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1 State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China
2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
3 Laboratory of Virology, Wageningen University, 6709 PD Wageningen, The Netherlands
Correspondence
Zhihong Hu
huzh{at}wh.iov.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Phylogenetic studies have indicated that NPVs can be subdivided into two groups, I and II (Bulach et al., 1999; Herniou et al., 2003; Zanotto et al., 1993). During the infection cycle of lepidopteran baculoviruses, two distinct viral phenotypes are produced, the occlusion-derived virus (ODV) and budded virus (BV) (Volkman & Summers, 1977). ODVs are encapsulated in polyhedra that dissolve in the alkaline environment of the midgut, release the virions and initiate infection in midgut columnar epithelial cells. The ODV is associated with the spread of the virus from insect to insect, whereas BVs are not occluded and are adapted to disseminate infection from cell to cell and are responsible for the systemic infection in larvae and in cell cultures. Although ODVs and BVs differ in the composition of their envelopes, the nucleocapsids of the two viral phenotypes are similar in structure and function (Funk et al., 1997). The functions of many of the structural virion proteins, however, are unknown.
Helicoverpa armigera single nucleocapsid NPV (HearNPV, also called HaSNPV) is a highly infectious pathogen of the cotton bollworm H. armigera and related heliothines (Sun et al., 1998). The virus was isolated from diseased H. armigera larvae in the Hubei province of China in 1975 (Zhang, 1989). HearNPV has been adapted for production as a biological pesticide and has been applied to control insect pests in China and other countries on cotton and vegetables. The genome sequence of the HearNPV G4 strain has been determined in its entirety (Chen et al., 2001). Computer-assisted analysis has revealed 135 putative open reading frames (ORFs) among which 100 ORFs have homologues in the Autographa californica multicapsid NPV (AcMNPV), 15 have homologues in other baculoviruses and the remaining 20 ORFs are so far unique to HearNPV and its genotypic variant Helicoverpa zea SNPV (HzSNPV) (Chen et al., 2002).
Ha107 is one of the 15 ORFs with homologues in other baculoviruses and is currently uncharacterized. The Ha107 gene has been predicted to encode a protein of about 51 kDa. A homologue of this ORF is present in the HearNPV Cl strain (Zhang et al., 2005) and in HzSNPV (Chen et al., 2002). Moreover, homologues of Ha107 are also found in a limited number of baculovirus species, such as Agrotis segetum multicapsid nucleopolyhedrovirus (AgseNPV; Jakubowska et al., 2006), Spodoptera exigua MNPV (SeMNPV; IJkel et al., 1999), S. litura MNPV (SpltNPV; Pang et al., 2001) and A. segetum granulovirus (AgseGV; unpublished GenBank accession no. NC_005839
[GenBank]
), but not in group I NPVs.
In this study, the transcription characteristics of Ha107 and the expression and location of its encoded protein were investigated. The HA107 protein appeared to be a nucleocapsid-associated protein of both BV and ODV. In addition, Ha107 was not essential for virus replication, but deletion of this gene appeared to increase the BV titre and the median lethal dose (LD50) of the recombinant virus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was maintained in Grace's medium with 10 % fetal bovine serum. HearNPV strain G4 (Sun et al., 1998) was used for experiments and was propagated in HzAM1 cells. A culture of H. armigera insects was maintained according to Sun et al. (1998) for HearNPV occlusion body production and bioassays.
Total RNA isolation and 3' and 5' rapid amplification of cDNA ends (RACE) analysis.
HzAM1 cells were infected with BVs of HearNPV at an m.o.i. of 10. Total RNA was isolated from infected and mock-infected cells at 0, 4, 8, 12, 24, 36, 48, 72 and 96 h post-infection (p.i.) by Trizol extraction (Invitrogen). RT-PCR was performed with 1 µg purified total RNA as template per time point. First-strand cDNA synthesis was performed using AMV reverse transcriptase (Promega) and an oligo(dT) anchor primer to the 3' end of Ha107 (5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTTV-3'). The cDNA mixtures were amplified using an anchor primer (5'-GACCACGCGTATCGATGTCGAC-3') and Ha107-specific forward primer 1 (5'-GTTGCCCACTCGCGACATAAG-3'). The PCR products obtained from RNA at 48 h p.i. were cloned into the pGEM-T Easy vector (Promega) and sequenced to determine the 3' end of the Ha107 transcripts. The 3'RACE PCR of ORF106 of HearNPV (Ha106) was amplified with the oligo(dT) anchor primer and Ha106-specific forward primer 2 (5'-GCGTGGATTGCACGGTATACAC-3').
