 (115)
96 (117)
97 (119)
98 (120)
99 (122)
100 (123)
101 (124)
102 (125)
103 (126)
104 (127)
105 (128)
106 (129)
107 (130)
108 (131)
109 (132)
110 (133)
111
112 (135)
113 (136)
114 (137)
115 (138)
116 (139)
117 (141)
118 (142)
119 (143)
120 (144)
121 (145)
122 (146)
123 (147)
124 (148)
125 (149)
126 (150)
127 (151)
128 (153)
129 (154)
130 (1)
131 (2)
132
133 (4)
134 (5)
135 (6)
Gene
2 h.p.i. 6 h.p.i. 12 h.p.i.
3
2
4
Pk2
2
lef-7
Chitinase
v-cath
gp64/67
p24
gp16
pp34
1
2
1
7
13
1
2
alk-exo
p35
p26
p10
p74
me53
ie-0
1
ie-1
odv-e56
1
3
1
2
3
2
5
16
1
3
9
4
1
1
odv-e18
odv-ec27
ie-2
pe38
2
3
ptp
bro-d
bro-e
2
1
4
2
2
lef-2
2
Total EST number
2
30
369
Note. Each ORF sequence of BmNPV was searched by BLASTN against all EST data. Number in parentheses indicates AcMNPV ORF
corresponding to BmNPV ORF (Gomi et al., 1999).
Expression profiles of host genes
We analyzed the 757 ESTs obtained from the BmN
library to determine the host gene expression profiles of
uninfected cells. No significant homologies were evident
for 342 ESTs. The remainders were classified into eight
major categories according to the functions of corresponding genes as proposed in the literature (Adams et
al., 1995; Table 2). Three major ESTs, however, could not
be classified into any of the eight categories. The first,
found 24 times in the BmN library, did not have homology
with known proteins in the protein database. A BLASTN
search of the public DNA database using this sequence
revealed homology with BMC1, a class of B. mori-specific
repetitive sequences (Ogura et al., 1994). The two other
EST DATABASE OF BmNPV-INFECTED BmN CELLS
351
TABLE 2
Summary of Analyzed and Classified ESTs Derived from Host Genes from Four cDNA Libraries
Library
BmN
No. of EST
species
Gene/protein expression
Cell structure/mobility
Cell/organism defense
Metabolism
Cell signaling
Cell division
Unclassified
BMC1
Tymovirus-like ESTs
Unmatched
Total host EST number
71
19
15
48
10
5
70
1
2
No. of EST
135 (18.4%)
28 (3.8%)
30 (4.1%)
60 (8.2%)
10 (1.4%)
5 (0.7%)
74 (10.1%)
24 (3.3%)
49 (6.7%)
342
757
2 h.p.i.
No. of EST
species
33
20
13
38
20
7
73
1
1
6 h.p.i.
No. of EST
78 (14.0%)
29 (5.2%)
27 (4.9%)
70 (12.6%)
22 (4.0%)
8 (1.4%)
84 (15.1%)
2 (0.4%)
7 (0.4%)
229
556
No. of EST
species
51
19
12
44
18
5
56
—
2
No. of EST
129 (19.7%)
40 (6.1%)
27 (4.1%)
76 (11.6%)
22 (3.4%)
6 (0.9%)
65 (9.9%)
—
14 (2.1%)
277
654
12 h.p.i.
No. of EST
species
33
12
6
27
4
2
35
—
—
No. of EST
45 (16.2%)
12 (4.3%)
6 (2.2%)
57 (20.6%)
4 (1.4%)
2 (0.7%)
38 (13.7%)
—
—
113
277
Note. Percentages were calculated by no. of categorized EST/total number of host-derived EST.
ESTs were a tymovirus-like RNA replicase homologue
(14 ESTs: P value, where P is the smallest sum of probability in a Poisson distribution ⫽ 8.0e⫺44, 81% similarity
in 133 amino acids) and a tymovirus-like coat protein
homologue (35 ESTs: P value ⫽ 3.8e⫺11, 50% similarity
in 112 amino acids). These three major EST sequences
were the most frequent ESTs in this library, comprising
10% of the total number of ESTs obtained.
