Journal of General Virology (2003), 84, 3021–3027
DOI 10.1099/vir.0.19291-0
Open reading frame 94 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus encodes a novel conserved occlusion-derived virion protein, ODV-EC43 Minggang Fang,1 Hanzhong Wang,1 Hualin Wang,1 Li Yuan,1 Xinwen Chen,1 Just M. Vlak2 and Zhihong Hu1 Correspondence Zhihong Hu
[email protected]
1
Join-Laboratory of Invertebrate Virology and Key Laboratory of Molecular Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, People’s Republic of China
2
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Received 12 April 2003 Accepted 11 July 2003
Open reading frame 94 (Ha94) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is 1086 bp long and a homologue of Autographa californica multiple NPV ORF109. The gene is conserved among all baculoviruses whose genomes have been completely sequenced so far and is thus considered a baculovirus core gene. Ha94 transcripts were detected from 24 to 96 h post-infection (p.i.) of HzAM1 cells with HaSNPV. Polyclonal antiserum raised to a GST–HA94 fusion protein recognized a 43 kDa protein, HA94, in infected cell lysates from 36 to 96 h p.i., suggesting that Ha94 is a late gene. Western blot analysis of proteins present in budded virus and occlusion-derived virus (ODV) showed that Ha94 encodes a structural component of ODV. When ODVs were fractionated further into nucleocapsid and envelope components, Western blot analysis indicated that the encoded protein was associated with both the nucleocapsid and the envelope. In summary, data available indicated that Ha94 encodes a novel ODV-specific protein of HaSNPV, designated ODV-EC43.
INTRODUCTION The Baculoviridae, a diverse family of more than 600 viruses, encompasses two genera, the Nucleopolyhedroviruses (NPVs) and the Granuloviruses (GVs) (Blissard et al., 2000). These viruses are very specific and mainly infect insects of the orders Lepidoptera, Hymenoptera and Diptera, but always within the phylum Arthropoda. The family is characterized by the occlusion of virions into large proteinaceous capsules or occlusion bodies (OBs). NPVs have two morphotypes, SNPV and MNPV, depending on the single (S) or multiple (M) packaging of the nucleocapsids (NCs) into the envelope (E). Based on phylogenetic analysis, NPVs have been divided into groups I and II (Zanotto et al., 1993; Herniou et al., 2001). In a single infection cycle, two forms of virions are produced, the budded virus (BV) and the occlusion-derived virus (ODV). BVs are responsible for the spread of the virus from cell to cell in the larva host and ODVs initiate the infection in a susceptible host and are responsible for the horizontal spread of the virus in an insect population (Blissard et al., 2000). The structural components of BVs and ODVs, except for the circular double-stranded DNA, are different to accommodate their respective 0001-9291 G 2003 SGM
Printed in Great Britain
functions in an infection cycle (Braunagel & Summers, 1994). The molecular biology of baculoviruses has developed rapidly in the last two decades. To date, the complete sequences of 17 baculovirus genomes have been reported; they range in size from 101 to 179 kbp and are predicted to encode 109 to 181 open reading frames (ORFs). These viruses are Autographa californica MNPV (AcMNPV) (Ayres et al., 1994), Bombyx mori NPV (BmNPV) (Gomi et al., 1999), Culex nigripalpus NPV (CuniNPV) (Afonso et al., 2001), Epiphyas postvittana NPV (EppoNPV) (Hyink et al., 2002), Helicoverpa armigera SNPV (HaSNPV) (Chen et al., 2001), Helicoverpa zea SNPV (HzSNPV) (Chen et al., 2002), Lymantria dispar MNPV (LdMNPV) (Kuzio et al., 1999), Mamestra configurata NPV A (MacoNPV A) (Li et al., 2002b), MacoNPV B (Li et al., 2002a), Orgyia pseudotsugata MNPV (OpMNPV) (Ahrens et al., 1997), Rachiplusia ou NPV (RaouNPV) (Harrison & Bonning, 2003), Spodoptera exigua MNPV (SeMNPV) (IJkel et al., 1999), Spodoptera litura NPV (SpltNPV) (Pang et al., 2001), Cydia pomonella GV (CpGV) (Luque et al., 2001), Phthorimaea operculella GV (PhopGV) (GenBank accession no. NC_004062), Plutella xylostella GV (PxGV) (Hashimoto 3021
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METHODS
et al., 2000) and Xestia c-nigrum GV (XcGV) (Hayakawa et al., 1999). Genome comparisons of all of the baculoviruses completely sequenced so far revealed 30 conserved genes (Table 1), most of which are related either to DNA replication/gene expression or to virion structure (Herniou et al., 2003). However, there are still several genes whose functions remain unknown, including Ac109.
