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
Correspondence
Zhihong Hu
huzh{at}pentium.whiov.ac.cn
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ABSTRACT |
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INTRODUCTION |
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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 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.
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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.
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METHODS |
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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 µl water and quantified by optical density measurements (wavelength, 260 nm). Mock-infected HzAM1 cells were used as controls.
RT-PCR was performed using 1µg 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 (5'-GAATTCCAATATGACGTCTCC-3'). PCR products were analysed by agarose gel electrophoresis.
Expression of Ha94 in Escherichia coli and generation of an HA94-specific antibody.
Two primers, 94up and 94down (5'-CTCGAGGCATAATTAACGTATCACA-3') 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 EcoRIXhoI fragment. This generated plasmid pGEX-Ha94, in which Ha94 is in-frame and fused with GST at the C terminus. E. coli DH5
cells containing pGEX-Ha94 were grown to an OD600 of 0·4 and then induced with 1 mM IPTG. After 3 h at 37 °C, cells were harvested and lysed with lysozyme, sonicated and centrifuged at 5000 g for 10 min at 4 °C. 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).
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RESULTS |
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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 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-X-S/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.
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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 mostly in the NC fraction and a minor part of this protein was located in the E fraction of ODVs (Fig. 3C
).
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DISCUSSION |
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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 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 April 2003;
accepted 11 July 2003.
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