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Institute of Insect Science, Zhejiang University, Hangzhou 310029, PR China
Correspondence
Chuan-Xi Zhang
chxzhang{at}zju.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
A supplementary table showing oligonucleotide PCR primers that were designed for and used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The two viral forms are essential for natural propagation of baculoviruses.
Open reading frame 56 (ORF56 or Bm56) of Bombyx mori nucleopolyhedrovirus (BmNPV) is considered to be a core gene (Herniou et al., 2003), with homologues existing in all baculoviruses that have had their genomes sequenced to date, including lepidopteran NPVs, lepidopteran granuloviruses, hymenopteran NPVs and a dipteran baculovirus. It was reported that Culex nigripalpus nucleopolyhedrovirus (CuniNPV) ORF58, which is homologous with Bm56, encodes a structural protein for ODV, as determined by the nano-electrospray quadrupole time-of-flight mass spectrometry (GeLC-MS/MS) method (Perera et al., 2007). These data led to the proposal that Bm56 probably plays a defined role in viral replication. However, knowledge about this common gene is still very limited.
In this study, we showed that Bm56 is transcribed at 12 h post-infection (p.i.) and that its encoded protein could be detected at 16 h p.i. with polyclonal serum against glutathione S-transferase (GST)–Bm56. Confocal microscopy demonstrated that Bm56 was mainly distributed in the outer nuclear membrane and intranuclear region in BmNPV-infected cells. Western blot analysis showed that Bm56 is a structural component of the ODV nucleocapsid. Moreover, we utilized a bacmid of BmNPV that replicates in Escherichia coli to delete Bm56 and evaluated the replication of the deletion bacmid in cultured cell lines. There was no difference observed between the Bm56 deletion bacmid and BmNPV bacmid with respect to BV production. Additionally, in an in vivo assay, no significant difference in the 50 % lethal dose (LD50) was observed between the Bm56-deleted bacmid and the wild type. However, the 50 % lethal time (LT50) of the Bm56-deleted bacmid was 16–18 h longer than that of the wild-type BmNPV bacmid in this study. Thus, our data suggested that Bm56 is a structural component of ODVs that facilitates efficient virus production in vivo. However, Bm56 is not essential for BV production in vitro.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Bacterial strains, bacmid DNA and plasmids.
E. coli strain DH10B and pFastBac1 were purchased from Invitrogen. E. coli strain BW25113 (pKD46) was kindly provided by Dr Mary Berlyn (Yale University, New Haven, CT, USA); plasmid pKD46 contains the phage 
Red system under the control of an arabinose promoter. E. coli strain DH10Bac (Invitrogen) was used to isolate the helper plasmid (pMON7124), which encodes a transposase. E. coli strain BmDH10B, containing BmNPV bacmid (BmBac) DNA, was kindly provided by Dr Enoch Y. Park (Shizuoka University, Shizuoka, Japan). The pRADZ3 plasmid, containing the chloramphenicol-resistance gene (CmR), was kindly provided by Dr Hua Yuejin (Zhejiang University, Zhejiang Province, China). All strains were cultured in Luria–Bertani (LB) medium with appropriate antibiotics. Plasmid pFastBacGFP (Wu et al., 2006), containing the green fluorescence protein gene (gfp) under the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter, was kindly provided by Dr Pang Yi (Sun Yat-sen University, Guangdong Province, China).
Expression of Bm56 and preparation of antibody.
The Bm56 coding region was amplified from BmNPV genomic DNA by PCR with primers Bm56F and Bm56R (see Supplementary Table S1, available in JGV Online), which were synthesized based on the genomic sequence of BmNPV T3 (GenBank accession no. NC_001962
[GenBank]
). Bm56 was subcloned into the expression vector pGEX4t-2 with GST at the N terminus. Fusion protein GST–Bm56 was expressed in E. coli under induction by 0.1 mM IPTG at 37 °C, and retrieved after SDS-PAGE. Anti-GST–Bm56 serum was prepared by using standard techniques (Harlow & Lane, 1988). Purified GST–Bm56 protein (about 2 mg) in complete Freund's adjuvant was injected subcutaneously to immunize New Zealand white rabbits, followed by two booster injections in incomplete Freund's adjuvant, with a gap of 2 weeks before exsanguinations. The polyclonal rabbit antibody against GST–Bm56 was used for immunoassay.
