Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor (PIF-2)

Baculoviruses are large, double-stranded DNA viruses pathogenic to a variety of insect species. Naturally occurring and modified baculoviruses are potentially viable as environmentally benign biological-control agents against economically important forest and agricultural insect pests (Wood & Granados, 1991). The baculovirus Helicoverpa armigera nucleopolyhedrovirus [HearNPV, also called Helicoverpa armigera single nucleocapsid polyhedrovirus (HaSNPV)] is a naturally occurring pathogen of cotton bollworm and was first isolated from diseased larvae in the Hubei province of China. It has been developed as a successful commercial pesticide for the control of cotton bollworm in China (Zhang, 1994).

To understand the genetic properties of HearNPV and further improve its effectiveness, the complete nucleotide sequence and genetic organization of HearNPV have been elucidated (Chen et al., 2001). The virus was modified genetically by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene from its genome or by inserting an insect-selective scorpion toxin gene that has been shown to improve the viral insecticidal property (Chen et al., 2000; Sun et al., 2004). We are now focusing on studying other genes that may contribute to the insecticidal property of the virus. Per os infectivity factors are among the genes of interest, as they may serve as targets for future genetic engineering to enhance the oral infectivity of baculoviruses.

Recently, Se35 has been identified as encoding a per os infectivity factor (PIF-2) of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) (Pijlman et al., 2003). It has been known that the PIF-2 homologue (Ac22) in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a structural component of the occlusion-derived virus (ODV) (Braunagel et al., 2003). Although the gene is conserved in baculoviruses, its functionality in viruses other than SeMNPV has not yet been elucidated. NPVs are designated single (S) or multiple (M) NPVs based on whether the ODV that initiates primary midgut infections contains single or multiple nucleocapsids. Washburn et al. (2003) revealed that, in orally inoculated larvae of Heliothis virescens, Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) initiated primary infections quicker and in greater numbers than AcMNPV, implying differences between SNPVs and MNPVs in primary midgut infection.

In this report, we characterized open reading frame (ORF) 132 of HearNPV (Ha132), a homologue of Se35, and studied its function by deleting it from the virus. RT-PCR was performed to detect transcription and Western blot analysis was used to identify whether HA132 was a component of the virus structure. Recombinant viruses HearNPVΔ132 with deletion of Ha132 were constructed from wild-type (wt) HearNPV. The infectivity of budded virus (BV) and ODV was examined in vivo. The data presented in this manuscript ascertain that Ha132 is functional in HearNPV.

Computer-assisted analysis.
A similarity search of the Ha132 gene and its deduced amino acid sequences was performed against updated GenBank/EMBL, SWISS-PROT and PIR databases by using BLASTP, FASTA and PSI-BLAST programs (Pearson, 1990; Altschul et al., 1997). HA132 was analysed by using software of the ExPASy server (Appel et al., 1994) to predict domains and motifs (Reinhardt & Hubbard, 1998). Multiple sequence alignments were performed with CLUSTAL_X (Thompson et al., 1997). Alignment editing was performed with GeneDoc software (Nicholas et al., 1997). The following motifs were searched as potential transcription start sites of Ha132: early promoter motifs TATAA, ATCA(G/T)T and CGTGC (Blissard & Rorhmann, 1990), baculovirus late transcription start site DTAAG (Blissard & Rorhmann, 1989) and downstream activating elements (A/T)CACNG (Friesen, 1997).

Insect cells and virus.
The Helicoverpa zea cell line HzAM1 (McIntosh & Ignoffo, 1983) was maintained at 28 °C in Grace's medium supplemented with 10 % fetal bovine serum. HearNPV strain G4, the genome of which has been sequenced entirely (GenBank accession no. AF271059[GenBank] ; Chen et al., 2001), was deemed as wt and propagated in HzAM1 cells.