Insect cells, insects and virus. The H. zea cell line HzAM1
(McIntosh & Ignoffo, 1983) was maintained at 28 uC in Grace’s medium supplemented with 10 % FBS. A culture of H. armigera insects was maintained according to Sun et al. (1998). An in vivocloned strain of HaSNPV (HaSNPV-G4) (Sun et al., 1998; Chen et al., 2001) was used as wild-type virus and propagated in H. armigera.
Helicoverpa armigera SNPV (HearNPV, also called HaSNPV) was isolated initially from diseased larvae in the province of Hubei. It has been developed as a commercial pesticide and has been used successfully to control cotton bollworm in China (Zhang, 1994). The genome of HaSNPV contains 135 ORFs of 50 aa or more (Chen et al., 2001). We have embarked on the functional characterization of HaSNPV genes, with particular emphasis on genes common to all baculoviruses or unique to HaSNPV. Several HaSNPV genes, such as polyhedrin (Chen et al., 1997a), ecdysteroid UDP-glucosyltransferase (egt) (Chen et al., 1997b), late gene expression factor 2 (lef-2) (Chen et al., 1999), basic DNA-binding protein (BDBP) (Wang et al., 2001) and Ha122 (Long et al., 2003), have been characterized so far. Ha122 is unique to HaSNPV and its gene product is an ODV protein.
Computer-assisted analysis. HA94 was analysed using the EXPASY server (Appel et al., 1994) for the prediction of signal peptides, transmembrane regions and motifs. Protein comparisons with entries in the updated GenBank/EMBL, SWISS-PROT and PIR databases were performed using the BLASTP, FASTA and PSI-BLAST programs (Pearson, 1990; Altschul et al., 1997). Multiple sequence alignments were performed using the PILEUP program, with gap creation and extension penalties set to 8 and 2, respectively (Devereux et al., 1984). Alignment editing was done using the GENEDOC software.
Transcription analysis. 106 HzAM1 cells were infected with
HaSNPV-G4 BV at an m.o.i. of 5 p.f.u. per cell. Total RNA was isolated at 0, 4, 8, 16, 24, 48, 72 and 96 h post-infection (p.i.) with Trizol, according to the manufacture’s guidelines (Gibco-BRL). RNA was dissolved in 50 ml water and quantified by optical density measurements (wavelength, 260 nm). Mock-infected HzAM1 cells were used as controls. RT-PCR was performed using 1mg total RNA as template for each time-point. First-strand cDNA synthesis was performed with AMV reverse transcriptase (Promega) and a 15 nt oligo(dT) primer, according to the manufacturer’s instructions. cDNA mixtures were amplified by PCR using the oligo(dT) primer and a gene-specific forward primer, 94up (59-GAATTCCAATATGACGTCTCC-39). PCR products were analysed by agarose gel electrophoresis.
HaSNPV ORF94 (Ha94), a homologue of Ac109, is one of the conserved or core genes among baculoviruses (Herniou et al., 2003), although its function remains elusive. By transcriptional analysis, protein identification and localization, we present evidence that HA94 is a newly assigned structural protein of baculoviruses.
