|
Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(6):
615-620
CN 31-1300/Q |
Sequence
Analysis of the BamHI-J Fragment of the Spodoptera litura
Multicapsid Nucleopolyhedrovirus
( State Key Laboratory for Biocontrol
and Institute of Entomology, Zhongshan University, Guangzhou 510275,
China )
In
Autographa californica MNPV genome, nineteen genes are required for the
transactivation of late gene expression such as vp39 and p6.9
genes, the very late polyhedrin and p10 genes, including ie-1, ie-2,
lef-1-lef-12,
dnapol, p143, p47, p35 and 39k etc[10].
These genes are referred to as late gene expression factor (LEF) genes. Four of
the transcription-specific LEFs, LEF-8, LEF-9, LEF-4, and P47, are components
of the virus-encoded RNA polymerase[11]. In addition to AcMNPV, lef-8
gene has also been identified in Orgyia pseudotsugata MNPV[12],
Bombyx mori NPV[13], Spodoptera exigua MNPV[14],
Lymantria dispar MNPV[15], Helicoverpa armigera single
nucleocapsid NPV[16,17], Helicoverpa zea single nucleocapsid
NPV (GenBank accession No.U67265), Spodoptera littoralis MNPV[18],
Xestia c-nigrum granulovirus(XcGV)[19], and Plutella
xylostella granulovirus (PxGV)[20]. But lef genes have
not been studied in SpltMNPV.
Molecular
chaperones occur ubiquitously and many of them are classified as stress
proteins, although they have essential functions under normal growth
conditions, such as Hsp70 and Hsp40[21]. Proteins of the Hsp40
family typically contain one or more blocks of sequence homology to E.coli
DnaJ, a 41 kD protein with a clear domain structure[21]. The DnaJ
protein family shares a modular organization called J domain, which is highly
conserved within 70 amino acids near the N-terminus. Several eukaryotic DnaJ
proteins have been identified in various organisms[22-26], but none
have been reported in baculoviruses.
In
a previous investigation, a detailed physical map of SpltMNPV was constructed
for five restriction enzymes (data not shown). In this paper, we identified and
analysed a 5.6 kb BamHI-J fragment located between map units 25.8 and
29.9 of the SpltMNPV genome. The homologs of proteins of SpltMNPV were compared
with other organisms. In addition, the organization of this fragment was used
to investigate the ancestral relationships of SpltMNPV to other
baculoviruseses.
SpltMNPV
genotype strain G2 was isolated from ZSU strain following a modification of the
in vivo method described by Smith et al[27]. The viral
DNA was extracted according to the method described by O'Reilly et al[28].
The DNA was digested with restriction enzyme (New England) and the fragments
were separated by electrophoresis in 1% agarose gel at 40 V (1.5 V/cm) for 20-22
h. The BamHI-J fragment was recovered using Gel Extraction Kit (Qiagen),
and transferred into pUC18 according to the methods of Sambrook et al[29].
Plasmid DNA was purified using Plasmid Extraction Kit (Qiagen) according to the
handbook. The termini of the fragment were sequenced using the universal
primers. Further sequencing were done by primer walking using specific
oligonucleotide primers. Each DNA strand was sequenced three or more times in
each direction using different primers. Sequencing reactions were run on a
MegaBACE1000 sequencer using tetra-color-fluorescently-labeled terminater
method. DNA sequence analysis was analyzed using DNASIS and DNASTAR. Deduced
amino acid sequences were compared with the updated GenBank data using BLAST
programs[30]. The analyses of domains in proteins were carried out
by SMART online[31,32].
2.1
Organization of the SpltMNPV BamHI-J fragment
SpltMNPV
DNA was isolated from purified virions and digested with BamHI. The size
of fragments ranged from about 27 to 1.5 kb, and the total size of the SpltMNPV
genome was estimated to be about 139 kb(Fig.1).
Fig.1 Electrophoresis in 1% agarose
gel of the SpltMNPV DNA isolated from purified virus particles and digested
with BamHI
M1,
Lambda DNA/HindIII; S, SpltMNPV genomic DNA digested with BamHI;
M2, 1 kb DNA ladder (New England); J, BamHI-J fragment.
Fig.2 The localization of BamHI-J
fragment of SpltMNPV and the analysis of four ORFs
(A) BamHI map of the SpltMNPV genome.