RNA isolation and RT-PCR.
HzAM1 cells were infected by wt HearNPV at an m.o.i. of 5 and total RNA was isolated with TRIzol (Gibco-BRL) at 0, 4, 8, 16, 24, 48 and 72 h post-infection (p.i.). RT-PCR was performed with 1 µg total RNA as template per time point. First-strand cDNA synthesis was performed by using AMV (avian myeloblastosis virus) reverse transcriptase (Promega) and a 15mer oligo-dT primer (Takara) according to the manufacturer's instructions. The cDNA mixtures were amplified with PCR by using the 132down primer (5'-GGGAAGCTTTTACGACGGCAAATCCCTACG-3') (the HindIII site is underlined and the italic sequence is complementary to nt 127750127770 in the HearNPV G4 genome) and a primer internal to Ha132 (132in) containing an EcoRI site (underlined) (5'-GAATTCAAAATATGAGTCAGG-3') (the italic sequence corresponds to nt 127122127137 in the HearNPV G4 genome).

Western blot analysis.
Monolayers of HzAM1 cells were infected by wt or recombinant HearNPV at an m.o.i. of 5. Infected cells were harvested at 0, 8, 16, 24, 36, 48 and 72 h p.i. BV and ODV were purified according to IJkel et al. (2001). Samples of total cell proteins, BV and ODV were separated by SDS-PAGE and transferred onto a Hybond-N membrane (Amersham Biosciences) for Western blotting. The primary antibody was a polyclonal, HA132-specific antiserum generated from rabbits immunized with purified HA132glutathione S-transferase expressed in Escherichia coli. Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Gibco-BRL) was used as the secondary antibody. The signal was detected by using a BICP/NBT kit (Sino-America).

Deletion of Ha132 from HearNPV.
To construct an Ha132 deletion mutant, a transfer vector was constructed as follows: the upstream flanking sequence of Ha132 was amplified by PCR with the primers P1 containing a HindIII site (underlined), 5'-GGGAAGCTTTGTTGCGGGGTTACGAAGAGC-3' (the italic sequence corresponds to nt 125131125151 in the HearNPV G4 genome) and P2 with a PstI site (underlined), 5'-CCCCTGCAGCAATAGCAGCCAGATCAACAT-3' (the italic sequence is complementary to nt 126599126621 in the HearNPV G4 genome). The downstream flanking sequence of Ha132 was obtained by PCR with the primers P3 containing a KpnI site (underlined), 5'-GGGGGTACCTTCGTAGGGATTTGCCGTCGT-3' (the italic sequence corresponds to nt 127748127768 in the HearNPV G4 genome) and P4 containing an EcoRI site (underlined): 5'-GGGGAATTCAAACGAAACATTGGATTGAACTT-3' (the italic sequence is complementary to nt 129309129331 in the HearNPV G4 genome). The two PCR products were first cloned into pUC19 to generate p132LR. The lacZ gene was cloned into p132LR to generate p132LR-LacZ. Co-transfection of wt HearNPV and p132LR-LacZ in HzAM1 was performed as described by King & Possee (1992). Recombinant plaques were identified by blue colour and polyhedron formation. The deletion mutant HearNPVΔ132 was purified by three rounds of plaque purification and identified by restriction-enzyme analysis.

In vivo infectivity assay.
The infectivity of BV was examined by injecting 10 µl supernatant with a titre of 10⁵ TCID₅₀ ml¹ into the third-instar larvae of H. armigera. Grace's medium was used as a negative control. For the oral-infectivity assay, polyhedra of wt HearNPV and HearNPVΔ132 were purified from diseased larvae as described by Sun et al. (1998). The infectivity was assayed in neonate and third-instar H. armigera larvae by diet contamination, using 10⁶ occlusion bodies (OBs) per larva. Infected larvae were reared individually in 24-well plates and monitored daily until all larvae had either pupated or died.

Sequence analysis of HearNPV ORF132 and its homologues
The Ha132 gene is located in the HindIIID fragment of the HearNPV genome. A late baculoviral transcript motif TTAAG was found 13 nt upstream of the putative translational start site, suggesting that Ha132 may be transcribed late in infection. A polyadenylation signal, AATAAA, was identified at nt 249254 downstream of the TAA stop codon.