The 5' initial sites of the Ha107 transcripts were determined using a 5'/3' RACE kit (Roche) with 2 µg purified total RNA (48 h p.i.) as a template. Briefly, first-strand cDNA synthesis was performed with a gene-specific primer (5'-CACAGAACCCGCAGCAACG-3'). cDNAs were then purified using a High Pure PCR purification kit (Roche) and a poly(A) tail was added to the 3' ends using terminal transferase with dATP. The tailed cDNAs were amplified using the oligo(dT) anchor primer and the first nested Ha107-specific primer (R1 : 5'-AACAAGGGCGGTATAAGTTGAAC-3'). A second PCR was performed using the PCR anchor primer and the second nested primer (R2 : 5'-GTCGCTTTGCTAGTGCTGCTAC-3'). The obtained PCR products were gel purified, cloned into pGEM-T Easy and sequenced.
Generation of polyclonal antibody against HA107.
A truncated fragment of Ha107 (nt 101017–102391) (Chen et al., 2001) encoding the hydrophilic loops (aa 136–304) (Fig. 1) was PCR-amplified using two primers, ExF (5'-GGGGGATCCGATTCGGTAATTGCTTTAATGTAC-3') (BamHI site underlined) and ExR (5'-GGGGAATTCAGCGAGTGGGCAACATTATCGT-3') (EcoRI site underlined). The purified PCR product was cloned into the pGEX-KG expression vector (Guan & Dixon, 1991), giving pGEX-EX107 and expressed as truncated HA107 protein fused to glutathione S-transferase (GST–EX107) in E. coli BL21 cells. The purified GST–EX107 proteins from gel slices were injected into a rabbit (300 µg per injection for initial as well as booster) to generate anti-HA107 antibodies.
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. Membranes were allowed to react with the anti-HA107 antiserum diluted 1 : 1500 and bands were detected after incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma) using a SuperSignal West Femto Maximum Sensitivity Substrate kit as described by the manufacturer (Pierce).
Purification of BV and ODV fractions and Western blot analysis.
The envelope and nucleocapsid fractions of BV and ODV were separated after treatment with NP-40 as previously described (Long et al., 2003). Proteins of purified BVs and ODVs, as well as their nucleocapsid and envelope fractions, were separated by 12 % SDS-PAGE and transferred onto Hybond-N membranes (Amersham). Anti-HA107 antiserum was used for Western blot analysis and polyclonal anti-VP39, anti-ODV-E18 and anti-HaF1 antibodies were used as controls for nucleocapsid-, ODV envelope- and BV envelope-specific proteins, respectively.
Immunoelectron microscopy (IEM) analysis.
The IEM protocol was according to Tsai et al. (2006). Suspensions of purified ODVs or their nucleocapsid fractions were diluted and adsorbed to Formvar-supported, carbon-coated nickel grids (250 mesh) for 30 min at 4 °C. The grids were blocked in blocking buffer [4 % BSA, 50 mM Tris/HCl (pH 7. 5), 200 mM NaCl] for 30 min at 37 °C and then incubated with anti-HA107 antiserum or pre-immune serum (1 : 20 dilution) in incubation buffer [0.1 % BSA, 50 mM Tris/HCl (pH 7.5), 200 mM NaCl] for 2 h at 37 °C. After incubation and several washes with incubation buffer, the grids were incubated with goat anti-rabbit secondary antibody conjugated with gold particles (1 : 20 dilution in incubation buffer, 12 nm diameter; Jackson ImmunoResearch) for 1 h at 37 °C. The grids were then washed extensively with incubation buffer and negatively stained with 2 % phosphotungstate. Specimens were examined with a transmission electron microscope (H-7000 FA; Hitachi).