The position and orientation of the SpltMNPV polyhedrin are indicated. (B) The
gene organization of the BamHI-J fragment of the SpltMNPV is shown.
m.u., map unit.
The
ORF570 encoded a polypeptide of 189 aa with a deduced molecular mass of 21.636
kD. BLAST analysis indicated the predicated protein showed 31% identity to the
helicase-2 of LdMNPV, overlapping only 43 aa and it was smaller than the
homolog of LdMNPV (LdMNPV helicase-2 molecular mass 99.211 kD)[15].
BLAST analysis showed ORF570 shared 25% identity to PxGV ORF107.
The
ORF165 encoded a polypeptide of 54 aa with a deduced molecular mass of 6.225
kD. Motif search found a late promoter motif TAAG located at -12 to -15 nt and
a TATA box located at -14 to -17 nt upstream of the translational start codon.
ORF165 did not show homology at the amino acid level to any other protein from
GenBank and was unique to SpltMNPV. The function of these two ORFs remained to
be investigated.
2.3
Baculovirus J domain protein gene (bjdp)
The
bjdp ORF of 909 bp encoded a predicted protein of 302 amino acids (aa)
with a molecular mass of 34.631 kD. Its orientation was the same as the
polyhedrin gene. The 5′
noncoding region contained an early transcription motif CAGT located at -75--78
nucleotides (nt), and two TATA boxes located at -32--35
nt and -40--43
nt, respectively, upstream of the translational start codon. No polyadenylation
signal site was found within the noncoding region downstream of the translation
termination codon. BLAST homology search revealed that the predicated product
BJDP showed identities of various degrees to the homologs of DnaJ proteins in
other organisms at the N-terminus (position: 13-68
aa) and 31% identity to the homolog of SeMNPV ORF111 (overlapping about 96 aa,
position: 147-242
aa). However, SeMNPV ORF111 lacked the J domain at the N-terminus, and the
function of SeMNPV ORF111 was unknown.
DnaJ
protein was first purified from E.coli, and has been shown to possess a dimeric
form with the molecular mass of 76 kD under native conditions[35].
DnaJ protein is involved with DNA replication, stimulating the capacity of DnaK
(the homolog of Hsp70 in E.coli) to form a replication-competent complex
at the phage origin of replication. Alignment of the J domain of BJDP in
SpltMNPV and J domain of different organisms revealed the highly conserved
tripeptide histidine-proline-aspartate (HPD) existed in a loop between helix II
and helix III(Fig.3). HPD is absolutely conserved within the J domain of all
known DnaJ homologs, substitution mutations in any of the residues render the J
domain defective in activating Hsp70[36,37].
Fig.3 Amino acid sequence alignment of
J domain and the conserved J domain present in different organisms
The J domain of SpltMNPV BJDP is shown at
the top, followed by the sequence in other prokaryotic and eukaryotic J domain
of DnaJ family proteins. The number in parentheses to the left of each sequence
indicates the position in the polypeptide for the first residue listed. The
shaded regions represent 100% identity among sequences. The polypeptide
sequences were obtained from GenBank data bases by accession number as follows:
Aquifex aeolicus, E70361; Bacillus stearothermophilus, JC4739; Haemophilus
influenzae Rd, C64112; Methylovorus sp.SS1, AAC95379.1; Arabidopsis
thaliana, AAD39315; Saccharomyces cerevisiae, S48085; Thermus
thermophilus, AAB04678.1; Escherichia coli, AAC73126.1.
Besides
the J domain, SMART analysis of BJDP domains revealed it possessed a coiled
coil region between 132-159
aa. The coiled-coil motif played an important role in the oligomerization and
fusion activity of other viral glycoproteins[40].
2.4
lef-8 gene
The
SpltMNPV lef-8 gene was adjacent to hr in BamHI-J
fragment, and the orientation was opposite to the polyhedrin gene [Fig.2(B)]. No
promoter motif [TATA box, CAGT or (A/G/T) TAAG] was found within 210 bp
upstream of the translation initiation codon. The possible polyadenylation site
(AATAAA) was present immediately downstream of the translation termination
codon (2 nt). The gene product of SpltMNPV lef-8 predicted a polypeptide
of 106.196 kD, which was similar to that of the ten baculoviruses to which it
was compared (Table 1). BLAST homology search revealed the SpltMNPV LEF-8 amino
acid sequence showed high identities to homologs of other baculoviruses, with
the maximal identity reaching 86% (SpliMNPV, Table 1). The sizes of LEF-8 and
the high identity suggested the LEF-8 was well-conserved in baculoviruses.
