1 Joint Laboratory of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2 Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Correspondence
Just Vlak
just.vlak{at}viro.dpw.wau.nl
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ABSTRACT |
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Introduction |
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Baculoviruses are used widely as bioinsecticides to control heliothine insects in cotton and vegetable crops around the world (Ignoffo, 1973; Cunningham, 1998
). Helicoverpa zea SNPV (HzSNPV) was registered as one of the first commercial baculovirus pesticides (Virion-H, Biocontrol-VHZ, Elcar) in the 1970s and has been used extensively to control the cotton bollworm in the USA and elsewhere (Hsieh, 1989
; Cunningham, 1998
). HaSNPV, isolated in 1975 in the province Hubei, People's Republic of China, has been used successfully in China for over almost 25 years to control H. armigera in cotton and vegetable crops in areas of well over 100 000 hectares (Zhang, 1994
).
HaSNPV has a DNA genome of 135 kb and its sequence has been determined in its entirety (Chen et al., 2001). Some 135 open reading frames (ORFs) of 50 aa or larger in size have been determined for HaSNPV. Twenty ORFs were present only in HaSNPV and have elusive functions; some may be responsible for the unique features of this virus, such as host specificity. These 20 unique ORFs were also present in the closely related HzSNPV, a genotypic variant (Chen et al., 2002
).
This report is part of an effort to elucidate the function of the unique ORFs of HaSNPV. Eight ORFs were pre-selected for analysis based on their size (>100 aa) and the presence of known baculovirus consensus transcription initiation and termination signals (Blissard et al., 2000). In this paper, we analysed the putative ORF 122 (Ha122) (Fig. 1
A) and demonstrated by transcriptional analysis and protein identification and localization that Ha122 is a functional gene expressed at late times post-infection (p.i.). Ha122 encodes a 21 kDa protein and is present in nucleocapsids of occlusion-derived virus (ODV) but not in budded virus (BV).
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Methods |
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Computational analysis.
Ha122 was analysed using the software EXPASY (Appel et al., 1994) for the prediction of domains, motifs, transmembrane regions and subcellular localization domains (Reinhardt & Hubbard, 1998
). Protein comparisons with entries in the updated GenBank/EMBL, SWISS-PROT and PIR databases were performed with BLASTP and PSI-BLAST programs (Altschul et al., 1997
). Sequence alignment was performed with MEGALIGN and alignment editing was performed with the GENEDOC software.
Transcription analysis.
Total RNA was isolated from mock-infected and HzAM1 cells infected with BVs of HaSNPV strain G4 (m.o.i. of 5 TCID50 units per cell) at 0, 4, 8, 12, 24, 48 and 72 h p.i. Cells were resuspended in 500 µl Trizol (Gibco-BRL) and 100 µl chloroform, incubated for 8 min and centrifuged at 14 000 g for 15 min at 4 °C. The RNA present in the water fraction was precipitated using isopropanol, centrifuged at 14 000 g for 10 min at 4 °C, washed with 70 % ethanol and resuspended in 50 µl water. The RNA solutions were incubated at 55 °C for 10 min and the RNA quantified by optical density measurements at 260 nm.
RT-PCR was performed using a 3'RACE kit (Roche) employing 1 µg of purified total RNA as template per time-point. First-strand cDNA synthesis was performed using AMV reverse transcriptase and an oligo(dT) anchor primer, according to the manufacturer's instructions. The cDNA mixtures were amplified with PCR using the PCR oligo(dT) anchor primer and the gene-specific forward primer F1 (5'-ATGGAAACGGTACGCACATTCATTCTG-3') (Fig. 1B). The PCR products obtained were analysed by agarose gel electrophoresis. The amplified RT-PCR products from 48 h p.i. were gel-purified, cloned into pGEM-T (Gibco-BRL) (Sambrook et al., 1989
) and sequenced with T7 primers to determine the 3' end of the Ha122 transcript.