Table 1. Genes conserved among members of the Baculoviridae The conserved genes were the ORFs that exist in all 17 baculoviral genomes sequenced so far are listed. The sources were: Ayres et al. (1994) for AcMNPV; Gomi et al. (1999) for BmNPV; Afonso et al. (2001) for CuniNPV; Hyink et al. (2002) for EppoNPV; Chen et al. (2001) for HaSNPV; Chen et al. (2002) for HzSNPV; Kuzio et al. (1999) for LdMNPV; Li et al. (2002b) for MacoNPV A; Li et al. (2002a) for MacoNPV B; Ahrens et al. (1997) for OpMNPV; Harrison & Bonning (2003) for RaouNPV; IJkel et al. (1999) for SeMNPV; Pang et al. (2001) for SpltNPV; Luque et al. (2001) for CpGV; GenBank accession no. NC_004062 for PhopGV; Hashimoto et al. (2000) for PxGV; and Hayakawa et al. (1999) for XcGV. Expression/replication-related genes
Structural genes
lef-1 (Ac14) lef-2 (Ac6) lef-4 (Ac90) lef-5 (Ac99) lef-8 (Ac50) lef-9 (Ac62) dna-pol (Ac65) helicase (Ac95) p47 (Ac40) vlf-1 (Ac77)
pif-2 ld130 vp1054 gp41 vp91 vp39 p6.9 pif-1 p74 odv-ec27 odv-e56
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(Ac22) (Ac23) (Ac54) (Ac80) (Ac83) (Ac89) (Ac100) (Ac119) (Ac138) (Ac144) (Ac148)
Others
Unknown genes
alk-exo(Ac133)
Ac68 Ac81 Ac92 Ac96 Ac98 Ac109 (Ha94) Ac115 Ac142
Journal of General Virology 84
HaSNPV ORF94 encodes a novel ODV protein, ODV-EC43
Expression of Ha94 in Escherichia coli and generation of an HA94-specific antibody. Two primers, 94up and 94down
(59-CTCGAGGCATAATTAACGTATCACA-39) were designed to amplify the entire Ha94 ORF from the HaSNPV-G4 genome. The PCR product was cloned first into pGEM-T-Easy (Promega) and then into the expression vector pGEX-KG (Guan & Dixon, 1991) as an EcoRI–XhoI fragment. This generated plasmid pGEX-Ha94, in which Ha94 is in-frame and fused with GST at the C terminus. E. coli DH5a cells containing pGEX-Ha94 were grown to an OD600 of 0?4 and then induced with 1 mM IPTG. After 3 h at 37 uC, cells were harvested and lysed with lysozyme, sonicated and centrifuged at 5000 g for 10 min at 4 uC. The fusion protein present in the pellet was separated in 12 % SDS-polyacrylamide gels and purified. Antisera were generated by immunizing rabbits with purified protein (Sambrook et al., 1989) and tested by Western blot analysis. Western blot analysis. HzAM1 cells were infected with HaSNPV-
G4 at an m.o.i. of 5 p.f.u. per cell. Samples of total cell proteins were prepared from infected cells harvested at 0, 12, 16, 24, 36, 48, 72 and 96 h p.i. The BVs and ODVs of HaSNPV were purified and the E and NC fractions of ODVs were separated according to IJkel et al. (2001). Protein samples were separated by 12 % SDS-PAGE and the separated proteins transferred onto Hybond-N membranes (Amersham) by semi-dry electrophoresis transfer (Ausubel et al., 1994). HA94-specific antiserum and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Sino-American) were used as the primary and secondary antibodies, respectively. The signal was detected using a BCIP/NBT kit (Sino-American).
RESULTS
homologues (data not shown) indicates that CuniNPV, which was isolated from a dipteran host, is distantly related to baculoviruses isolated from the Lepidoptera. No signal peptide sequence was predicted on the putative HA94 protein (Fig. 1) by PSORT and SIGNALP and no transmembrane region was identified by TMPRED at the EXPASY server (Appel et al., 1994). HA94 contained three potential N-linked glycosylation consensus sequences (N-XS/T), but we have not investigated whether these sites are glycosylated. Only one of these, at amino acid position 80, was predicted by the EXPASY server to be glycosylated. Alignment of the putative HA94 peptide with its homologues in other baculoviruses indicated that 13 aa were absolutely conserved (Fig. 1). These conserved amino acids may be important for the function of the HA94 homologues. Transcription of Ha94 Transcription of Ha94 was examined by RT-PCR, using total RNA isolated from HzAM1 infected with HaSNPV at different time points as template and primers 94up primer and oligo(dT). A single band of 1?2 kbp was first detected at 24 h p.i. and continued to be present until 96 h p.i., indicating that Ha94 is a late gene (Fig. 2A). The predicted size from 94up to the poly(A) signal site AATAAA is 1114 nt, which is in agreement with the size of the transcript detected.
