Laboratory of Insect Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
Correspondence
Yuanyang Hu
yyhu{at}whu.edu.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number of the nucleotide sequence reported in this paper is AY665654.
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MAIN TEXT |
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The members of the genus Densovirus have an approximately 6 kb genome with a similar ambisense genomic organization and long inverted terminal repeats (ITRs; >500 bp). They contain open reading frames (ORFs) encoding non-structural and structural proteins located on the 5'-halves of the genome strands, such as Galleria mellonella densovirus (GmDNV; Tijssen et al., 2003) and Junonia coenia densovirus (JcDNV; Dumas et al., 1992
). The members of the genus Pefudensovirus, possess an approximately 5·5 kb genome with ITRs of about 200 nt, have coding sequences on 5'-halves of ambisense genome. The minus' strand contains no less than two ORFs encoding structural proteins (Guo et al., 2000
). The densoviruses in the genus Brevidensovirus have genomes of about 4 kb with Y-like terminal hairpin structures, but lacking ITRs, and ORFs of non-structural and structural proteins are located on the same strand. Examples are Aedes albopictus parvovirus (AalDNV; Boublik et al., 1994
) and Aedes densonucleosis virus (AaeDNV; Afanasiev et al., 1991
). So far, the genus Iteravirus only consists of two members: Bombyx mori densovirus-1 (BmDNV-1; Li et al., 2001
) and Casphalia extranea densovirus (CeDNV; Fédière et al., 2002
). The genomic DNAs of BmDNV-1 and CeDNV are 5076 and 5002 nt long, respectively. The CeDNV genome organization shows high similarity to that of BmDNV-1. The ORFs encoding non-structural proteins are on the 5'-half of the plus' strand whereas the ORF encoding structural proteins is on the 3'-half of the same strand. Unlike other densoviruses and vertebrate parvoviruses, the terminal sequences of their ITRs can be folded into an imperfect J-shaped palindrome. Like other parvoviruses, the conserved PGY motif is located within the unique part of VP1 of CeDNV and BmDNV-1, which contains the conserved motifs of phospholipase A2 (PLA2) domain (Zádori et al., 2001
). Their PLA2 activity had been confirmed using the mixed micells assay (Li et al., 2001
; Fédière et al., 2002
). The tissue tropism of CeDNV and BmDNV-1 is different to that of members of the other three genera. CeDNV and BmDNV-1 can replicate almost exclusively in the columnar cells of midgut epithelium, whereas the other densoviruses replicate in most larval tissues except for the midgut (Tijssen & Bergoin, 1995
).
Dendrolimus punctatus larvae (pine caterpillar) are the most destructive defoliator of massopine forests. Recently, a non-enveloped icosahedral DNA virus has been isolated from dead larvae of Dendrolimus punctatus in the Xinxian Forestry Center, Henan province, China. This virus is likely to be an important pathogen of Dendrolimus punctatus. Virus particles are about 22 nm in diameter and contain a single-stranded DNA genome of approximately 5·0 kb. In the present study, we report the nucleotide sequence of this new densovirus from Dendrolimus punctatus. The results indicate that it is a new virus that should be classified within the genus Iteravirus of the subfamily Densovirinae and we have tentatively named it Dendrolimus punctatus densovirus (DpDNV).
Virus was isolated from dead larvae by the method described previously (Jousset et al., 2000). In order to obtain viral DNA, virus was dissociated by incubation at 56 °C for 30 min in a buffer containing 10 mM Tris/HCl (pH 7·5), 100 mM NaCl, 15 mM MgCl2, 0·5 % SDS and 50 µg Proteinase K ml1. Next, a sample was extracted with an equal volume of phenol, followed by several back extractions of the phenol phase plus interphase with a NaCl solution (100 mM). The pooled, aqueous sample was extracted once with phenol/chloroform (1 : 1), and once with an equal volume of chloroform. The DNA was precipitated from the supernatant with ethanol and resuspended in water. After treatment with RNase A, the viral nucleic acids migrated as a single 5 kb band in 0·7 % agarose gel electrophoresis.