RT-PCR analysis.
For RT-PCR analysis, total RNA was extracted from mock- or BmNPV-infected cells at various time intervals (3, 6, 12, 16, 24, 48 and 72 h p.i.). Total RNA was purified by incubating with DNase I (Worthington Biochemical) to remove potential genomic DNA contamination. Purified RNA was examined by PCR with primers Bm56F and Bm56R (see Supplementary Table S1, available in JGV Online). RT-PCR was performed by using a RevertAid First Strand cDNA Synthesis kit (Fermentas) with 1 µg purified RNA as the template. First-strand cDNA was synthesized with avian myeloblastosis virus reverse transcriptase and oligo-p(dT)18 primer. Subsequently, a nested PCR product was amplified with primers Bm56F and Bm56R (see Supplementary Table S1). PCR products were analysed on a 1.0 % agarose gel.
Temporal expression of Bm56 in infected BmN cells.
For time-course analysis, 1x106 BmN cells were infected with BmNPV at an m.o.i. of 10. Cells were harvested at the designated times (4, 8, 12, 16, 24, 48 and 72 h) and washed with 1x PBS (0.14 M NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) three times. The protein concentration of the cell extracts was determined by Bradford's method (Bradford, 1976). Cell lysates (20 µg) were analysed by SDS-PAGE (10 % gel) and subsequently subjected to Western blot assay.
Immunodetection of the Bm56 protein in ODVs and BVs.
Preparation of ODVs and BVs and the fractionation of ODVs into envelope and nucleocapsids were performed as described previously (Xu et al., 2006). The purified ODVs and BVs and the ODV nucleocapsid and envelope preparations were used for Western blot assay.
Immunofluorescence microscopy.
BmN cells were infected with BmNPV at an m.o.i. of 5 and collected at 48 h p.i. The harvested cells were rinsed three times with 1x PBS and fixed in cold methanol : acetone (1 : 1) for 15 min, followed by three washes with 1x PBS. To detect the localization of Bm56, cells were incubated with anti-GST–Bm56 polyclonal antibody (1 : 400 dilution) in 1x PBS for 2 h at room temperature. Primary antibody was removed by washing three times with 1x PBS. The cells were incubated with protein G fused to EGFP for 2 h and the nucleus (DNA)-specific DAPI stain (Sigma) for 1 h. Subsequently, the cells were observed and photographed under a Zeiss LSM 510 confocal laser-scanning microscope.
Preparation of a linear fragment for homologous recombination.
To generate Bm56-knockout virus by recombination in E. coli (Fig. 1a), we constructed a transfer vector (pET-ufs/CmR/dfs) in which the CmR gene was introduced to disrupt the Bm56 coding region (corresponding to nt 54277–54280), and 219 bp of the 5' end and 182 bp of the 3' end were retained so that the deletion would not affect transcription of the adjacent genes (lef-3 and orf57). Briefly, the transfer vector was constructed as follows. First, a 750 bp (nt 53527–54276) upstream flanking sequence (ufs) was PCR-amplified from BmNPV bacmid genomic DNA with primers De56UF and De56UR (see Supplementary Table S1, available in JGV Online) and cloned into pET-2 to generate pET-ufs. Second, a 1009 bp (nt 54281–55289) downstream flanking sequence (dfs) was amplified with primers De56DF and De56DR (see Supplementary Table S1) and cloned into pET-ufs to generate pET-ufs/dfs. Eventually, using the pRADZ3 plasmid as template, a 948 bp CmR sequence was amplified with primers CmRF and CmRR (see Supplementary Table S1) and cloned into pET-ufs/dfs to generate pET-ufs/CmR/dfs. The reconstructed vector was verified by sequencing.
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Generation of the Bm56-deleted bacmid.