Sequence analysis of Ha94 and its homologues Ha94 is located in the HindIII-A fragment of the HaSNPV genome (nt 87 300–88 385) (Chen et al., 2001). Sequence analysis indicated that Ha94 is 1086 bp and encodes a protein of 362 aa with a predicted molecular mass of 41?5 kDa. A late baculoviral transcription initiation motif, ATAAG, was found 65 nt upstream of the putative translational start site of Ha94, suggesting that Ha94 may be a ‘late’ gene of HaSNPV. A polyadenylation signal (AATAAA) was identified 14 nt downstream of the TAA stop codon. Searches of databases showed that HA94 is conserved among baculoviruses and is shared by all baculoviruses whose complete genome sequences have been published so far. The homologues of HA94 are AcMNPV ORF109, BmNPV ORF92, CuniNPV ORF69, EppoNPV ORF95, HzSNPV ORF97, LdMNPV ORF107, MacoNPV A ORF79, MacoNPV B ORF79, OpMNPV ORF109, RaouNPV ORF104, SeMNPV ORF59, SpltNPV ORF96, CpGV ORF55, PhopGV ORF50, PxGV ORF43, and XcGV ORF53. A homologue was also reported in another baculovirus, Leucania separata NPV (LeseNPV), as LS109. Analysis indicated that, apart from HzSNPV, HA94 shared amino acid sequence identities ranging from 58 % with LdMNPV ORF107 to 10 % with CuniNPV ORF69. HA94 is related most closely to HzSNPV ORF97, with 99 % identity, consistent with the previous suggestion that HzSNPV and HaSNPV are different strains of the same virus (Chen et al., 2002). The very low identity of CuniNPV ORF69 with all other baculoviral HA94 http://vir.sgmjournals.org
Immunodetection of HA94 in infected cells and virus particles To obtain a polyclonal rabbit antibody against this protein, HA94 was expressed in E. coli as a GST–HA94 fusion protein (data not shown) and purified by gel separation and electroelution. Specific antiserum was generated by immunizing rabbits with purified protein. When HaSNPVinfected HzAM1 cell extracts were analysed by SDS-PAGE and Western blot, the HA94 antibody detected a specific protein of molecular mass 43 kDa (Fig. 2B). This protein was first detectable at 36 h p.i. and continued to accumulate until 96 h p.i. This suggested, in agreement with the analysis of transcripts, that HA94 was synthesized at a late stage of infection, possibly as part of OB morphogenesis. The size of the 43 kDa protein is in agreement with the predicted 41?5 kDa based on its sequence, which suggests that no major post-translational modification occurs. To investigate if HA94 is a structural component of HaSNPV virions, Western blot analysis of BV and ODV proteins was conducted. HA94 was detected in preparations of ODVs but not BVs (Fig. 3A), indicating that HA94 is a structural component of ODVs. The location of HA94 in ODVs was determined further by Western blot analysis of the NC and E fractions of ODVs. The ODV NC and E fractions were analysed by SDS-PAGE and the purity of nucleocapsids was checked by electron microscopy. The protein profiles of the NC and E were distinct (Fig. 3B). Western blot analysis indicated that HA94 was located 3023
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HaSNPV ORF94 encodes a novel ODV protein, ODV-EC43
Fig. 2. Analysis of the transcription and expression of Ha94. (A) RT-PCR of HaSNPV Ha94 transcripts. Total RNA was extracted at different time-points (h p.i.) from HzAM1 cells, which were infected with HaSNPV at an m.o.i. of 5 p.f.u. per cell. Reverse transcription was performed using an oligo(dT) primer and PCR amplification was done using the 94up primer and oligo(dT). M, DNA marker DL2000. (B) Western blot analysis of HA94. HzAM1 cells were infected with HaSNPV at an m.o.i. of 5 p.f.u. per cell and cells were collected from 0 to 96 h p.i. About 15 mg total protein was loaded per lane, separated in a 12 % gel, transferred to a Hybond membrane and reacted with HA94-specific antibody. Fig. 3. Detection of HA94 in HaSNPV BVs and ODVs. (A) About 10g of purified BVs and ODVs were separated by SDS-PAGE and analysed by Western blot analysis using anti-HA94 serum as a probe. (B) SDS-PAGE analysis of purified ODV, nucleocapsid (NC) and envelope (E) of ODV. About 15g of ODV and NC, and 10g of E were loaded and separated on a 12 % gel. The gel was stained with Coomassie brilliant blue R250. M, Protein standard. (C) Western blot analysis of HA94 in ODV, NC and E fractions of ODVs with HA94 antibody. The amounts of proteins used were the same as in (B).