Genes involved in gene or protein expression (gene/
protein expression category; Table 3) was the largest
category in the BmN library, occupying 18.4% (135 ESTs).
The metabolism category comprised 8.2% (60 ESTs), with
48 distinct EST species. The cell/organism defense category made up 4.1% (30 ESTs), with 15 species, and the
cell structure/mobility category took up 3.8% (28 ESTs),
with 19 species. Cell signaling comprised 1.4% (10 ESTs)
with 10 species, and cell division comprised 0.7% (5
ESTs) with 5 species.
The ESTs from the other three libraries were also
categorized to study the change of expression profiles of
host cells during BmNPV infection. As discussed below,
most of the categories did not show significant proportional changes during infection progression, except the
metabolism category, and the BMC1 and tymovirus-like
ESTs (Table 2). The 229, 277, and 113 ESTs that were
obtained from the 2, 6, and 12 h.p.i. libraries, respectively,
had no homologues in the public database. These represented approximately the same proportion of the total
ESTs from each library.
Host gene expression gradually decreased as viral
replication progressed (Fig. 1). To compare the expression profiles among the four stages of infection, host
genes that appeared at least three times in any single
library were examined (Table 3). The ESTs of these major
transcripts decreased significantly in number with the
progression of BmNPV infection. Especially notable, the
most abundant ESTs in the BmN library appeared only
once in the 12 h.p.i. library (Table 3). The ESTs of three
major cDNAs, BMC1, tymovirus RNA replicase, and coat
protein homologues, were significantly decreased in
number and are represented, respectively, 2, 7, and 1
times in the 2 h.p.i. library; 0, 1, and 10 times in the 6 h.p.i.
library; and 0, 1, and 1 times in the 12 h.p.i. library.
Ribosomal protein genes, of which there were 39 species in the BmN library, also declined during virus replication. For example, the 60S-ribosomal protein L1, L4,
L7A, and 40S-ribosomal protein S2 were the most frequently appearing ESTs of ribosomal protein genes in
the BmN library. L4 was found 14 times in the 2 h.p.i.
library and 13 times in the 6 h.p.i. library, but it was seen
only once in the 12 h.p.i. Most of the genes appeared
only once in the 12 h.p.i. library, even if they were identified several times in the BmN and early infection stage
libraries.
In contrast, the ESTs in the metabolism category increased from 8.2% in the BmN library to 20.6% in the
12 h.p.i. library. This increase was caused by a significant increase in the number of mitochondrial-related
genes. The cytochrome c oxidase polypeptide 1 (COI)
gene, which is encoded by mitochondria, appeared only
once in the 756 ESTs from the BmN library. This EST,
however, appeared 5, 8, and 18 times in the 2, 6, and
12 h.p.i. libraries, respectively, increasing its proportion
from 0.13% of the ESTs in the BmN library to 6.5% in the
12 h.p.i. library. The cytochrome C oxidase polypeptide 3
(COIII) gene appeared 3 times in the 6 h.p.i. library and 6
times in the 12 h.p.i. library, although it did not appear in
either the BmN or the 2 h.p.i. libraries. The NADH-ubiquinone oxidoreductase chain 1 gene, which is encoded by
mitochondrial genome, appeared 3 times in the 12 h.p.i.
352
OKANO ET AL.
TABLE 3
Expression Profiles of Major Host Genes from Four cDNA Libraries
Library
Putative definition
Gene/protein expression
RNA helicase
RNA binding protein SQUID
Transcriptional regulatory protein ALGP
EF-1-␣
EF-1-␣-S
EF-2
EIF-5A
60S ribosomal protein L1
60S ribosomal protein L4
60S ribosomal protein L7A
40S ribosomal protein S2
26S proteasome regulatory subunit S2
Cell structure/mobility
Actin-5C
Tublin ␤-4
Importin ␤-3
Verprolin
Cell defense/organism defense
HSP2
HSC4
T complex protein 1 ETA subunit
Metabolism
S-adenosylmethionine synthetase
Mitochondrial phosphate carrier protein
ADP/ATP translocase
Cytochrome C oxidase poly1
Cytochrome C oxidase poly3
NADH-ubiquinone oxidoreductase chain1
Unclassified
GPI-anchored protein
Repetitive sequence or virus-like sequences
BMC1
Tymovirus-like COAT protein
Tymovirus-like RNA replicase
Accession no. a
BmN
2 h.p.i.