AcMNPV
LEF-8 possesses a conserved motif, GXKX4HGQ/NKG, which is found in
the DNA-directed RNA polymerase from a diverse range of organisms including
bacteria, yeasts, plants, invertebrates and vertebrates[41]. In
every case, the location of this motif is positionally conserved (at the
C-terminus) and is thought to be an essential component at the catalytic site
of the polymerase[42]. A similar motif GIKICGIHGQKG was also found
at the C-terminus of SpltMNPV LEF-8 (position: 764-776
aa). As well as the GIKICGIHGQKG motif, the alignment of the amino acid
sequence of SpltMNPV LEF-8 and other viral LEF-8 showed that these LEF-8
proteins exhibited a number of conserved regions (data not shown), especially
near the C-terminus. This indicated that the conserved regions were necessary
for the function as a component of virus-encoded RNA polymerase. The presence
of the LEF-8 conserved motif in all the baculoviruses compared suggested that
baculovirus RNA polymerase could be different to the counterparts of other organisms.
A
phylogenic tree was constructed to estimate evolutionary relationships
according to the LEF-8 sequence (Fig.4). Based on the phylogenic tree, the
baculoviruses were divided into GV and NPV groups. The NPV group included two
clades. The first clade was comprised of two closely related subclades:
SpltMNPV and SpliMNPV. The second clade had two subclades: Group I and Group II[43].
This result indicated that SpltMNPV and SpliMNPV were not clustered in either
Group I or Group II, and it coincided with the result found using polyhedrin
sequences (data not shown) and the study of Levin et al[44].
In contrast, Bulach et al[45] did include SpltMNPV and
SpliMNPV in Group II by using the DNA polymerase gene sequence.
Fig.4 A phylogenetic tree of
baculoviruses based on LEF-8
The
tree was carried out by DNASIS.
In
summary, although the bjdp gene showed 31% identity to SeMNPV ORF111, it
contained a J domain that had not been identified in other baculoviruses to
date. SpltMNPV LEF-8 shared conserved motif GI(V)KICG(S)I(V)HGQKG with other
baculoviruses and it may play a key role in baculoviruses replication and
transcription. The phylogenetic analysis of LEF-8 demonstrated that SpltMNPV
was closely related to SpliMNPV. In addition, the position of lef-8 gene
in genome was comparable to that of other NPVs. Overall results of this study
demonstrate that although SpltMNPV had some characteristics analogous with
other NPVs it also displayed some distinct differences in some respects.
Acknowledgments The authors thank Dr.
Debbie Rae for her comments on the manuscript.
1 Pang Y. Study and application
of insect pathogens. In: Bao J Z et al eds. Biological Control in
China, Taiyuan: Shanxi Science & Technology Press, 1998, 369-494
2 Wei
YJ, Long QX, Chen SW, Wang XZ. Restriction patterns and nucleotide sequence of
the polyhedrin gene of Spodoptera litura nuclear polyhedrosis virus. Microbiology,
1999, 26(2): 88-92
3 Yan
QS, Pang Y, Yang J, Nong G, Ouyang XG, Dai XJ. Characterization of the
ecdysteroid UDP-glucosyltranstferase gene of Spodoptera litura
multinucleocapsid nuclear polyhedrosis virus. Chinese Journal of
Biotechnology, 1999, 15(2): 176-182
4 Zheng
J, Li Z, Long QX, Wei YJ, Wang XZ. Nucleotide sequence of a 4730 base pairs
region of the Spodoptera litura nucleopolyhedrovirus genome. Acta
Biochimica et Biophysica Sinica, 2000, 32(5): 445-450
5 Wei
YJ, Long QX, Chen SW, Wang XZ. Nucleotide sequance and characterization of the p10
gene of Spodoptera litura nuclear polyhedrosis virus. Acta Biochimica
et Biophysica Sinica, 1998, 30(6): 550-555
6 Wei