BW25113/pKD46 competent cells were made according to the method described by Datsenko & Wanner (2000). The BmNPV bacmid DNA was electrotransformed into BW25113/pKD46 competent cells to generate bacterial strain BW25113 containing pKD46 and BmNPV bacmid, designated BW25113/pKD46/BmBac.
Red system-induced BW25113/pKD46/BmBac electrocompetent cells were made as described by Pijlman et al. (2002). Briefly, the ufs/CmR/dfs fragment (100 ng) was mixed with 40 µl competent cells on ice. Electroporation was then performed by use of a Bio-Rad Gene Pulser II (2.5 kV, 25 
and 25 µF) and a 2 mm diameter cuvette, according to the manufacturer's instructions. Next, these cells were mixed with 800 µl pre-heated SOC medium (Sambrook & Russell, 2001) and incubated for 4 h at 30 °C with gentle shaking. The cells were collected and spread onto LB plates with kanamycin (50 µg ml–1) and chloramphenicol (7 µg ml–1) for another 48 h. Finally, recombinant bacmid DNA was extracted and identified by PCR with primers iF and iR (see Supplementary Table S1, available in JGV Online). The identified BmNPV bacmid with the Bm9 deletion was temporarily named BmBac
56 (Fig. 1d
).
BmBac56 DNA was extracted and electrotransformed into E. coli strain DH10B, designated DH10B/BmBac56. Then, the helper plasmid (pMON7124) was chemically transformed into DH10B/BmBac56 to generate DH10B cells containing the Bm56-deleted bacmid and the helper plasmid, designated DH10B/BmBac56/helper, and subsequently used for marker-gene insertion.
Construction of BmNPV bacmid and Bm56-deleted bacmid containing gfp and polyhedrin.
To facilitate examination of virus infection, we introduced donor plasmid pFB1-PH-GFP, which was generated by inserting the polyhedrin and gfp genes into pFastBac1 plasmid under the control of the polyhedrin promoter and the AcMNPV ie-1 promoter, respectively (Wu et al., 2006), by Tn7-mediated transposition in the Bac-to-Bac system (Invitrogen). pFB1-PH-GFP was transformed into BmDH10B and DH10B/BmBac56/helper competent cells to generate BmBacWT-PG (Fig. 1c) and BmBacKO-PG (Fig. 1d), respectively. Successful transposition was verified by PCR with pUC/M13 forward and reverse primers.
BV growth curve.
To determine the BV growth curve, BmN cells were infected with BmBacWT-PG and BmBacKO-PG at an m.o.i. of 5, then the supernatant was harvested at various times p.i. (8, 12, 16, 24, 48, 72, 96 and 120 h). BV titration was performed using an end-point dilution assay (TCID50) (O'Reilly et al., 1992).
Electron microscopy.
The BmN cell monolayer was infected with BmBacKO-PG at an m.o.i. of 5. At 96 h p.i., cells were harvested and the pellet was fixed in 2.5 % glutaraldehyde for 1 h at 4 °C, followed by fixation with 1 % osmium tetroxide for 1 h at room temperature. After the fixed cells were dehydrated in graded ethanol (50–100 %) and then soaked in acetone for 20 min, infiltration in graded Spurr resin (50–100 %) (Sigma) and incubation for 16 h at 70 °C were performed. After staining with uranyl acetate and lead citrate, ultrathin sections were viewed under a JEM-1230 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV.
B. mori larval bioassay.
LD50 and LT50 values of BVs were determined by injection into the haemocoel of B. mori larvae, within 8 h of moulting to the fifth instar, of different doses of BVs (50, 500, 5000 and 50 000 p.f.u.) diluted in PBS. Twenty-five larvae per dose were used and each dose was repeated in triplicate. Mortality was determined every 4 h.
LT50 and LD50 values were estimated by using the DPS data processing system for practical statistics (Tang & Feng, 2002). Probit analysis (Finney, 1971) was adopted in the statistics.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Thus, these results, coupled with the late consensus initiation sequence (ATAAG), indicate that Bm56 is a late-transcribed gene.