Appropriate searches of protein databases showed that the putative HA132 protein and homologues were highly conserved among all baculoviruses whose genomes have been sequenced so far. Alignment of HA132 homologues from SeMNPV (a group II MNPV), AcMNPV (a group I MNPV), Xestia c-nigrum granulovirus (XcGV), Neodiprion sertifer nucleopolyhedrovirus (NeseNPV; a hymenopteran NPV) and Culex nigripalpus nucleopolyhedrovirus (CuniNPV; a dipteran NPV) revealed that the predicted amino acid sequence of HA132 shared identity ranging from 66 % with SeMNPV ORF35 to 44 % with NeseNPV ORF55 (Fig. 1). As expected, HA132 is related most closely to ORF97 of HzSNPV with 100 % identity, as HzSNPV is a substrain of HearNPV (Chen et al., 2002). The similarity was distributed throughout the sequence and there were several regions where the sequences were more highly conserved. One striking phenomenon was that all 14 cysteine residues of HA132 were completely conserved among this group of proteins (Fig. 1). This indicates that the protein can form multiple disulfide bonds. The N terminus of HA132 was found to be highly hydrophobic, with 13 of the first 19 aa consisting of Leu, Ile and Val. This hydrophobic N-terminal region was conserved in all of the HA132 homologues (Fig. 1). This region was also predicted as a transmembrane domain by TMPRED (Hofmann et al., 1993).

(114K): Fig. 1. Alignment of HearNPV ORF132 and its homologues. The 14 absolutely conserved cysteines are marked with asterisks. GenBank accession numbers are: AAG53875 for HearNPV ORF132 (Ha132), AAF33565 for Se35, AAA66652 for ORF22 of AcMNPV (Ac22), AAF05159.1 for ORF45 of XcGV (Xc45), AAQ96432 for ORF55 of NeseNPV (Nese55), AAK94116 for ORF38 of CuniNPV (Cuni38). GeneDoc software was used for similarity shading.

Transcription analysis of the Ha132 gene
RT-PCR was performed to study the transcription of Ha132. One primer internal to Ha132 (132in) and primer 132down were used to amplify a fragment of 650 bp. Consistently, an Ha132-specific sequence was amplified at 24 h p.i. and increased at 48 and 72 h p.i. (Fig. 2). The data demonstrate that Ha132 is a late gene, as the replication of viral DNA in HzAM1 cells started at 7 h p.i. (W. T. Dai, personal communication). Sequence analysis of the upstream and downstream regions indicated that there was a baculovirus late promoter motif TTAAG 13 nt upstream of the start codon ATG. As it has been shown previously by primer extension that the p6.9 gene of HearNPV used ATAAG as its late promoter (Wang et al., 2001), this TAAG motif upstream of Ha132 is likely to be functional.

(36K): Fig. 2. Transcription analysis of the Ha132 gene. RT-PCR analysiswas performed on total RNA extracted from HearNPV-infected HzAM1 cells at different time points p.i. by using primers 132down and 132in.

Immunodetection of HA132 in the infected cells and in BV and ODV
Western blot analysis of extracts of HearNPV-infected HzAM1 cells revealed a specific protein band of 43 kDa from 36 to 72 h p.i. (Fig. 3a). The size of the 43 kDa protein is close to the predicted 44.5 kDa size of the putative Ha132 translational product, suggesting no major post-translational modifications.

(30K): Fig. 3. Western blot analysis of HA132. (a) Western blot analysis of HA132 in HearNPV-infected HzAM1 cells from 0 to 96 h p.i. Lane numbers correspond to time p.i. (b) Western blot analysis of HA132 in the purified BV and ODV. Size standards are indicated on the left; the single 43 kDa immunoreactive protein is indicated on the right.

To investigate whether HA132 is a structural component of HearNPV, Western blot analysis of BV and ODV was conducted. Results showed that HA132 was detected in preparations of ODV, but not in BV (Fig. 3b), suggesting that HA132 is a structural component of ODV.