YJ, Long QX, Chen SW, Wang XZ. Partial nucleotide sequence of protein kinase
gene of Spodoptera litura nuclear polyhedrosis virus. Acta
Scientiarum Naturalium Universitatis Sunyatseni, 1998, 37(5): 119-121
7 Chen
SW, Wei YJ, Long QX, Xu AL, Wang XZ. Cloning and sequencing of p74 gene
of Spodoptera litura nuclear polyhedrosis virus. Acta Scientiarum
Naturalium Universitatis Sunyatseni, 1998, 37(5): 65-69
8 Hu
GD, Pang Y, Yang K, Li CB, Su DM. Cloning and sequance analysis of the
chitinase gene of Spodoptera litura nuclear polyhedrosis virus. Acta
Biochimica et Biophysica Sinica, 2000, 32(5): 537-540
9 Li
Z, Long QX, Zhang YG, Wang XZ, Pang Y. Cloning and sequence analysis of p49
gene from Spodoptera litura nucleopolyhedrovirus (SpltNPV). Acta
Scientiarum Naturalium Universitatis Sunyatseni, 2000, 39: 73-76
10 Rapp
JC, Wilson J A, Miller LK. Nineteen baculovirus open reading frames, including
LEF-12, support late gene expression. J Virol, 1998, 72: 10197-10206
11 Jin
J, Dong W, Guarino LA. The LEF-4 subunit of baculovirus RNA polymerase has RNA
5′-triphosphatase and ATPase
activities. J Virol, 1998, 72: 10011-10019
12 Ahrens
CH, Russell RLQ, Funk CJ, Evans JT, Harwood SH, Rohrmann GF. The sequence of
the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus
genome. Virology, 1997, 229: 381-399
13 Gomi
S, Majima K, Maeda S. Sequence analysis of the genome of Bombyx mori
nucleopolyhedrovirus. J Gen Virol, 1999, 80: 1323-1337
14 IJkel
WFJ, van Strien EA, Heldens JGM, Broer R, Zuidema D, Goldbach RW, Vlak J M.
Sequence and organization of the Spodoptera exigua multicapsid
nucleopolyhedrovirus genome. J Gen Virol, 1999, 80: 3289-3304
15 Kuzio
J, Pearson MN, Harwood S H, Funk C J, Evans J T, Slavicek JM, Rohrmann GF.
Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria
dispar. Virology, 1999, 253: 17-34
16 Chen
X, IJkel WFJ, Tarchini R, Sun X, Sandbrink H, Wang H, Peters S et al.
The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus
genome. J Gen Virol, 2001, 82: 241-257
17 Zhang
CX, Wu JC. Genome structure and the p10 gene of the Helicoverpa
armigera nucleopolyhedrovirus. Acta Biochimica et Biophysica Sinica,
2001, 33(2): 179-184
18 Faktor O, Kamensky B. Genomic
localization and nucleotide sequence of a lef-8 gene of the Spodoptera
littoralis nucleopolyhedrovirus. Virus Genes, 1997, 15: 9-15
19 Hayakawa
T, Ko R, Okano K, Seong SI, Goto C, Maeda S. Sequence analysis of the Xestia
c-nigrum granulovirus genome. Virology, 1999, 262: 277-297
20 Hashimoto
Y, Hayakawa T, Ueno Y, Fujita T, Sano Y, Matsumoto T. Sequence analysis of the Plutella
xylostella granulovirus genome. Virology, 2000, 275: 358-372
21 Hartl
FU. Molecular chaperones in cellular protein folding. Nature, 1996, 381:
571-580
22 Luke
MM, Sutton A, Arndt KT. Characterization of SIS1, a Saccharomyces cerevisiae
homologue of bacterial dnaJ proteins. J Cell Biol, 1991, 114: 623-638
23 Raabe
T, Manley JL. A human homologue of the Escherichia coli DnaJ heat-shock
protein. Nucleic Acids Res, 1991, 19: 6645
24 Preisig-Muller
R, Kindl H. Plant dnaJ homologue: Molecular cloning, bacterial
expression, and expression analysis in tissues of cucumber seedlings. Arch
Biochem Biophys, 1993, 305: 30-37
25 Brightman
SE, Blatch GL, Zetter BR. Isolation of a mouse cDNA encoding MTJ1, a new murine
member of the DnaJ family of proteins. Gene, 1995, 153: 249-254
26 Iliopoulos
I, Torok I, Mechler BM. The DnaJ60 gene of Drosophila melanogaster
encodes a new member of the DnaJ family of proteins. Biol Chem, 1997, 378:
1177-1181
27 Smith
IRL, Crook NE. In vivo isolation of baculovirus genotypes. Virology,
1988, 166: 240-244
28 O'Reilly