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). This band was detectable as early as 16 h p.i., increased to high levels at 24 h p.i. and lasted until 72 h p.i. (Fig. 2b). However, the molecular mass of the detected protein (42 kDa) was larger than the predicted putative Bm56 gene product (15.8 kDa). This might be due to post-translational modifications, a protein complex or some other processing functional form.
Immunodetection of the Bm56 protein in ODVs
To determine whether the Bm56 protein is a structural component of BmNPV, Western blot analysis for purified BVs and ODVs was performed. The ODV fraction showed a reactive band of 42 kDa (Fig. 3a) and the size was in agreement with that detected in lysates from infected cells. In contrast, no band was detected in the BV fraction (Fig. 3a). Thus, the Bm56 protein appeared to be a structural protein specific for ODVs. To locate Bm56 precisely within ODVs, the ODV fraction was further separated into ODV nucleocapsid protein (ODV-NC) and ODV envelope protein (ODV-E) fractions. When ODV-NC and ODV-E were analysed with the antiserum against GST–Bm56, Bm56 was found only in the ODV-NC fraction and not in the ODV-E fraction. The efficacy of the fractionation was examined by immunoassay with antibody against Bm79 (ODV-E28, an ODV envelope-specific protein) (Xu et al., 2006) and GP64 (a BV-specific protein). The results showed that positive bands of 28 and 64 kDa were detected only in the ODV-E fraction and BV sample, respectively (Fig. 3a). Thus, the separation of ODV-NC, ODV-E and BV was considered to be pure. Hence, the above results suggest that the Bm56 gene encodes a structural protein associated with the nucleocapsid of ODVs.
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). In contrast, no fluorescence was detected in uninfected cells (Fig. 3c).
Construction of BmBacKO-PG and BmBacWT-PG
To delete the Bm56 gene in the BmNPV bacmid via Red recombination, we constructed a linear donor fragment (ufs/CmR/dfs) containing the ufs, CmR and dfs (Fig. 1a). The donor fragment was electrotransformed into BW25113/pKD46/BmBac competent cells to produce the Bm56-knockout bacmid BmBac56. To verify that the deletion was derived precisely from the Bm56 locus in the BmNPV bacmid genome, PCR analysis was performed with two specific primers (iF and iR) located at the extending region of the ufs and dfs, respectively (Fig. 1b). As expected, PCR products of 3.05 and 2.09 kb were amplified from BmBac56 and BmNPV bacmid genomic DNA, respectively. Thus, the PCR results apparently confirmed that Bm56 was deleted successfully from the Bm56 locus in BmNPV bacmid DNA.
To facilitate observation of viral infection, the donor plasmid pFB1-PH-GFP, containing AcMNPV polyhedrin under the control of the AcMNPV polyhedrin promoter and gfp under the control of the AcMNPV ie-1 promoter, was transposed into the polyhedrin locus in the BmNPV bacmid and Bm56-deleted bacmid to produce BmBacWT-PG (Fig. 2c) and BmBacKO-PG (Fig. 2d), respectively. A PCR assay with pUC/M13 forward and reverse primers confirmed successful transposition.
Bacmid infection and BV growth curve
To determine the effect of Bm56 deletion upon virus replication, BmN cells were transfected with BmBacKO-PG and BmBacWT-PG. GFP expression by BmN cells with viral propagation was examined. At 96 h post-transfection, fluorescence was observed from the majority of cells with both types of bacmid transfection and the supernatants were collected for pass-through infection. Subsequently, fluorescence was observed from the second infection, indicating that DNA replication was occurring normally. The supernatants were then collected and BV titres were determined by TCID50 assay. We observed that BmBacKO-PG achieved a titre equivalent to that for BmBacWT-PG.
To assess the effect of the Bm56 deletion on viral replication quantitatively, we generated viral growth curves for BmBacWT-PG and BmBacKO-PG. Cells infected with BmBacWT-PG had growth kinetics similar to those of BmBacKO-PG, both reaching 109 TCID50 ml–1 at 120 h p.i. (Fig. 4). The above data demonstrated that Bm56 was not essential for BV production in cultured cells.