mostly in the NC fraction and a minor part of this protein was located in the E fraction of ODVs (Fig. 3C).
DISCUSSION The conservation of HA94 in baculoviruses may imply an important function of this protein in baculovirus multiplication. To elucidate its function, we analysed the transcription and translation of Ha94 in HaSNPV-infected H. armigera cells and investigated whether it was a structural component of the virions. Transcriptional analysis of Ha94 by RT-PCR showed that Ha94 transcription started at 24 h p.i. and continued until at least 96 h p.i. (Fig. 2A). This result suggests that Ha94 is a late gene and probably uses the late transcription initiation ATAAG at 65 nt upstream of the translational start site. Western blot analysis confirmed this observation, as HA94 was detected predominantly from 36 to 96 h p.i., relatively late in the infection cycle (Fig. 2B). This is compatible with the observation that the 43 kDa protein is present in ODVs but not in BVs (Fig. 3A). Western blot analysis revealed further that HA94 was an ODV-specific structural protein
and is present in both the NC and E fractions of ODVs (Fig. 3C). Therefore, HA94 is a novel structural ODV protein, which we have named ODV-EC43. The conservation of ODV-EC43 in baculoviruses suggests that the protein plays an important role in ODV morphogenesis and/or ODV structure, or in the infection process. Considerable progress has been achieved in the identification of baculovirus genes that are likely to encode virion structural proteins. At the start of our research, nine structural virion proteins had been identified as conserved in all baculoviruses. These include three proteins that exist in both BVs and ODVs: the basic DNA-binding protein p6.9 (Wilson et al., 1987), the major capsid protein VP39 (Blissard et al., 1989) and VP1054 (Olszewski & Miller, 1997). One protein exists in BVs: LD130 (Pearson et al., 2000). In addition, five proteins that are specific for ODVs have been identified: P74 (Kuzio et al., 1989), GP41 (Whitford & Faulkner, 1992a, b), ODV-EC27 (Braunagel et al., 1996b), ODV-E56 (Braunagel et al., 1996a) and VP91 (Russell & Rohrmann, 1997). Recently, two novel structural virion proteins that are involved in oral
Fig. 1. Alignment of HA94 and the homologues of baculoviruses. The sources of the sequences were the same as those described in Table 1, except for LS109 of LeseNPV (PIR accession no. T00200). GENEDOC software was used for homology shading. Two shading levels were set: black for 100 % identity and grey for 80 % identity. http://vir.sgmjournals.org
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infectivity, PIF-1 (Kikhno et al., 2002) and PIF-2 (Pijlman et al., 2003), have been identified, although their exact location in the ODV has not been established yet. The identification of ODV-EC43 brings the growing number of conserved structural proteins to 12. These 12 proteins are likely to form the core structure of the baculovirus virions or may be important for infection. Deletion of Ha94 from the HaSNPV genome is under way, to allow further functional analysis of HA94 in terms of its interaction with other structural proteins and its role in virus infection.
ACKNOWLEDGEMENTS This research was supported in part by grants from the National Natural Science Foundation of China (NSFC) (grant nos 30025003 and 30070034), the Chinese Academy of Sciences (CAS) (grant nos Kscx21-02 and Kscx2-SW-301-09), an 863 project of China (grant no. 2001AA214031) and a joint grant from the CAS and the Royal Netherlands Academy of Sciences (grant no. 01CDP023).
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