6 h.p.i.
12 h.p.i.
P19109
Q08473
P12676
P29520
P13549
P13060
P10159
P09180
P36578
P46223
P31009
Q13200
9
5
1
13
5
1
1
6
7
5
3
4
20
—
3
—
1
2
2
1
14
—
—
—
23
1
4
15
5
8
6
3
13
1
2
—
7
—
1
—
—
—
1
—
1
—
1
—
P10987
P30883
O00410
P37370
1
2
—
7
3
5
—
—
8
7
6
2
1
—
—
1
P54652
P11147
P80313
7
5
2
11
2
—
10
1
5
1
1
1
P40320
P16036
Q27238
P34838
P33508
P34846
1
—
8
1
—
—
3
8
10
5
—
—
3
4
6
8
3
—
1
1
4
18
6
3
Q14444
—
7
—
—
AB008780 b
P20124
P10358
24
14
35
2
1
7
—
1
10
—
1
1
Note. Among the four stages, the major host genes that appeared at least three times in any single library, were examined. —, not detected in the
sequenced cDNAs in each library.
a
Accession numbers of PIR and SWISS-PROT.
b
Accession number of GenBank/EMBL/DDBJ.
library, but was absent from mock and earlier infection
stage libraries.
On the other hand, ESTs for ADP/ATP translocase and
mitochondrial phosphate carrier protein, which are localized in the mitochondria but encoded by the host genome, decreased in parallel with other host genome
encoded genes. ADP/ATP translocase EST appeared 8,
10, 6, and 4 times, whereas mitochondrial phosphate
carrier protein EST appeared 0, 8, 4, and 1 times in the
BmN, 2, 6, and 12 h.p.i. libraries, respectively. Northern
hybridization analysis was performed to confirm the expression profiles of COI, ADP/ATP translocase, and Tublin ␣ for a representative nuclear genome-encoded gene
at 2, 6, 12, 18, and 24 h.p.i. (Fig. 2). The expression of COI
was stable until 24 h.p.i.; however, the expression of
ADT/ATP translocase and Tublin ␣ gradually decreased
until 12 h.p.i. and then dramatically decreased after
18 h.p.i.
Apoptosis-related genes in the EST database
Apoptosis is employed as a host defense mechanism
against viral infection. The p35 genes of Autographa
californica multinucleocapsid NPV (AcMNPV) and BmNPV are inhibitors of the caspase family, which plays a
central role in apoptotic cell death (Clem et al., 1991;
Kamita et al., 1993). The inhibitor of apoptosis (iap) genes
isolated from Cydia pomonella granulosis virus (CpGV)
and Orgyia pseudotsugata NPV (OpMNPV) are able to
complement the antiapoptotic function of the p35 gene
EST DATABASE OF BmNPV-INFECTED BmN CELLS
353
homologue. The other gene potentially associated with
apoptosis was a homologue of human death-associated
protein 1, DAP-1 (Levy-Strumpf and Kimchi, 1998). The
EST NV120764, which was found in the 12 h.p.i. library,
had high homology to DAP-1 (Accession No. PIR, I37274;
P value: 1.4e⫺18) by BLASTX homology search, but no
other significant homologue (Fig. 3B).
DISCUSSION
FIG. 2. Northern blot analysis of selected BmN genes. 5 ␮g of total
RNA per lane from mock-infected (M) and infected cells (2, 6, 12, 18,
and 24 h.p.i.) was separated on 1.2% agarose gel. Genes to which the
probes correspond are identified to the right of the images. The ribosomal RNA signals are shown as a loading control.