Construction of Ha107 knockout HearNPV bacmid.
The Ha107 knockout HearNPV bacmid was constructed by using a modified 
phage Red recombination system (Datsenko & Wendell, 2000). Two primers were designed to generate by PCR a linear fragment containing a chloramphenicol resistance gene (CmR) and with 43 bp flanking sequences targeting the Ha107 region on the HearNPV genome. The forward primer was 5'-TGTACGATGTGTCATCGTAACGTTATTGGCGCTCGCGACAGTGGGCACCAATAACTGCCT-3' (nucleotides homologous to Ha107 are underlined). A stop codon (in bold) was introduced in the primer. The reverse primer was 5'-TAATTTGTACACTAAAGTTTGCATTAAAGCGCCAGCAATTGGACCTGTGCGACGGTTACG-3' (nucleotides homologous to Ha107 are underlined). The 3' end of the primers anneal to the CmR gene of pBeloBac11 (Shizuya et al., 1992). After DpnI digestion, the linear PCR fragment was electrotransferred into competent DH10B cells carrying HearNPV bacmid HaBacHZ8 and the helper plasmid pKD46 (Hou et al., 2002). The resulting recombinant bacmid was designated HaBac
Ha107.
The Ha107 truncation and CmR gene insertion in HaBacHa107 was confirmed by EcoRI digestion and PCR analysis with two pairs of primers. The first pair was used to confirm insertion of the CmR gene cassette into the Ha107 locus of the HearNPV bacmid: one primer corresponded to sequences within the inserted CmR gene (CM: 5'-GTATGGCAATGAAAGACGGTGAG-3') and the other (EnF: 5'-GGGATGTCTTCTGTACGATGTGTCAT-3', underlined nucleotides correspond to the virus genome nt 101017–101039) was from the HearNPV genome outside the sequence used for recombination. With this pair of primers, a PCR product of 530 bp should be amplified from HaBacHa107 but not from the control bacmid HaBacHZ8. The second pair of primers (ExF and ExR) was within the Ha107 cassette and was used to confirm the absence of the Ha107 gene. These two primers should not produce a PCR product in HaBacHa107 but should produce a 524 bp product in the parental HaBacHZ8. As the inserted CmR gene contained an extra EcoRI site, this enzyme was used to identify the bacmids. According to software-predicted results, a 5.99 kb EcoRI fragment from HaBacHZ8 should be replaced by two smaller EcoRI fragments of 2.91 and 2.92 kb, respectively, in HaBac107.
Construction of knockout, repair and control HearNPV bacmids containing the polyhedrin and enhanced green fluorescence protein genes.
To insert the polyhedrin gene (ph) and the enhanced green fluorescence protein gene (egfp) into HaBacHa107 and HaBacHZ8, a donor plasmid, pFastBac-eGFP-PH, was constructed. The egfp ORF was amplified using primer pair GFP-F (5'-GACCTCGAGATGGTGAGCAAGGGCGAGGA-3') and GFP-R (5'- GCCGCTAGCTTACTTGTACAGCTCGTCCATG-3') containing XhoI and NheI (underlined) sites, respectively, from the pEGFP-N1 vector (Invitrogen). The digested PCR product was cloned into the pFastBacDual vector (Invitrogen), giving pFastBac-eGFP. The HearNPV ph gene and its own promoter were PCR amplified with primers BacpolhF (5'-GGGGATATCGTTGAAGGAGCGTACGTGCCA-3') and BacpolhR (5'-GGGGAATTCAATCGCAAGTTTAATATGCAGGAC-3'), which contained EcoRV and EcoRI (underlined) sites, respectively. After sequence verification, the ph gene was inserted into Bst1107I/EcoRI-restricted pFastBac-eGFP vector, resulting in pFastBac-eGFP-PH.