The 5' end of the Ha122 transcript was determined using the 5'RACE kit (Roche) employing 1 µg of purified total RNA as template and isolated at 4 and 48 h p.i. Briefly, first-strand cDNA synthesis was performed with the gene-specific reverse primer R1 (5'-GGGGAATTCCTGCTTTCTGGAATTGTCCAATCAACTT-3') (Fig. 1B). The cDNA was purified with the High Pure PCR Purification kit (Roche) and a poly(A) tail was added to the 5' end using terminal transferase and dATP. The tailed cDNAs were amplified by PCR using the oligo(dT) anchor primer (forward) and the nested, gene-specific reverse primer R2 (5'-ACTAACATTATACACTTGGTCACCG-3') (Fig. 1B
). A second PCR was performed using the PCR 5' oligo(dT) anchor primer and the nested reverse primer R3 (5'-CTTGTTCCTTGGCTAGATTACAAACG-3') (Fig. 1B
). The PCR products obtained were gel-purified, cloned into pGEM-T and sequenced with T7 primers.
Generation of anti-Ha122 antiserum.
The complete Ha122 ORF (588 nt) was amplified by PCR and subcloned into the vector pGEX-KG, in-frame with the C-terminal glutathione S-transferase (GST) tag present in this plasmid (Guan & Dixon, 1991). The plasmid, designated pGST-Ha122, was transfected into Escherichia coli BL21 cells and fusion protein expression was induced by incubation in the presence of 1 mM IPTG when the optical density measurement at 600 nm reached
0.7. After 3 h, the cells were harvested and lysed by sonication in ice-cold PBS. The lysate was centrifuged at 3000 g for 5 min. The pellets (inclusion bodies containing the highly expressed fusion protein) were washed twice with 100 mM Tris/HCl, pH 8.0, and were dissolved in the above buffer containing 5 % SDS (sonication was required to dissolve the pellets). This fusion protein solution was separated on a continuous-elution, preparative 12·5 % SDS-PAGE gel (Model 491 PrepCell) (BioRad). The eluted fractions were collected and electrophoresed and the bands were visualized by Coommassie brilliant blue staining. The proteins were concentrated with a Centriprep 10 kDa filter device (Amicon) and the concentrated solution was dialysed overnight with two changes of PBS. This dialysed protein sample was used as an immunogen to raise Ha122-specific antiserum in 8-month-old, egg-laying chickens.
Chickens were injected intramuscularly, each with 100 µg purified GSTHa122 fusion protein using Freund's incomplete adjuvant. The chickens were boosted after 2 weeks with 100 µg of purified protein. Eggs were collected every day for 4 weeks and serum was collected 12 weeks after the boost injection. Western blot analyses using E. coli BL21 cell extracts expressing GSTHa122 and column-purified GSTHa122 were used to test the batches of specific antisera.
Western blot analysis.
Monolayers of HzAM1 cells were mock- and HaSNPV-infected at an m.o.i. of 5 TCID50 units per cell. Cells were harvested at 0, 24, 48 and 72 h p.i., pelleted at 3000 g, resuspended in PBS and lysed in SDS-PAGE loading buffer by boiling for 10 min. Protein samples were separated by SDS-PAGE and transferred onto an Immobilon-P nitrocellulose membrane (Millipore) by semi-dry electrophoresis transfer (Ausubel et al., 1994). The membranes were incubated overnight in 2 % skimmed milk powder in TBS at 4 °C. The membranes were allowed to react with Ha122 antiserum diluted 1 : 1000 for 1 h at room temperature and treated further as described by IJkel et al. (2000)
.
HzAM1 cells (106 per Petri dish in 2 ml medium) were infected with HaSNPV at an m.o.i. of approximately 5 TCID50 units per cell and incubated at 27 °C. Cells were harvested at 0, 8, 16, 24, 48 and 72 h p.i., washed three times in PBS and lysed in SDS-PAGE loading buffer. Uninfected cells were taken as a control. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Duplicate blots were prepared and processed for immunoblotting. One blot was incubated in the presence of pre-immune serum (dilution 1 : 1000) and the other in the presence of the anti-Ha122 antiserum (dilution 1 : 1000), as detailed by IJkel et al. (2000).
Purification of HaSNPV BV and ODV fractions.