Extraction of viral DNA, in the presence of high salt concentrations, resulted in double-stranded DNA indicating that the plus and minus strands are packaged in separate virus particles. We failed to obtain recombinant plasmids of this double-stranded DNA, even after blunt-ending with Klenow. Therefore, we cloned PstI fragments into PstI-digested pUC18, whereas the terminal fragments were cloned by a method reminiscent of the 5'-RACE method for mRNA cloning. In this method, the single-stranded DNA of DpDNV, obtained after heating for 5 min at 94 °C and chilling for 1 min on ice, was polyadenylated with terminal deoxynucleotidyl transferase and then amplified by PCR with 5'-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC(T)15-3' primer and the viral sequence-specific primers. The clones were sequenced on an Applied Biosystems automated sequencer, model 377, using universal sequencing and walking primer methods.
We determined the nucleotide sequence of the DpDNV genome (GenBank accession no. AY665654). The genome of DpDNV was 5039 nt in length with J-shaped hairpin structures (Fig. 1) and its organization is similar to that of members in the genus Iteravirus (Fig. 2a
). Three large ORFs were found in the plus strand, encoding the non-structural proteins within the 5'-half and the structural proteins within the 3'-half. DpDNV genome sequence shared about 60 % identity with that of both CeDNV and BmDNV-1. The base composition of the plus strand of the genome was A/T rich (35·56 % A, 18·49 % C, 19·07 % G and 26·87 % T), similar to other densoviruses (for example, CeDNV 36·34 % A, 25·80 % T; GmDNV 32·43 % A, 31·18 % T; IHHNV, Infectious hypodermal and hematopoietic necrosis virus 20·68 % A, 36·28 % T).
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A direct repeat of 45 nt, containing a putative TATA-box, has been observed between the NS1 and VP ORFs of the BmDNV-1 but not for CeDNV (Li et al., 2001). The DpDNV sequence also revealed a direct repeat between the NS1 and VP ORFs, but smaller (23 nt between 2656 and 2691), did not contain the putative TATA-box. The corresponding sequence of DpDNV and BmDNV-1 contains a conserved TCTAATC.
Three potential promoters were detected for the putative NS transcript of DpDNV according to the characteristics of invertebrate transcription initiation (Cherbas & Cherbas, 1993; Purnell et al., 1994
) (Fig. 2b
). The third putative NS promoter at nt position 356419, which contained the typical downstream promoter element (DPE) (Kutach & Kadonaga, 2000
) and the motif ten element (MTE) (Ohler et al., 2002
; Lim et al., 2004
) of invertebrate promoters, was most likely to be the functional promoter, named by P7 (at map unit 7). The first AUG (nt 494) in this transcript had been suggested to act as the initiation codon for NS2 protein translation. Since this initiation site conformed poorly to the Kozak consensus sequence (A/GCCaugG) (Kozak, 1987
, 1999
) (Fig. 2b
), a leaky scanning may occur as with the other densoviruses. When an AUG codon is flanked by A3or by G3 and G+4, the rest of the consensus sequence contributes only marginally (Kozak, 1999
). Consequently, some 40S ribosomal subunits may bypass the AUG (nt 494) and then initiate at the next downstream in-frame AUG (the left frame) at position 799, producing NS1 protein. Moreover, a potential promoter (P54) upstream from the right ORF contained a TATA-box (TATAAAT) at nt position 2700, and an initiator (Inr; TCAGT) at nt 2730 might be responsible for the transcription of mRNA encoding viral capsid proteins. Nine AATAAA sites, specific sequences for polyadenylation signal, were found downstream of the left and right ORFs. However, only two AATAAA sites, at nt positions 2704 and 4747, were most likely to be polyadenylation sites for DpDNV pre-mRNAs since those were followed by a CAYTG sequence and G/T-rich sequence, which are typical for eukaryotic transcription terminators (Birnstiel et al., 1985
). The AATAAA site (nt 2704) at the end of the left ORF was followed by CATTC (nt 2727) and TTGCCGGGT (nt 2754), while another AATAAA site (nt 4747) at the end of the right ORF was flanked by CAATA (nt 4804) and GTGTGTGGTG (nt 4951). Interestingly, the polyadenylation signal at position nt 4747 overlapped the stop codon of the right ORF.