(Crook et al., 1993; Clem and Miller, 1994). Moreover, the
expression of viral DNA synthesis machinery or transient
expression of the viral transactivator IE-1 of AcMNPV is
sufficient to induce apoptosis in transfected cells (Lu and
Miller, 1995; Prikhod’ko and Miller, 1996). These facts
suggest a significant role for apoptosis during baculovirus infection.
Searching the EST database based on 2645 cDNAs
described here, we found two host-derived cDNAs that
might participate in the apoptotic pathway of BmN cells
following BmNPV infection (Fig. 3). One gene was a
homologue of the iap gene of baculoviruses. Homologues of the iap family are widely conserved from virus
to human (Miller, 1999). The EST NV021896 appeared in
the 2 h.p.i. library and showed the highest homology to
CpGV IAP (P value: 3.9e⫺13; Fig. 3A), but relatively lower
homology with the Drosophila homologue, DIAP1 (Accession No. PIR: A45679, P value: 7.5e⫺07; Hay et al.,
1995) and the human homologue, hIAP-2 (Accession No.
SWISS-PROT: Q13490, P value: 3.9e⫺08; Rothe et al.,
1995), despite the fact that this gene is a B. mori IAP
Baculovirus infection of a host cell is a complex interaction whereby the virus is dependent upon the integrity
of a variety of host systems, yet the ultimate goal is to
maximize virion production which leads to the destruction of the host cell. One approach to elucidate this
interaction is to examine the expression of host and viral
gene products during a time course of the infection. In
this report, we describe the construction of an EST database to investigate the differential gene expression
profiles of BmN cells infected with BmNPV. In contrast to
the many ongoing EST database projects, our EST database is the first one constructed to analyze large-scale
changes in the gene expression of host cells due to
infection with a large DNA virus.
NPV genes are classified into immediate early, delayed early, late, and very late hyperexpressed genes
(Blissard and Rohrmann, 1990; Friesen and Miller, 1986),
and host protein synthesis is significantly curtailed in
6–18 h.p.i. However, the specific genes involved in the
early stages of infection are not well characterized. Furthermore, expression analysis has not been conducted
for over half of all ORFs in the BmNPV genome.
From our EST database analysis, it was evident that
host gene expression decreased and BmNPV gene expression increased dramatically between 6 and 12 h.p.i.
Since the assembly of BmNPV replication factories occurs at around 8 h.p.i. (Okano et al., 1999), the major
increase of BmNPV transcripts might reflect their association in the production of these structures.
FIG. 3. Alignment of BmN ESTs with apoptosis-related proteins. Shown are BLASTX homology search results for NV021896 and NV120764. (A)
NV021896 has significant homology with CpGV IAP (Crook et al., 1993; PIR: A45679): P value: 3.9e⫺13, 68% identity and 84% similarity. (B) NV120764
has significant homology with DAP-1 (Levy-Strumpf and Kimchi, 1998; PIR: I37274): P value: 1.4e⫺18, 53% identity and 65% similarity. * indicates an
identical amino acid. ⫹ indicates a conservative amino acid substitution.
354
OKANO ET AL.
Of the viral ESTs in the 2 h.p.i. library, the only immediate early genes found were ie-0 and ie-2. Neither ie-1
nor pe38 were identified. By colony hybridization of the
2 h.p.i. library, we found that the frequency of ie-1 and
pe38 and ie-2 were 3, 1, and 7 in over 10,000 colonies
screened (data not shown). Therefore, although these
genes are considered to be the earliest baculovirus
genes expressed, in the context of the total mRNA
present, their frequency is very low. In the 6 h.p.i. library,
several genes associated with viral DNA replication, late
transcription, and some structural protein genes were
found, but ie-1 and pe38 were still not found at this stage.
There could be several causes of the differences for
these observations. The level of expression of ie-1 and
pe38 may be lower than that of the replication/transcription and structural protein genes. It is also possible that
these transcripts are less stable. Furthermore it is also
possible that the differences between these cDNA is not
statistically significant. Future more detailed studies with
a larger sample size or incorporating gene-array technology should clarify these issues.