DR, Miller LK, Luckow VA. Baculovirus Expression Vectors-A Laboratory Manual,
New York: W.H. Freeman and Company, 1986, 142-142
29 Sambrook
J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd
ed, New York: Cold Spring Harbor Laboratory Press, 1989
30 Altschul,
SF, Madden TL, Schffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST
and PSI-BLAST: A new generation of protein database search programs. Nucleic
Acids Res, 1997, 25: 3389-3402
31 Schultz
J, Milpetz F, Bork P, Ponting CP. SMART, a simple modular architecture research
tool: Identification of signaling domains. Proc Natl Acad Sci USA, 1998,
95: 5857-5864
32 Schultz
J, Copley R R, Doerks T, Ponting CP, Bork P. SMART: A web-based tool for the
study of genetically mobile domains. Nucleic Acids Res, 2000, 28:
231-234
33 Gong
CL, Kobayashi J, Jin W, Wu XF. Inactivation analysis of HcNPV cysteine protease
gene and chitinase gene. Acta Biochimica et Biophysica Sinica, 2000, 32(2):
187-191
34 Gong
CL, Kobayashi J, Miyajima N, Jin W, Wu X F. Nucleotide sequence analysis of the
HcNPV cysteine protease gene. Acta Biochimica et Biophysica Sinica,
1998, 30(3): 307-310
35 Zylicz
M, Yamamoto T, McKittrick N, Sell S, Georgopoulos C. Purification and
properties of the dnaJ replication protein of Escherichia coli. J
Biol Chem, 1985, 260: 7591-7598
36 DeCaprio
JA. The role of the J domain of SV40 large T in cellular transformation. Biologicals,
1999, 27: 23-28
37 Tsai
J, Douglas M G. A conserved HPD sequence of the J-domain is necessary for YDJ1
stimulation of Hsp70 ATPase activity at a site distinct from substrate binding.
J Biol Chem, 1996, 271: 9347-9354
38 Kelley
WL, Landry SJ. Chaperone power in a virus? Trends Biochem Sci, 1994, 19:
277-278
39 Kelley
WL, Georgopoulos C. The T/t common exon of simian virus 40, JC, and BK polyomavirus
T antigens can functionally replace the J-domain of the Escherichia coli
DnaJ molecular chaperone. Proc Natl Acad Sci USA, 1997, 94: 3679-3684
40 Watanabe S, Takada A, Watanabe T, Ito
H, Kida H, Kawaoka Y. Functional importance of the coiled-coil of the Ebola
virus glycoprotein. J Virol, 2000, 74: 10194-10201
41 Passarelli
AL, Todd JW, Miller LK. A baculovirus gene involved in late gene expression
predicts a large polypeptide with a conserved motif of RNA polymerases. J
Virol, 1994, 68: 4673-4678
42 Schultz
P, Celia H, Riva M, Sentenac A, Oudet P. Three-dimensional model of yeast RNA
polymerase I determined by electron microscopy of two-dimensional crystals. EMBO
J, 1993, 12: 2601-2607
43 Zanotto
PM, Kessing BD, Maruniak JE. Phylogenetic interrelationships among
baculoviruses: Evolutionary rates and host associations. J Invertebr Pathol,
1993, 62: 147-164
44 Levin
DB, Whittome B. Codon usage in nucleopolyhedroviruses. J Gen Virol,
2000, 81: 2313-2325
45 Bulach
DM, Kumar CA, Zaia A, Liang B, Tribe DE. Group II nucleopolyhedrovirus
subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase
gene sequences. J Invertebr Pathol, 1999, 73: 59-73
46 Ayres
MD, Howard SC, Kuzio J, Lopez-Ferber M, Possee RD. The complete DNA sequence of
Autographa californica nuclear polyhedrosis virus. Virology,
1994, 202: 586-605
Received: June 14, 2001 Accepted:
July 23, 2001
This work was partially supported by the
National Natural Science Foundation of China (No.39730030) and the Special Funds
for Major State Basic Research (973) of China (No. G2000016209)
The nucleotide sequence database reported
in this paper have been submitted to the GenBank nucleotide sequence database
and have been assigned the accession number AF325155
*Corresponding author: Tel,
86-20-84113860; Fax, 86-20-84037472; e-mail, [email protected]