To generate a Ha107 repair HearNPV bacmid, a donor plasmid, pFastBac-eGFP-PH-Ha107, was constructed as follows. A 238 bp NotI–AflII fragment containing the SV40 poly(A) tail addition signal was obtained from the vector pEGFP-N1. A 1.7 kb AflII–XbaI fragment containing the coding region of Ha107 and its proximal promoter region was amplified using forward primer 5'-GGGCTTAAGCGACACCAGCAACGGATGTA-3' and reverse primer 5'-GCGTCTAGATTAGCAGTGTATATCTTTTAACCA-3'. After purification and sequence verification of the PCR product, the Ha107 insert and digested SV40 poly(A) were inserted into NotI/XbaI-digested vector pFastBac-eGFP-PH at the same time to give the donor plasmid pFastBac-eGFP-PH-Ha107.
Bacmid transpositions were performed according to the Bac-to-Bac manual (Invitrogen) to insert the donor sequences into the various bacmids. The resulting recombinant bacmids were named: HaBacHZ8-eGFP-PH (the control bacmid HaBacHZ8 containing ph and egfp), HaBacHa107-eGFP-PH (Ha107 knockout bacmid containing ph and egfp) and HaBac-rHa107-eGFP-PH (Ha107-repaired bacmid containing ph and egfp) (see Fig. 4a). These recombinant bacmids were used to transfect HzAM1 cells to obtain the corresponding viruses.
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Ha107-eGFP-PH or HaBac-rHa107-eGFP-PH at an m.o.i. of 10 and BV titres were determined by end-point dilution assay at 0, 8, 16, 24, 36, 48, 72, 96 and 120 h p.i. Green fluorescence was used as a marker to determine positive viral infection. Each virus infection was carried out in triplicate. BV titres at different time p.i. were analysed by two-way analysis of variance in GLM (SPSS Inc.) with virus and time as factors. Mean BV titres of virus types were separated by Fisher's least significant difference if significant effects were found. BV titres were logarithm transformed before analysis.
Bioassay of recombinant viruses.
LD50 values of the recombinant HearNPVs (HaBacHZ8-eGFP-PH, HaBacHa107-eGFP-PH and HaBac-rHa107-eGFP-PH) in third-instar H. armigera larvae were determined with five different doses (20, 60, 200, 600 and 2000 polyhedra per larva). Second-instar larvae were starved for 16 h at 28±1 °C and allowed to moult into third instar. The starved larvae (4.61±0.34 mg) were fed on a diet plug with 1 µl polyhedra suspension. The larvae that consumed the entire plug within 24 h were transferred to new diet. For each dose of each virus, 48 larvae were inoculated. Inoculated larvae were maintained at 28±1 °C. Mortality was scored daily until surviving larvae in each treatment had either pupated or were in the pre-pupal stage. LD50 values and their standard deviations were determined by probit analysis using SPSS (SPSS Inc.). LD50 values of viruses were further compared using two-sided z-tests (Snedecor & Cochran, 1989).
Electron microscopy of recombinant virus-infected cells.
HzAM1 cells were infected with HaBacHZ8-eGFP-PH or HaBacHa107-eGFP-PH at an m.o.i. of 5. Infected cells were fixed at 60 h p.i. and processed for electron microscopic analysis as described previously (Wang et al., 2003).
Computational analysis.
The HA107 protein sequence was analysed using the ExPASy server (Appel et al., 1994) (http://us.expasy.org) and CBS server (Jensen et al., 2003) (http://www.cbs.dtu.dk) for prediction of signal peptide, transmembrane domain and potential post-translational modification sites and subcellular localization. Homologues were explored using the BLAST network service on ExPASy in the Swiss-Prot and TrEMBL databases (lambda, 0.327; K, 0.136; H, 0.405). The topology of the HA107 protein homologues was predicted using TMHMM software. The topology was drawn according to the predicted results.
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RESULTS |
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) (Fig. 2a). The regions upstream of Ha107 were analysed for the presence of putative transcriptional initiation sites, such as baculovirus consensus early CAG/TT, late DTAAG promoter motifs and other transcriptional regulation elements, such as GATA and CACGTG. No consensus motif was found upstream of the Ha107 translational start codon ATG. A polyadenylation signal sequence (A)ATAAA was located 20 nt downstream of the putative translation stop codon TAA.