In the 12 h.p.i. library, a large number of transcripts of
bro genes was identified. The bro genes are highly
conserved and often present as multiple copies in the
baculovirus genome (Kuzio et al., 1999). The transcription
of BmNPV bro genes begins by 2 to 4 h.p.i. and is
maximal at 8 h.p.i. (Kang et al., 1999). Interestingly, we
found that the five bro genes constituted 12% of the ESTs
from the 12 h.p.i. library, suggesting that bro genes play
important roles in early baculovirus infection. Moreover,
31 of the 90 identified ORFs in this stage represented
genes predicted from the BmNPV sequence analysis
(Gomi et al.,1999), but for which evidence for expression
was lacking.
BMC1, tymovirus RNA replicase, and tymovirus coat
protein homologues were highly expressed in host BmN
cells. It has been shown that 5000 copies of BMC1 exist
in the B. mori genome and were expressed in fat body
(Ogura et al., 1994). By searching the entire B. mori EST
database, BMC1 was found to be expressed not only in
BmN cells, but also in many tissues, including the embryo, fat body, midgut, and others (data not shown). The
analyzed tymovirus-like ESTs contained multiple stop
codons in three frames. Smear signals were detected
with genomic Southern blot of BmN cells and silk glands
of B. mori (data not shown). These indicate that the
virus-like sequences are also novel repetitive sequences
encoded by the host genome.
The detailed analysis of host-derived ESTs showed
that most of the major host genes became reduced
upon virus infection. The BMC1 and novel virus-like sequences also appeared to decrease quickly as BmNPV
infection proceeded. The expression of the mitochondrial-encoded genes COI and COIII increased in the EST
database with the progression of infection (Table 3). COI
was transcribed at the same level from 0 to 24 h.p.i. by
Northern blot hybridization (Fig. 2). Northern blotting also
showed the same expression patterns for COIII and the
cytochrome b oxidase gene, which is encoded by mitochondrial genome (data not shown). Consistent with this,
a previous report showed that expression of the Spodoptera frugiperda COIII gene was stable until at least
24 h.p.i. during AcMNPV infection (Ooi and Miller, 1988).
As shown in Fig. 1, the ratio of host-derived transcripts
gradually decreased during the infection. In addition, the
ATP/ADP translocase and mitochondrial phosphate carrier protein genes, which are encoded by the host nuclear genome and play important roles in mitochondrial
metabolism, showed decreasing expression during infection (Table 3 and Fig. 2). These observations indicate
that BmNPV infection does not affect mitochondrial-specific transcription during the BmNPV infection process.
Consequently, the increasing number of ESTs of COI and
COIII genes might be attributed to an overall general
reduction in host gene expression, as a direct result of
increased BmNPV gene expressions during infection.
Our study revealed two new apoptosis-related genes
of BmN cells. Interestingly, one of the genes was a CpGV
iap homologue. Homologues of iap have been isolated
from yeast, nematode, mammals, and insects (Miller,
1999). Until recently, the Drosophila iap homologues
DIAP1 and DIAP2 (Hay et al., 1995) were the only ones
isolated from insects. However Tn-IAP1 and SfIAP have
recently been isolated from Trichopusia ni and Spodoptera frugiperda, respectively (Seshagiri et al., 1999; Huang
et al., 2000). NV021896, an EST that is a partial cDNA
sequence of a B. mori iap homologue, is more similar to
SfIAP and Tn-IAP1 than to CpGV iap. Furthermore, this
study shows that this B. mori IAP exists during the
BmNPV infection process. Although the sequence of the
B. mori iap homologue is incomplete, we speculate the
baculovirus iap was derived from the lepidopteran iap
during baculovirus evolution, because lepidoptera is the
major host of these viruses and viral iap homologues
have only been found in members of the baculoviridae.
