Centre de recherche de microbiologie et biotechnologie, INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, QC, CanadaH7V 1B71
Department of Bioscience, Faculty of Agriculture, Hokkaido University, Sapporo, Japan2
Center of Virology-IRD, Faculty of Agriculture, University of Cairo, Giza, Cairo, Egypt3
Author for correspondence: Peter Tijssen. Fax +1 450 686 5626. e-mail peter.tijssen{at}inrs-iaf.uquebec.ca
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Bombyx mori DNV (BmDNV) was isolated from silkworms with flacherie disease in Ina City (Nagano Prefecture, Japan; Shimizu, 1975 ). The Ina isolate (BmDNV-1) differed from the Yamanashi and Saku isolates, which were designated BmDNV-2, and the Zhenjiang (China) isolate, which was designated BmDNV-3. Both BmDNV-2 and BmDNV-3 were found to have a split genome with a genome organization unlike parvoviruses and no terminal hairpins (Bando et al., 1992
; Gao & Cai, 1994
). Hence it was proposed that BmDNV-2 and -3 belong to a new family, the Bidensoviridae (Tijssen & Bergoin, 1995
).
The nucleotide sequence and genome organization of BmDNV-1 were the first to be reported for DNVs (Bando et al., 1987b , 1990
; GenBank M15123, M60583, M60584). Two large ORFs (430 and 887 codons, respectively) were found on one strand and a small ORF (167 codons) on the complementary strand. The total length of the BmDNV-1 sequence was reported to be 5048 nt with inverted terminal repeats (ITRs) of 225 nt and terminal hairpins of 153 nt. Its unique genome organization prompted ICTV to classify it in a separate genus with the name Iteravirus (itera to indicate the presence of ITRs).
Several points led us to reassess the sequence of BmDNV-1. Although the restriction map of the Casphalia extranea DNV (CeDNV) genome shows little resemblance to that of BmDNV (Fédière et al., 1991 ), sequencing revealed a close relationship (unpublished results). The genome organization, however, was found to be different. The ORF for the VPs of BmDNV was reported to be much larger than required and contains motifs, for example ATPase/helicase, found for other parvoviruses in the NS ORFs. Finally, a phospholipase A2 (PLA2) motif, recently identified in porcine parvovirus VP1 (Zádori et al., 2000
), was also present in the published sequence of BmDNV-1, but mostly out of the coding frame.
BmDNV-1 genomic DNA was cloned as described by Bando et al. (1987a) using the Ina isolate after further propagation in B. mori. The sequence of both strands of two independent clones [pN215BB4.3 (without extremities) and pIN919, in a pBluescript SK- vector] was determined with an ABI 310 automatic sequencer by the primer-walking procedure. The sequence and genome organization of the clones (Fig. 1A
) was identical for their 4·3 kb overlap and similar to that of the CeDNV and has been submitted to GenBank (accession no. AY033435). Compared to CeDNV (GenBank AF375296), the coding sequence for BmDNV-1 NS1 was 754 codons (vs 753), for NS2 it was 451 (vs 452) and for VP it was 672 (vs 678). For the original BmDNV-1, a single NS of 455 codons and a VP of 885 codons was reported (Bando et al., 1987a
, 1990
). Sequencing of another clone from that original cloning experiment (pCBg42, in a pHC79 vector) revealed, with respect to the CeDNV and new BmDNV-1 sequences, at least 100 insertions, deletions or inversions without significant ORFs and was deemed to be aberrant, like the previously reported sequence.
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The differences in length between the genomes of BmDNV-1 and CeDNV (5076 vs 5002 nt) could be partly accounted for by differences in the lengths of the noncoding sequences between the ITRs and the viral genes (Fig. 1B). However, the main reason was the occurrence of a direct repeat of 45 nt between the NS1 and VP sequences of BmDNV-1 but not of CeDNV (Fig. 1B
). This intergenic sequence probably contains promoter elements for VP. The lack of this direct repeat in CeDNV suggests that BmDNV has developed strategies to prevent conflict between these repeat elements. Interestingly, the analogous sequence of CeDNV demonstrated four differences with both repeats of BmDNV-1 and a supplementary four differences with the upstream repeat only. Among the latter, two to three are in a putative TATA box and could thus make the first TATA box less attractive. Kutach & Kadonaga (2000)
observed that, for invertebrates, less than 50% of the class II gene promoters use either TATA boxes or the equivalent downstream promoter elements (DPE) but that initiator (Inr) elements, or a CATG box, are always conserved at the transcription start. This core motif, with the consensus (G/A/T)(C/T)A(G/T)TG, enables RNA polymerase II to function even in the absence of a TATA box (Cherbas & Cherbas, 1993
; Purnell et al., 1994
) and is better conserved when the TATA and DPE boxes are less prominent (Kutach & Kadonaga, 2000
). The conserved Inr box at position 26972702 (Fig. 1B
) may also restrict the TATA box for the VP transcription to the sequence at position 26672673.
The sequences of the termini of the viral genome (Bando et al., 1990 ) were also corrected. Almost all clones that were obtained had a 5 nt deletion, with respect to CeDNV, which resulted in many unpaired bases in the ITR (Bando et al., 1990
). Sequencing of pIN919 corrected this gap and demonstrated that the flip/flop sequence contained only 1 nt difference with CeDNV. The ITR could now be folded into a J-form hairpin (Fig. 1C
) and contained side-arms or bubbles as seen with other parvoviruses such as porcine parvovirus (PPV) (Bergeron et al., 1993
, 1996
).