The left ORF (nt 7992655) encoded a putative NS1 of 618 aa, which had high identities with NS1 of two members of Iteravirus (CeDNV 44 %; BmDNV-1 44 %). The mid ORF (nt 4941855) was located entirely within the left (NS1) ORF but in a different reading frame and encoded a putative NS2 of 453 aa with unknown function. It had 47 % homology to putative NS2s of CeDNV and BmDNV-1, members of Iteravirus, and shared no similarity with that of other parvoviruses. The right ORF spanned from nt 2745 and terminated at nt 4751. Translation from the first in-frame ATG would produce a predicted 668v, 74 kDa protein. The amino acid sequence of the protein showed low identities (<15 %) with that of members of the Densovirus, Pefudensovirus and Brevidensovirus genera but 76 and 72 % with VP1s of CeDNV and BmDNV-1, respectively. Additional structural proteins (VP24) could be synthesized by a leaky scanning mechanism by initiation of translation from downstream AUGs.
The amino acid sequence of NS1 was further found to share the functional domains of the replication initiator and of the DNA-dependent ATPase/helicase with other parvoviruses, which may be involved in the initiation of DNA replication (llyina & Koonin, 1992; Koonin, 1993
). Motif I (H&H&&&) for metal binding site and motif II (Y&.K/R) for cleavage-ligation reaction (Nüesch et al., 1995
), characteristic for the replication initiator domain, were located at aa 207212 and 254257, respectively (Fig. 3a
). The C-terminal sequence (aa 461580) contained the typical sequences of superfamily III-type ATPase/helicase (Koonin, 1993
) (Fig. 3b
). The sequence of the DNA-dependent ATPase/helicase domain of DpDNV shared 58·7 % identity with that of CeDNV and BmDNV-1. Motif A and B diverged slightly from the conserved sequences (GKN; &&&&D/ED/E) of the tripartite superfamily III-type ATPase/helicase motifs. So far, the biological functions of NS1 protein of densoviruses have not been investigated in detail as vertebrate parvoviruses. However, it was reported that NS1 of AaeDNV could stimulate expression of the viral protein gene (Afanasiev et al., 1994
; Ward et al., 2001
) and JcDNV NS1 possesses activities common to the superfamily of rolling-circle replication initiator proteins especially parvovirus replication proteins (Ding et al., 2002
).
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Recently, a conserved PLA2 domain, resembling the conserved motifs of secreted PLA2 was identified by sequence alignment in the VP1 unique region of parvoviruses (Zádori et al., 2001). The conserved PGY motif was located between aa 2 and 55 in VP1 of DpDNV. This motif contained YIGPG (aa 913) and HDLAY(x)12D (aa 3249) sequences corresponding to the consensus sequences of the conserved Ca2+ binding loop and catalytic site of secreted PLA2 domain, respectively (Fig. 3c
) (Zádori et al., 2001
; Fédière et al., 2004
). Moreover, they demonstrated parvovirus PLA2 (pvPLA2) activity of Porcine parvovirus (PPV) and Erythrovirus B19 (B19), both in expressed viral proteins as well as in the infectious clones. Knock-out mutants displayed, by in situ hybridization, a defect in the transfer of the viral genome to the nucleus and cumulated in a perinuclear accumulation of virions (Zádori et al., 2001
; Girod et al., 2002
). Furthermore, pvPLA2 domain in VP1 of BmDNV-1, CeDNV and GmDNV has been demonstrated to be PLA2 activity (Li et al., 2001
; Fédière et al., 2002
; Tijssen et al., 2003
). Therefore, the conserved motifs of pvPLA2 domain of DpDNV may play a similar role in viral infectivity as other parvoviruses.
In conclusion, similarities in the genome organization, structural characteristics of the genome and sequence identities all suggest that DpDNV is a new third member of the genus Iteravirus of the subfamily Densovirinae. These results further support the classification of the Iteravirus as a separate genus within the subfamily Densovirinae.
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ACKNOWLEDGEMENTS |
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Received 15 January 2005;
accepted 22 April 2005.
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