HzAM1 cells were infected with BVs of HaSNPV G4 at an m.o.i. of 0.1 TCID50 units per cell. After 3 days, the cell culture supernatant was collected and clarified at 2000 g for 10 min at 4 °C. The supernatant containing the BVs was passed through a 045 µm pore-size filter. BVs in the filtrate were pelleted through a 25 % (wt/wt) sucrose cushion made up in 0·1 TE (10 mM Tris/HCl, pH 7·5, and 1·0 mM EDTA) by centrifugation at 100 000 g for 90 min at 4 °C, and resuspended in 0·1 TE. ODVs were purified from HaSNPV G4 polyhedra derived from infected H. armigera fourth-instar larvae as described previously (Chen et al., 2001). Separation of envelope and nucleocapsid fractions after treatment with NP-40 was done as described by IJkel et al. (2000)
. The purity of the nucleocapsids was checked by electron microscopy.
Construction of a transient expression plasmid.
To construct plasmids that allow gene expression in H. zea cell lines upon transfection, a vector (pHaie-1-GFP) with a HaSNPV ie-1 promoter-driven green fluorescent protein (GFP) gene was constructed. The HaSNPV ie-1 promoter (nt 1117011355) (Chen et al., 2001) was amplified by PCR using two specific primers (primer1, 5'-CGCGAGCTCATAACAGTATTTGGTCCTTCGTTCTG-3', and primer2, 5'-CGAGGATCCCTTGCCGTAAAGTTTAATTTCAATAA-3'). The restriction sites for BamHI and SstI are shown in italics. The PCR product was cloned into the multiple cloning site of pGEM-T EASY (Gibco-BRL) and the HaSNPV ie-1 promoter insert was sequenced to make sure that there were no mutations in the promoter region. SstI/BamHI digestion was used to isolate the promoter fragment, which was then cloned into a p166AcV5-GFP (IJkel et al., 2000
) derivative, where the Opgp64 promoter and the AcV5 segment were removed by SstI/BamHI digestion, to give pHaie-1.
The Ha122-encoding sequence containing the ATG but not the TAA codon was amplified by PCR. Two restriction sites, BamHI and EcoRI, were introduced at either end by the primers. These two restriction sites were used to insert Ha122 downstream of the Haie-1 promoter and upstream of GFP to render the fusion vector pHaie-1-ORF122-GFP.
Fluorescence microscopy.
Hz2E5 cells (1x105) were grown on glass cover slips in Petri dishes and transfected with 5 µg of plasmid DNA using Cellfectin (Gibco-BRL). At 60 h p.i., the cells were examined under a Zeiss LSM510 (confocal) laser scanning microscope for fluorescence using an excitation wavelength of 488 nm and an emission band pass filter of 505530 nm.
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Results |
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The regions upstream of Ha122 were analysed for the presence of putative transcription start sites, such as baculovirus consensus early CA(G/T)T and late DTAAG promoter motifs, downstream activating elements and host factor binding sites, such as GATA and CACGTG. A late gene transcription motif (TATAANATAAG, tata-1) was found 74 nt upstream of the putative translation start codon ATG of Ha122. Another TATA box (tata-2) was located 175 nt upstream of the start codon. An early transcription motif CAGT was observed 39 nt upstream of the ATG codon of Ha122. Additionally, a palindrome of 12 nt was found 18 nt upstream of the putative translation start site but downstream of putative transcription start sites. This palindrome might be involved in the transcriptional or translational control of Ha122. Two polyadenylation signal sequences (stop-1 and stop-2) were located 8 and 59 nt downstream, respectively, of the putative translation stop codon TAA.
Transcriptional analysis
To see whether Ha122 was transcribed, 3'RACE analysis was performed with total RNA purified from mock-infected and HaSNPV-infected HzAM1 cells at various times p.i. (Fig. 2). A product of the expected size (600 bp) was detected as early as 8 h p.i., which increased in amount up to 72 h p.i., suggesting that Ha122 is transcriptionally active predominantly late in infection. The 3'RACE products from 48 h p.i. were cloned into pGEM-T and sequenced. The obtained sequences mapped the 3' end of the Ha122 transcript 27 nt downstream of the putative translation stop codon TAA and 14 nt downstream of the last A in the first AATAAA sequence. No evidence was obtained to show that the downstream transcription termination signal stop-2 (Fig. 1B
) was used.