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). An N-terminal signal peptide was predicted and a potential cleavage site was found between aa 22 and 23 (YGA
FK). Five potential N-linked glycosylation sites (aa 45–48, 145–148, 146–149, 223–226 and 331–334) were found using NetNglyc prediction. According to PSORTII analysis (Horton & Nakai, 1997), the putative HA107 protein is probably localized in or associated with membrane structures. Only four homologous genes of Ha107 were found in the available baculovirus genomes (Fig. 1a) including ORF105 of SpltNPV (Pang et al., 2001; GenBank accession no. NC_003102), ORF22 of AgseNPV (Jakubowska et al., 2006; GenBank accession no. NC_007921), ORF22-23-24 of SeMNPV (IJkel et al., 1999; GenBank accession no. NC_002169) and ORF39 of AgseGV (unpublished data, GenBank accession no. NC_005839
[GenBank]
). The amino acid identity among these homologous proteins was low (20.1–47.4 %), but the predicted topological structure was rather conserved among the proteins from NPVs (Fig. 1b).
Transcriptional mapping of the 3' and 5' end of Ha107 transcripts
To determine whether Ha107 was transcribed, 3'RACE analysis was performed with total RNA purified from mock-infected and HearNPV-infected HzAM1 cells at various times p.i. (Fig. 2b). A product of the expected size (620 bp) was first detected at 24 h p.i., which increased in amount up to 96 h p.i., suggesting that Ha107 is transcriptionally active, predominantly late during infection. The 3'RACE products from 48 h p.i. were cloned into pGEM-T Easy and sequenced. The obtained sequences mapped the 3' end of the Ha107 transcript at 38 nt downstream of the putative translation stop codon TAA and 13 nt downstream of the last A of the polyadenylation signal sequence AATAAA (Fig. 2c).
5'RACE was performed using total RNA extracted at 48 h p.i. from infected HzAM1 cells to determine the 5' end of the Ha107 transcripts. The lack of an initial PCR product using the first reverse primer (R1) necessitated the use of a nested primer (R2) to detect a PCR product (Fig. 2a). Five clones were sequenced and putative start sites were identified in two regions upstream of the Ha107 translational start site ATG. One start site was 33 nt upstream of the ATG codon of Ha107 at an A residue (Fig. 2d); the other was a C residue located at 11 nt upstream of the ATG codon of Ha106 (Fig. 2e). The presence of a 1.8 bp 5'RACE product also showed that Ha107 not only has its own transcription initiation site, but is also transcribed as one transcription unit together with Ha106 (sod).
Time-course analysis of the HA107 protein in HearNPV-infected cells
Synthesis of the HA107 protein in infected HzAM1 cells was followed by Western blot analysis using a polyclonal antiserum raised by immunization of a rabbit with a bacterially expressed truncated HA107 protein (GST–EX107). The results showed that anti-HA107 antiserum recognized two bands of 48 and 51 kDa, respectively, in infected cells (Fig. 3a). The major band (48 kDa) appeared at 24 h p.i. and reached maximum levels at 72 h p.i. The minor 51 kDa band, detected as early as 48 h p.i., was the same as the theoretical size of HA107. The protein levels remained relatively high throughout late times of infection. The size of the major protein form of HA107 was smaller than the predicted size of the putative Ha107 translation product, possibly as the result of removal of the signal peptide.
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). These data indicated that the HA107 protein is a structural component of the nucleocapsids of both BV and ODV. To study the location of HA107 further, IEM analysis was used and showed that the anti-HA107 antiserum recognized only the nucleocapsids and not ODVs with intact envelope (Fig. 3c). This observation further confirmed that HA107 is a nucleocapsid-associated protein.
Construction of Ha107 knockout and repair HearNPV bacmids
To investigate the role of Ha107 during the viral infection cycle, a Ha107 knockout virus (HaBacHa107) was generated from the HearNPV bacmid HaBacHZ8 (Wang et al., 2003). The phage Red homologous recombination system (Hou et al., 2002) was used to obtain the Ha107 knockout bacmid. PCR analysis and EcoRI digestion were used to verify the construct. As expected, with primers CM and EnF, a PCR product of 530 bp was amplified from HaBacHa107 but not from the control bacmid HaBacHZ8, and with primers ExF and ExR, a 524 bp PCR product was amplified from HaBacHZ8 but not from HaBacHa107 (Fig. 4c). EcoRI digestion profiles revealed that the 5.99 kb EcoRI fragment from HaBacHZ8 containing HA107 was replaced by two smaller EcoRI fragments of 2.91 and 2.92 kb in HaBac107 (Fig. 4b) indicating correct insertion of the CmR gene. Therefore, the Ha107 gene was successfully eliminated in HaBac107.
