Sequence
Analysis of the BamHI-J Fragment of the Spodoptera litura
Multicapsid Nucleopolyhedrovirus
WANG
Li-Hua, YU Jian-Xiu, HU Xiao-Hui, PANG Yi*
( State Key Laboratory for Biocontrol
and Institute of Entomology, Zhongshan University, Guangzhou 510275,
China )
Abstract
The BamHI-J fragment located at 25.8―29.9
map units of the Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV)
genome was sequenced. The fragment contained four ORFs, one partial ORF
potentially encoding C-terminal of chitinase gene and one partial homologous
region (hr). The four ORFs included lef-8 gene, J domain protein
gene (bjdp gene), ORF570 and ORF165. The ORF570 revealed 31% identity to
the helicase-2 of Lymantria dispar MNPV. The ORF165 was unique to the
SpltMNPV. The bjdp gene, reported here for the first time in
baculoviruses, was one of J domain family protein genes, and the predicated
amino acid sequence possessed a characteristic of J domain protein of other
DnaJ proteins at its N-terminus. The lef-8 showed high identities to the
homologs of reported baculovirus genomes. As a component of virus-encoded RNA
polymerase, the LEF-8 of SpltMNPV had the conserved motif GIKICGIHGQKG near the
C-terminal end. Analysis of the LEF-8 phylogenic tree demonstrated SpltMNPV was
very closely related to SpliMNPV.
Key
words Spodoptera litura
multicapsid nucleopolyhedrovirus; lef-8 gene; J domain protein; sequence
analysis
The
cotton leafworm, Spodoptera litura, is an important pest of crops and
vegetables in south China and Southeast Asia. S. litura multicapsid
nucleopolyhedrovirus (SpltMNPV) infects only a single host, S. litura,
and it has been successfully applied as commercial biological insecticide
against this pest in China[1]. The sequences of a number of SpltMNPV
genes including polyhedrin gene[2], egt gene[3], odv-e66
gene[4], p10 gene[5], pk gene[6],
p74 gene[7], chitinase gene[8] and p49 gene[9]
have been elucidated and characterized.
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.
1 Materials and Methods
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 Results and Discussion
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.
The
BamHI-J fragment located between map units 25.8 and 29.9 of the SpltMNPV
genome [Fig.2(A)], encompassed 5 620 bp in total. The fragment contained four
ORFs, one partial homologous region (hr) and one partial ORF that
potentially encoded the C-terminus of chitinase, which is involved in
liquefaction of virus-infected larvae[33,34]. The four ORFs included
lef-8 gene, J domain protein gene (designated as baculovirus J domain
protein gene, bjdp gene), ORF570 and ORF165 (named according to the
length of nucleotides) [Fig.2(B)].
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.
2.2 ORF570 and ORF165
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.
In
viruses, a number of functions of J domain protein have been reported[36,38,39].
The J domain motif exists within the N-terminal segment of T antigen in
polyomavirus family. The J domain of DnaJ proteins has been shown to activate
the intrinsic ATPase activity of associated Hsp70; ATP hydrolysis may
contribute to the ability of certain DnaJ homologs to modulate folding of the
multimeric protein complex[36]. In addition, the conservation of the
J domain motif suggests the J domain plays an essential role in the viral life
cycle[38,39]. But in baculoviruses, the function of the J domain
protein remains unclear.
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.
The
location of the lef-8 gene within genome also gave information on the
relatedness among the baculoviruses. Among NPV genomes reported, the lef-8
gene all located next to the homolog of AcMNPV ORF51 except LdMNPV genome. The
transcription orientations of the two ORFs were opposite[12-16, 46].
The SpltMNPV lef-8 gene was adjacent to the bjdp gene. However,
BLAST analysis revealed that bdjp did not show identity to the homologs
of Ac51 in AcMNPV, OpMNPV, BmNPV or HaSNPV genomes. In addition, a homologous
region was located downstream of the lef-8 gene in SpltMNPV, that was
similar to the corresponding SpliMNPV region (GenBank accession No.Y10669),
providing further evidence that SpltMNPV and SpliMNPV was closely related.
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.
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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]