In contrast to a single NS ORF in the original sequence, two overlapping NS genes were identified in the new sequence of BmDNV-1 (Fig. 1A). Generally, parvoviruses from vertebrates generate their multiple NS proteins by alternative splicing, whereas parvoviruses from insects use overlapping ORFs in the different reading frames to obtain their complement of NS proteins. Splicing of the RNAs from DNVs is rare, but not unknown (Bergoin & Tijssen, 2000
). The VP proteins are generated, using probably the first four or five AUGs in the ORF as initiator codons, by a leaky scanning mechanism, in a similar way to Galleria mellonella DNV (GmDNV) (unpublished results) (Fig. 1A
).
One of the reasons why the original BmDNV-1 sequence was suspected to be incorrect was the out-of-frame location of a VP domain that is highly conserved among almost all parvoviruses. Recently, we found that this domain contained a PLA2 motif (Zádori et al., 2000 ). As with other parvoviruses, this PLA2 motif was identified in the unique portion of the VP1 protein (VP1up; Figs 1A
and 2A
), which is a minor component of the BmDNV-1 capsid (Nakagaki & Kawase, 1980
).
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In order to confirm enzyme activity in this domain, BmDNV-1 VP1up (first 94 amino acids of VP1) was cloned into BglIIXbaI of the pBADTBX vector, which was then used to transform E. coli BL21-CodonPlus(DE3)-RIL (Stratagene) to obtain a fusion protein. pBADTBX was derived from the pBAD/TOPO Thiofusion expression vector (Invitrogen) by inserting a tobacco etch virus (TEV) protease site and a polylinker downstream of the thioredoxin gene. The expressed protein was purified using its His-tag on a Niagarose column and was then cleaved by TEV protease to remove the thioredoxin. PLA2 activity of the protein was not significantly affected by the presence of thioredoxin. Usual yields of recombinant protein were about 1 mg/l. PLA2 activity of expressed VP1up was established using the mixed micelles assay (Manjunath et al., 1994 ). The substrates (from New England Nuclear/DuPont) were 6 µM L-3-phosphatidylcholine,1,2-di[1-14C]oleoyl (sp. act. 110 mCi/mmol), L-3-phosphatidylinositol L-1-stearoyl-2-[1-14C]arachidonyl (sp. act. 48 mCi/mmol) or phosphatidylethanolamine L-1-palmitoyl,2-[1-14C]arachidonyl (sp. act. 54·6 mCi/mmol). Triton X-100 (1 mM) was used instead of deoxycholate and the total reaction volume was 50 µl. The reaction was stopped after 10 min by adding 80 µl chloroform/methanol (2:1) and 50 µl of saturated KCl solution. Separation was on silica gel TLC plates. Optimal separation of the phosphatidylcholine reaction products was obtained using a solvent solution containing chloroform, methanol and water (65:35:4, v/v). For phosphatidylethanolamine and phosphatidylinositol, chloroform and methanol (87:13, v/v) were used as solvent. The separated products were quantified using a Molecular Dynamics PhosphorImager SI after drying. The degree of hydrolysis was plotted against the amount of expressed protein and only the regression lines of activity vs dilution with a correlation coefficient r2 0·98 were used to calculate the amount of protein required to hydrolyse 50% of the substrate. Bee venom PLA2, purchased from Sigma-Aldrich (cat. no. P9279) at 1360 U/mg, was used as a positive control (Fig. 2 B
).
In this assay, 590 ng of VP1up was required for BmDNV-1 to obtain 50% hydrolysis of phosphatidylcholine. Its (kcat/Km)app was (1·1±0·2)x104 compared to (1·09±0·14)x105 M-1s-1 for bee venom secreted PLA2. The (kcat/Km)app values of B19 and GmDNV PLA2 were (2·5±0·2)x104 and (0·4±0·03)x104 M-1s-1, respectively. With phosphatidylethanolamine, 550 ng of VP1up was required to hydrolyse 50% of the substrate in this assay. Whereas for B19 and PPV VP1up the degree of hydrolysis of the three phospholipids was similar, phosphatidylinositol was at least 10-fold less hydrolysed by BmDNV VP1up than the other two phospholipids.
In the case of PPV, we found that site-directed mutagenesis of critical amino acids in the catalytic site decreased strongly both the enzyme activity and the virus infectivity (Zádori et al., 2000 ). When the critical amino acids HD in the PLA2 active site of the infectious clone of PPV were mutated, virus was still obtained upon transfection but was not infectious. Mutants bound to the cells and entered as efficiently as wild-type, colocalized with LAMP-2 protein in the late endosome/lysosome, but failed to release the viral genome from the final endosomal compartment into the nucleus (Zádori et al., 2001
).
An initiator (replicator) motif could be identified in the NS1 sequence, although this motif was less well defined (Fig. 2C; Tijssen & Bergoin, 1995
). The tripartite helicase/ATPase motif that was recognized in the putative structural protein in the original sequence was also identified in the NS1 sequence as with other parvoviruses (Fig. 2D
).
In conclusion, the corrected sequence of BmDNV-1 explains incongruencies that were previously noted (Tijssen & Bergoin, 1995 ). The new genomic organization of BmDNV-1 is identical to that of CeDNV, but distinct from other DNVs, indicating that both belong to a distinct genus, the Iteravirus.
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References |
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Received 17 May 2001;
accepted 19 July 2001.