Construction of HearNPVΔ132
HearNPVΔ132, an Ha132 deletion mutant, was constructed as described in Methods. HindIII restriction-digestion profiles of HearNPVΔ132 were compared with those of wt HearNPV (Fig. 4a). The data showed that the HindIIID fragment (12.9 kb) from the wt HearNPV genome had disappeared, with a concomitant appearance of two fragments of 8.1 and 7.2 kb in the HearNPVΔ132 profile. The digestion proved that the correct deletion mutant was produced.

(21K): Fig. 4. Analysis of Ha132 deletion recombinant HearNPVΔ132. (a) Restriction-enzyme digestion analysis of wt HearNPV (1) and HearNPVΔ132 (2) by HindIII. The different bands are indicated by arrows and their sizes are shown. (b) Western blot analysis of total cell proteins from HearNPVΔ132-infected cells using HA132 polyclonal antisera. The numbers above lanes represent different times post-infection (h p.i.). wt HearNPV-infected cells at 72 h p.i. were used as a positive control for Western blot.

Western blot analysis was performed to confirm further that total expression of Ha132 was eliminated in HzAM1 cells infected with HearNPVΔ132 (Fig. 4b). HA132 was detectable in wt HearNPV-infected cells at 72 h p.i., but was undetectable in HearNPVΔ132-infected cells at different times post-infection. This result confirmed that Ha132 was deleted in HearNPVΔ132.

HearNPVΔ132 lost its oral infectivity, but retained its BV infectivity
To study the infectivity of BV and ODV of HearNPVΔ132, three bioassay experiments were carried out (summarized in Table 1). Firstly, BVs of wt HearNPV and HearNPVΔ132 were injected into the haemolymph of third-instar H. armigera larvae. Mortalities due to HearNPVΔ132 and wt viruses were about 92 and 94 %, respectively (Table 1), whereas larvae injected with Grace's medium (negative control) survived (data not shown). OBs of wt HearNPV and HearNPVΔ132 were fed to neonate or third-instar H. armigera larvae by diet contamination. The results showed that HearNPVΔ132 was not infectious to H. armigera larvae by oral ingestion (Table 1). Therefore, the deletion of Ha132 resulted in the complete lost of per os infectivity, but did not affect BV infectivity.

Table 1. Comparison of infectivity of wt HearNPV and HearNPVΔ132

In this paper, we reported the sequence analysis, transcription, protein identification and function of Ha132, a homologue of per os infection factor 2, in HearNPV.

The HA132 antiserum identified a 43 kDa polypeptide from HearNPV-infected cell lysates (Fig. 3a). HA132 was first detected at 36 h and continued to be present at 96 h post-infection. This type of temporal expression is consistent with the finding that HA132 is an ODV structural protein. The N terminus of HA132 contains a hydrophobic region that is conserved in its homologues. It has been reported that some ODV proteins contain an N-terminal hydrophobic structure, such as OpMNPV P91 (Russell & Rohrmann, 1997), AcMNPV ODV-E66 and ODV-E25 (Hong et al., 1997). PIF-1 and SE35 also contain a similar structure (Kikhno et al., 2002; Pijlman et al., 2003). The hydrophobic domain of PIF-1, PIF-2, ODV-E66 and ODV-E25 is also rich in isoleucine, leucine and valine (Figs 1, 5; Hong et al., 1997; Kikhno et al., 2002; Pijlman et al., 2003). It has been reported that the N-terminal hydrophobic domains of ODV-E66 and ODV-E25 are uncleaved in the ODV envelope and they were sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the ODV envelope within baculovirus-infected cells (Hong et al., 1997). This sequence, called the sorting motif (SM), contains two features, a hydrophobic sequence and associated charged amino acids oriented on the cytoplasmic/nucleoplasmic face. It is proposed that, once inserted into the endoplasmic reticulum (ER), the SM protein interacts with the viral proteins FP25K and/or E26 during trafficking to the nuclear envelope (Braunagel et al., 1999, 2004; Rosas-Acosta et al., 2001). From the comparison (Fig. 5) of N-terminal sequences of PIF-1, PIF-2, PIF-3, ODV-E66 and ODV-E25, all of the PIFs possess the same characteristics of the SM as ODV-E25 and ODV-E66, suggesting that these PIFs are transported to the inner nuclear membrane and intranuclear vesicles by the same pathway as ODV-E25/ODV-E66. Whether the N-terminal hydrophobic domains of PIFs function in a manner similar to those of ODV-E66 and ODV-E25 needs to be investigated further.