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Immunodetection of the Ha122 protein in infected cells
The Ha122 protein has a predicted molecular mass of 21·6 kDa. To see whether a protein encoded by this ORF is made and to study its possible function, a polyclonal antibody was prepared by immunization of a chicken with a purified Ha122 protein. This protein was overexpressed in bacteria as a GSTHa122 fusion (52 kDa) and purified by column chromatography to almost homogeneity (Fig. 3). The chicken antiserum obtained reacted well against the purified fusion protein and not against bacterial proteins (data not shown).
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Localization of the Ha122 proteins in insect cells
The subcellular localization of the Ha122 protein was investigated using an N-terminal GFPHa122 fusion construct. The fusion construct was placed under the control of the Haie-1 promoter. Hz2E5 cells were transfected with plasmid constructs with GFP alone and with the GFPHa122 fusion insert, incubated for 48 h and examined for fluorescence by confocal laser scanning microscopy (Fig. 6). GFP alone showed homogeneous fluorescence in the cytoplasm and nucleus (Fig. 6A
). The GFPHa122 fusion protein was localized primarily in the cytoplasm (Fig. 6B
) and hardly present in the nucleus. However, when cells transfected with the GFPHa122 fusion construct were superinfected with HaSNPV, the fusion protein was localized both in the cytoplasm and in the nucleus (Fig. 6C
). A similar result was obtained with HzAM1 cells but the fluorescence was not as abundant as that seen in Hz2E5 cells.
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Discussion |
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Both Ha122 transcripts and the Ha122 product (21 kDa) are detected predominantly relatively late in the infection cycle and this is compatible with the observation that the 21 kDa protein is present in ODVs and thus required when ODVs are assembled. Analysis of the 5' ends of the Ha122 transcript support this conclusion further, as the major transcript starts close to the baculovirus consensus late motif RTAAG. This is a common feature of structural virion proteins (Funk et al., 1997). However, it cannot be excluded that Ha122 is expressed at earlier times after infection, as there is a putative early transcription motif (CAGT) preceded by two TATA boxes (Fig. 1B
) upstream of the baculovirus consensus late motif RTAAG. The necessity to use a nested PCR at late times after infection to detect the 5' end of the transcript suggests that the Ha122 transcript is not present in abundance in HaSNPV-infected cells. The Ha122 gene contains two downstream polyadenylation signals (AATAAA) at 8 and 59 nt relative to the TAA translation stop site. It is not clear why the second stop is preferred.
The functions and precise location of Ha122 in the virion have not been elucidated fully. The protein is associated with the ODV capsid but not with the BV capsid. This may suggest that the Ha122 protein either plays a role in the assembly of ODVs or contributes to the infection of midgut epithelial cells by ODVs. A similar situation exists for SeMNPV, where one of the unique ORFs, Se117, also encodes an ODV-specific protein (IJkel et al., 2001), whereas the Se117 product is present both in the nucleocapsid and in the envelope fractions. As for Ha122, this protein is associated predominantly with the ODV nucleocapsid. Further localization of the Ha122 protein could be obtained by immunogold electron microscopy and proteinprotein interaction studies. Ha122 lacks a nuclear import signal and this explains the inability of this protein to enter the nucleus on its own (Fig. 6
). Only in conjunction with a HaSNPV infection does Ha122 end up in the cell nucleus, suggesting that Ha122 needs most likely a viral chaperone protein or a host-induced/modified protein to get there. It would be of interest to learn which HaSNPV protein functions as a chaperone for Ha122, as this will enhance our understanding of the scaffolding of baculovirus ODVs. Further information of the function of Ha122 will come from deletion and mutagenesis studies using a HaSNPV bacmid constructed recently (H. Wang & J. M. Vlak, personal communication).
In the original HaSNPV isolate (Fig. 5, lane 3), another strong band was detected besides the 21 kDa band. This 23 kDa band reacts equally strongly with the 21 kDa antiserum. This may be a reflection of the polymorphism of the Ha122 gene, since the original isolate contains at least eight different genotypes (Sun et al., 1998
). Sequence analysis of the Ha122 regions of the various HaSNPV genotypes should give support to this supposition. However, the biological meaning of the two bands remains unclear. The antiserum against ORF122 might be used to identify or classify different strains or subtypes of HaSNPV.
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ACKNOWLEDGEMENTS |
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Received 26 March 2002;
accepted 11 September 2002.