The other apoptosis-related gene we isolated was a
homologue of the human DAP-1 gene. The DAP-1 gene
was originally isolated by expression screening to identify related apoptotic genes by the presence of cytokineinterferon-␥ (Levy-Strumpf and Kimchi, 1998). However,
its function remains unknown. This is the first report of an
insect DAP-1, perhaps indicating a high degree of conservation in various creatures. What roles these isolated
genes play in the baculovirus infection process are currently unclear. However, functional analysis of the IAP
and DAP-1 homologues will give new information on the
apoptotic pathway of the host as a defense mechanism
during viral infection.
This study demonstrated the EST analysis is a potentially powerful tool for the qualitative examination of differentially regulated mRNAs in host cells. DNA chips
permit genome-wide expression level studies such as
EST DATABASE OF BmNPV-INFECTED BmN CELLS
reported for the analysis of differential patterns of host
gene and human cytomegalovirus gene expression (Zhu
et al., 1998; Chambers et al., 1999). We are planning to
introduce DNA chip technology based on the B. mori EST
database for more detailed analysis of expression profiles of BmNPV and host genes and to clarify the molecular responses of NPV–host interactions.
MATERIALS AND METHODS
Cells and virus
The BmNPV T3 isolate was plaque-purified from a
virus preparation originally described by Maeda (1989).
The T3 isolate was propagated in a B. mori cell line as
described previously (Maeda, 1985; Maeda et al., 1989).
BmN cells were infected with BmNPV at a multiplicity of
infection (m.o.i.) of 10 PFU per cell. The infected BmN
cells were harvested at 2, 6, 12, 18, and 24 h.p.i.
cDNA library construction
Total RNA was isolated from mock-infected BmN cells
and from BmNPV-infected BmN cells at 2, 6, 12, 18, and
24 h.p.i. using a Rapid Total RNA Isolation kit (5 Prime 3
3 Prime). The mRNA was isolated from the purified total
RNA with a Micro-FastTrack kit (Invitrogen). Four infection-stage cDNA libraries, BmN and 2, 6, and 12 h.p.i.
libraries, were constructed from isolated mRNA with a
Zap-cDNA Synthesis kit (Stratagene) according to the
manufacturer’s instructions. In brief, first-strand cDNA
was synthesized from 5 ␮g of mRNA with the oligo d(T)
linker-primer, 5⬘-(GA) 10ACTAGTCTCGAG(T) 18-3⬘ and SuperScript II (Gibco BRL) instead of the MMLV reverse
transcriptase supplied in the kit. Second-strand cDNA
was synthesized with DNA polymerase I in the presence
of RNase H, and then the cDNA was blunt ended with Pfu
DNA polymerase. After EcoRI adapter ligation and digestion by XhoI, cDNA was fractionated by Sepharose CL-2B
gel filtration. The cDNA was directionally inserted into
the EcoRI–XhoI site of Uni-Zap phage vector. The average size of the insert cDNA was approximately 2 kb
among for four cDNA libraries. After mass in vivo excision by ExAssist helper phage infection to convert from
the Uni-Zap phage vector to a pBluescript vector, about
1000 white colonies were randomly isolated from each
library on LB-ampicillin plates containing X-gal.
Automatic DNA sequencing and EST database
construction
In all, 4100 colonies were inoculated into 2.5 ml of
LB-ampicillin medium and cultured for 10 h. The plasmid
DNA was purified using a PI-100 automatic DNA isolation system (Kurabo). The purified DNA was resuspended in 0.2 ml of TE buffer. A single-pass sequence
reaction was carried out from the 5⬘ end using a T3 dye
primer and 4 ␮l of purified DNA template. The reaction
355
products were electrophoresed on ABI377XL sequencers (ABI). The cDNA samples were named as follows:
N0001–N1100 from the BmN library, NV021001–
NV022000 from the 2 h.p.i. library, NV060001–NV061000
from the 6 h.p.i. library, and NV120001–NV121000 from
the 12 h.p.i. library. All of the edited ESTs were classified
into subgroups relative to each other by a BLASTN homology search and then compared to SWISS-PROT and
PIR database by a BLASTX homology search on a UNIXbased analysis system, DYNACLUST (Dynacom). An EST
sequence was defined as a homologue of a known gene
if it had at least 30% similarity over a minimum of 100
amino acids using the BLASTX. To identify ESTs derived
from BmNPV ORF, each ORF sequence of BmNPV was
searched by BLASTN against all the EST data. Sequence
data from this article have been deposited in DDBJ (DNA
Data Bank of Japan) under Accession Nos. AU002477–
AU003299 for N0001–N1100, AV398029–AV398029–
AV398586 for NV021003–NV022000, AV398587–AV399270
for NV060052–NV060991, and AV399271–AV399916 for
NV120002–NV120960.