The egfp and ph genes were inserted into HaBacHZ8 and HaBacHa107 to generate HaBacHZ8-eGFP-PH and HaBacHa107-eGFP-PH (Fig. 4a). To rescue the wild-type phenotype of the Ha107 knockout, a repair bacmid, HaBac-rHa107-eGFP-PH, was constructed. The Ha107 cassette with its own promoter was inserted into the polyhedrin locus of HaBacHa107 by Tn7-mediated transposition to give HaBac-rHa107-eGFP-PH (Fig. 4a). All insertion constructs were confirmed by PCR analysis (data not shown).
Viral replication in HzAM1 cells
To determine whether Ha107 is essential for viral replication, HzAM1 cells were transfected separately with HaBacHa107-eGFP-PH, HaBacHZ8-eGFP-PH and HaBac-rHa107-eGFP-PH bacmids and the transfection assay was monitored by eGFP expression. At 4 days post-transfection, green fluorescence and polyhedra formation were detected in all transfections. The supernatants were collected at 5 days post-transfection and used to infect fresh HzAM1 cells. After 3 days, green fluorescence was clearly observed in cells infected with each of the recombinant viruses (Fig. 5a). Normal polyhedra formation was also observed in the infected cells. Western blot analysis of purified BVs of HaBacHa107-eGFP-PH, HaBac-rHa107-eGFP-PH and HaBacHZ8-eGFP-PH was performed to confirm the effective removal of Ha107 (Fig. 4d). The HA107 protein was detected in BVs of both HaBac-rHa107-eGFP-PH and HaBacHZ8-eGFP-PH, but not in HaBacHa107-eGFP-PH. These results indicated that Ha107 is not essential for viral replication and propagation in cell culture.
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Ha107-eGFP-PH, HaBacHZ8-eGFP-PH or HaBac-rHa107-eGFP-PH at an m.o.i. of 10. Statistical analysis revealed that HaBacHa107-eGFP-PH produced BV to a significantly higher titre than HaBacHZ8-eGFP-PH (F=100.84, d.f.=1, 54; P<0.01) and the HA107 rescue virus HaBac-rHa107-eGFP-PH (F=100.84, d.f.=1, 54; P<0.01) (Fig. 5b).
Polyhedra formation and oral infectivity
At 60 h p.i., polyhedra were seen by light microscopy in the nuclei of HzAM1 cells infected with HaBacHa107-eGFP-PH, HaBacHZ8-eGFP-PH and HaBac-rHa107-eGFP-PH (Fig. 5a). Infected cells were harvested and analysed by electron microscopy, which revealed the formation of polyhedra containing large numbers of occluded virions (Fig. 6). Polyhedra with singly enveloped virions in HaBacHa107-eGFP-PH-infected cells had a similar shape and size to those of HaBacHZ8-eGFP-PH. These results indicated that the absence of Ha107 did not appear to influence the formation and structure of polyhedra or ODVs.
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). Third-instar larvae were fed polyhedra of HaBacHa107-eGFP-PH, HaBac-rHa107-eGFP-PH or HaBacHZ8-eGFP-PH and mortality was monitored daily. The results showed that HaBacHa107-eGFP-PH was infectious to H. armigera larvae, indicating that HA107 is not essential for infectivity in vivo. Notably, its LD50 was about 10 times higher than that of the control virus (z=4.972, P<0.01) and 15 times higher than that of the Ha107 repair virus (z=5.181, P<0.01) (Table 1). Statistically, the difference between the LD50 values of the control virus and Ha107 repair virus was not significant (z=1.598, P>0.05). The slopes of the regression lines between mortality (probit) and log (dose) of these three viruses were not significantly different from each other (z=0.885, P>0.05, for HaBacHa107-eGFP-PH and HaBacHZ8-eGFP-PH; z=1.656, P>0.05, for HaBacHa107-eGFP-PH and HaBac-rHa107-eGFP-PH; z=0.663, P>0.05, for HaBac-rHa107-eGFP-PH and HaBacHZ8-eGFP-PH).