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) and enveloped nucleocapsids (Fig. 5c). However, compared with wild-type bacmid-infected cells, many strip-like and de novo envelope-like structures, to which partial nucleocapsids were attaching, were observed in the nuclei of cells infected with the Bm56-deleted bacmid (Fig. 5b, c). Additionally, Bm56 deletion aborted occlusion-body formation, and only a polyhedron-like structure not containing ODVs was observed (Fig. 5a, d). Hence, the above observations suggested strongly that deletion of Bm56 influences occlusion-body morphogenesis.
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), respectively. However, this difference was not statistically significant. Thus, the LD50 assay indicated that the Bm56 deletion had no discernible effect on infectivity of BVs in B. mori larvae.
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). This observation suggested that the Bm56 deletion reduced the efficiency of BV spreading in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, Bm56 was further determined by Western blot analysis to be located on the ODV nucleocapsid. There is general agreement that genes encoding structural proteins are transcribed at the very late phase, with products accumulating within the nuclei of virus-infected cells (Lu & Miller, 1997). Here, we observed that Bm56 is transcribed at 12 h p.i. (Fig. 2a) and that its product (42 kDa) could be detected at 16 h p.i. (Fig. 2b), which indicated that Bm56 is a late-transcribed gene. In our primary experiments, when a Western blot assay was performed to determine the expression profile of Bm56, we detected a 16 kDa protein in addition to one of 42 kDa (data not shown). However, the 16 kDa protein could not be confirmed by repeating the assay, although its molecular mass is similar to the theoretical size (15.8 kDa). To confirm the specificity of the antibody against Bm56, BmN cells infected with the Bm56-deleted virus (BmBacKO-PG) were subjected to Western blot analysis. The results showed that no positive band was detected in BmBacKO-PG-infected cells (Fig. 3b), which excluded the possibility that the anti-Bm56 serum recognized a non-related viral protein in addition to Bm56. A likely explanation for this observation is that the 42 kDa protein was due to some other processing functional form. A similar phenomenon was also observed in several other structural proteins, such as ODV-EC56 (Braunagel et al., 1996a), ODV-EC43 (Fang et al., 2003) and ODV-E18 (Braunagel et al., 1996b). Additionally, confocal microscopy combined with immunoassay demonstrated that Bm56 localized to the outer nuclear membrane and the intranuclear region (Fig. 3c) where ODV formation occurred (Williams & Faulkner, 1997), assuming that Bm56 was transported from the cytoplasm into the nucleus after Bm56 synthesis. The property of transport has been reported in the ODV structural proteins ODV-E66 (Braunagel et al., 2004) and ODV-E25 (Hong et al., 1997). It has not been determined whether Bm56 shares this phenomenon.
With respect to BV production, no difference was observed between the Bm56-deleted bacmid and wild-type BmNPV bacmid (Fig. 4) in vitro. Also, an assay using larvae injected intrahaemocoelically with BV revealed that there was no statistically significant difference in LD50 between Bm56-deleted bacmid and BmNPV bacmid (Table 1). The above observations also applied to the gene encoding fibroblast growth factor (VFGF) in baculovirus (Detvisitsakun et al., 2006). In contrast to vfgf-deleted bacmid, which did not have any effects on the LT50 in susceptible larvae infected intrahaemocoelically (Detvisitsakun et al., 2007), Bm56-deleted bacmid had an LT50 that was 16–18 h longer than that for the wild-type BmNPV bacmid (Table 2). To examine the effects of Bm56 deletion on ODV and polyhedron formation, cells were prepared for electron microscopic observation. The results demonstrated that Bm56 deletion has an effect on occlusion-body morphogenesis. Polyhedron-like structures were observed in the nucleus, and they were incapable of infecting silkworm larvae via the midgut (data not shown). In general, the above results suggest that Bm56 is a structural component of ODVs, but is not essential for BV production in cultured cells. However, Bm56 has advantageous effects on BV infectivity in vivo.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 1 December 2007;
accepted 15 January 2008.
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