Northern hybridization
Total RNA, 5 ␮g per lane, was separated by electrophoresis on 1.2% agarose gel containing 2.2 M formaldehyde, transferred onto nylon membranes (Hybond-N;
Amersham), immobilized by UV cross-linking (GS Gene
Linker; Bio-Rad) and hybridized with 32P-labeled probes.
The DNA probes were labeled with [␣- 32P]dCTP with
Ready To Go DNA Labeling Beads (Pharmacia), and heat
denatured. The probe was added to the prehybridization
solution (5⫻ SSC/5⫻ Denhartds’/0.1% SDS) and incubated with the membrane at 65°C overnight. After incubation, the membrane was washed at room temperature
in 2⫻ SSC/0.1% SDS for 5 min and twice in 0.5⫻ SSC/
0.1% SDS at 65°C. Signals were detected using a BAS2000 imaging analyzer (Fuji Film) and X-ray films.
ACKNOWLEDGMENTS
This paper is dedicated to the memory of Susumu Maeda. The
authors thank Masaaki Kurihara for help with the culture of BmN cells.
We thank George Rohrmann, Marian Goldsmith, and Jennifer Ito for
critical reading of the manuscript. This work was partially supported by
a CREST award from Japan Science and Technology Corporation
(S.M.), Special Postdoctoral Researchers Program, RIKEN (K.O.), a
Grant-in-Aid for Scientific Research from the Japanese Society for
Promotion of Science (12760038) (K.O.), and the Bombyx genome research program in MAFF, Japan (T.S. and K.M).
REFERENCES
Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M.,
Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992).
Sequence identification of 2375 human brain genes. Nature 355,
632–634.
Adams, M. D., Kerlavage, A. R., Fleischmann, R. D., Fuldner, R. A., Bult,
C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D.,
White, O., et al. (1995). Initial assessment of human gene diversity
356
OKANO ET AL.
and expression patterns based upon 83 million nucleotides of cDNA
sequence. Nature 377(6547 Suppl), 3–174.
Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C., and Venter, J. C.
(1993). Rapid cDNA sequencing (expressed sequence tags) from a
directionally cloned human infant brain cDNA library. Nat. Genet. 4,
373–380.
Blissard, G. W., and Rohrmann, G. F. (1990). Baculovirus diversity and
molecular biology. Annu. Rev. Entomol. 35, 127–155.
Chambers, J., Angulo, A., Amaratunga, D., Guo, H., Jiang, Y., Wan, J. S.,
Bittner, A., Frueh, K., Jackson, M. R., Peterson, P. A., Erlander, M. G.,
and Ghazal, P. (1999). DNA microarrays of the complex human
cytomegalovirus genome: Profiling kinetic class with drug sensitivity
of viral gene expression. J. Virol. 73, 5757–5766.
Clem, R. J., Fechheimer, M., and Miller, L. K. (1991). Prevention of
apoptosis by a baculovirus gene during infection of insect cells.
Science 254(5036), 1388–1390.
Clem, R. J., and Miller, L. K. (1994). Control of programmed cell death by
the baculovirus genes p35 and iap. Mol. Cell. Biol. 14, 5212–5222.
Crook, N. E., Clem, R. J., and Miller, L. K. (1993). An apoptosis-inhibiting
baculovirus gene with a zinc finger-like motif. J. Virol. 67, 2168–2174.