(38K): Fig. 5. Comparison of N-terminal sequences of PIF-1, PIF-2, PIF-3, ODV-E25 and ODV-E66. GenBank accession numbers of these amino acid sequences are: AAA66724 for ODV-E25, AAA66676 for ODV-E66, AAA66749 for PIF-1 Ac119, AAM93424.1 for PIF-1 Spli7, AAA66652 for PIF-2 Ac22, AAF33565 for PIF-2 Se35, AAG53875 for PIF-2 Ha132 and AAA66745 for PIF-3 Ac115. Shaded amino acids indicate the strong hydrophobic domain, which is rich in valines (V), leucines(L) and isoleucines (I). Asterisks indicate charged amino acids.

So far, four conserved genes, p74, pif-1, pif-2 and pif-3, have been identified to be essential factors for per os infectivity of baculoviruses. P74 has been described previously in AcMNPV as a structural ODV protein required for infectivity of polyhedra (Kuzio et al., 1989; Faulkner et al., 1997). Deletion or disruption of the AcMNPV p74 gene results in the complete elimination of the per os infectivity of OBs, while virions purified from mutant OBs were infectious when injected into the haemocoel of Trichoplusia ni and Heliothis virescens larvae (Faulkner et al., 1997; Haas-Stapleton et al., 2004). Competing assays indicated that P74 might function as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut (Haas-Stapleton et al., 2004). Complementation assays revealed that the defect in oral infectivity of P74-null AcMNPV could be rescued by mixed infection of p74-null virus with wt AcMNPV (Zhou et al., 2005) or purified P74 protein (Yao et al., 2004). PIF-1 was first identified in Spodoptera littoralis nucleopolyhedrovirus (Kikhno et al., 2002). The virus with deletion of pif-1 (Spli7) was shown to be unable to infect S. littoralis larvae per os and the product of pif-1 is an ODV-specific structural protein (Kikhno et al., 2002). PIF-2 was first identified in SeMNPV, where deletion of pif-2 (Se35) resulted in the complete loss of per os infectivity to the host (Pijlman et al., 2003). It was speculated that pif-2 from SeMNPV would encode a structural protein of ODV (Pijlman et al., 2003). Recent research by Ohkawa et al. (2005) revealed that PIF-1 (Ac119), PIF-2 (Ac022) and PIF-3 (Ac115) are essential for oral infection of AcMNPV. The competing assay implicated that PIF-1 and PIF-2 might function as attachment proteins for ODV binding to primary target cells in the midgut, while PIF-3 mediates another unidentified, but critical, early event during primary infection (Ohkawa et al., 2005). In this study, we have ascertained that the homologue of pif-2 in HearNPV, Ha132, encodes a structural component associated with ODVs and that the deletion of Ha132 resulted in the complete elimination of per os infectivity of OBs. Therefore, like MNPVs, PIF-2 is also an essential factor for oral infection in SNPVs. It was speculated that P74, PIF-1 and PIF-2 might interact with each other (Kikhno et al., 2002; Pijlman et al., 2003) and with microvillar binding partners (Ohkawa et al., 2005). Further experiments would allow the determination of possible interactions of these ODV-specific structural proteins and how these proteins enable ODV to set up a successful infection in vivo. The work is supported by the 973 project (2003CB114202), an NSFC grant (30025003) and the 863 project (2003AA214050) from China and by Program Strategic Alliances projects from China and the Netherlands (2004CB720404 and 04-PSA-BD-02). We would like to thank Dr Basil M. Arif for scientific editing of the manuscript.

Footnotes

^,‡, Yingchao Nie^{1 ,‡}, Qian Wang¹ †These authors contributed equally to this work. ‡Present address: Department of Plant Science, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.