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DISCUSSION |
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) suggest that the Ha107 gene may have been derived from an ancestral virus. The observation that only one GV (AgseGV) has this gene might be explained by the fact that AgseGV and AgseNPV have the same host, A. segetum, and horizontal gene transfer may have occurred.
Ha107 potentially encodes a 51 kDa protein, but two forms of this protein (51 and 48 kDa) were found in infected cells. The smaller but major form (48 kDa) was present in nucleocapsids in both BV and ODV as elucidated by Western blot analysis (Fig. 3). This is in line with the possibility that the predicted 22 aa signal peptide is removed upon entry into the endoplasmic reticulum. Whether this is actually the case would require N-terminal sequencing of the HA107 protein and immunolocalization studies in the cell. The size difference could also be attributed to other post-translational modifications such as glycosylation.
On the basis of the predicted protein structure (Fig. 1), HA107 was predicted to have multiple transmembrane regions. However, HA107 was only found in the nucleocapsid fraction and not in the envelope fraction of BV or ODV, as evidenced by Western blot analysis (Fig. 3b). IEM of ODV and its nucleocapsid confirmed that HA107 is associated with the nucleocapsid and not with the envelope of intact virions (Fig. 3c). Whether HA107 is also a transmembrane component needs to be studied further.
Temporal transcription and expression analysis showed that Ha107 was active predominantly at the late stage in the infection cycle. This is in agreement with the observation that HA107 is a structural component of BV and ODV virions, which assemble relatively late after infection (Fig. 3a). Mapping of Ha107 transcripts by 5'RACE showed that Ha107 transcription was initiated at two distinct locations: one located at 33 nt upstream of the ATG of Ha107, within a sequence without a clear baculovirus consensus transcriptional initiation motif, and the other located 11 nt upstream of the ATG of Ha106 (sod). This suggests the presence of two types of transcript, a short transcript of Ha107 and a long transcript encompassing Ha106 and Ha107, co-terminating at the same 3' end. This observation was also supported by the absence of putative transcriptional termination signal sequences downstream of the Ha106 gene (Fig. 2e). This 3' co-terminal transcript was identified here for the first time in HearNPV, but is common in group I NPVs such as AcMNPV (Friesen & Miller, 1985). The repair virus HaBac-rHa107-eGFP-PH possessed similar replication characteristics (Fig. 5b) and LD50 values (Table 1) to the control virus HaBacHZ8-eGFP-PH, indicating that the function of Ha107 can be rescued by reintroduction of the Ha107 gene at a different locus in the HearNPV genome. These results also indicated that a short Ha107 transcript is enough to produce a functional protein and that the presence of a long transcript encompassing Ha106 and Ha107 does not appear to be necessary for the function of the Ha107 gene.
The Ha107 knockout mutant HaBac107-eGFP-PH was able to infect and propagate in cell culture producing polyhedra and infectious BV progeny (Fig. 5). Polyhedra were also able to infect H. armigera larvae. Interestingly, the BV titre of HaBac107-eGFP-PH was much higher than the control virus and the Ha107 repair virus (Fig. 5b), whilst the LD50 of HaBac107-eGFP-PH was significantly higher than that of the control and repair viruses. These results indicate that the HA107 protein is not essential for virus replication in vivo and in cell culture, but somehow directly or indirectly modulates virus infectivity.
It remains unknown why the deletion of Ha107 results in an increased BV titre and LD50 of the Ha107 knockout virus. It is also not known whether the function of HA107 as a structural component is related to its function affecting infectivity. The existence of Ha107 homologues in several baculoviruses (Fig. 1) suggest that the gene may have been derived from an ancestral virus, and it may still play a role in infection of viruses containing this gene.
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ACKNOWLEDGEMENTS |
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Received 8 August 2007;
accepted 23 August 2007.
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