Delseny, M., Cooke, R., Raynal, M., and Grellet, F. (1997). The Arabidopsis thaliana cDNA sequencing projects. FEBS Lett. 403, 221–224.
Friesen, P. D., and Miller, L. K. (1986). The regulation of baculovirus
gene expression. Curr. Top. Microbiol. Immunol. 131, 31–49.
Gomi, S., Majima, K., and Maeda, S. (1999). Sequence analysis of the
genome of Bombyx mori nucleopolyhedrovirus. J. Gen. Virol. 80,
1323–1337.
Hay, B. A., Wassarman, D. A., and Rubin, G. M. (1995). Drosophila
homologs of baculovirus inhibitor of apoptosis proteins function to
block cell death. Cell 83, 1253–1262.
Huang, Q., Deveraux, Q. L., Maeda, S., Salvesen, G. S., Stennicke, H. R.,
Hammock, B. D., and Reed, J. C. (2000). Evolutionary conservation of
apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of
apoptosis proteins are inhibitors of mammalian caspase-9. Proc.
Natl. Acad. Sci. USA 97, 1427–1432.
Kamita, S. G., Majima, K., and Maeda, S. (1993). Identification and
characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis. J. Virol. 67, 455–463.
Kang, W. K., Suzuki, M., Zemskov, E., Okano, K., and Maeda, S. (1999).
Characterization of baculovirus repeated open reading frames (bro)
in Bombyx mori nucleopolyhedrovirus. J. Virol. 73, 10339–10345.
Kuzio, J., Pearson, M. N., Harwood, S. H., Funk, C. J., Evans, J. T.,
Slavicek, J. M., and Rohrmann, G. F. (1999). Sequence and analysis of
the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253, 17–34.
Levy-Strumpf, N., and Kimchi, A. (1998). Death associated proteins
(DAPs): From gene identification to the analysis of their apoptotic and
tumor suppressive functions. Oncogene 17, 3331–3340.
Lu, A., and Miller, L. K. (1995). The roles of eighteen baculovirus late
expression factor genes in transcription and DNA replication. J. Virol.
69, 975–982.
Maeda, S. (1989). Expression of foreign genes in insects using baculovirus vectors. Annu. Rev. Entomol. 34, 351–372.
Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y.,
Sato, Y., and Furusawa, M. (1985). Production of human alpha-interferon in silkworm using a baculovirus vector. Nature 315, 592–594.
Miller, L. K. (1999). An exegesis of IAPs: Salvation and surprises from
BIR motifs. Trends Cell. Biol. 9, 323–328.
Ogura, T., Okano, K., Tsuchida, K., Miyajima, N., Tanaka, H., Takada, N.,
Izumi, S., Tomino, S., and Maekawa, H. (1994). A defective non-LTR
retrotransposon is dispersed throughout the genome of the silkworm, Bombyx mori. Chromosoma 103, 311–323.
Okano, K., Mikhailov, V. S., and Maeda, S. (1999). Colocalization of
baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to
viral replication factories. J. Virol. 73, 110–119.
Ooi, B. G., and Miller, L. K. (1988). Regulation of host RNA levels during
baculovirus infection. Virology 166, 515–523.
Prikhod’ko, E. A., and Miller, L. K. (1996). Induction of apoptosis by
baculovirus transactivator IE1. J. Virol. 70, 7116–7124.
Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V.
(1995). The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83,
1243–1252.
Seshagiri, S., Vucic, D., Lee, J., and Dixit, V. M. (1999). Baculovirusbased genetic screen for antiapoptotic genes identifies a novel IAP.
J. Biol. Chem. 274, 36769–36773.
Yamamoto, K., and Sasaki, T. (1997). Large-scale EST sequencing in
rice. Plant Mol. Biol. 35, 135–144.
Zhu, H., Cong, J. P., Mamtora, G., Gingeras, T., and Shenk, T. (1998).
Cellular gene expression altered by human cytomegalovirus: global
monitoring with oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95,
14470–14475.