Introduction

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a member of the Baculoviridae, which are large, enveloped, double-stranded DNA viruses with circular covalently closed genomes ranging in size from 80 to 180 kbp [1]. Baculovirus life cycle typically involves the production of two virus forms, budded virus (BV) and occlusion-derived virus (ODV). Mature BVs exit nucleus and bud through the plasma membrane of infected cells, which are highly infectious for tissues of the hemocoel and for cultured cells. ODVs are occluded in polyhedra or occlusion bodies and are required for the oral infection. AcMNPV is the most intensively studied member of baculovirus, with a genome of 134 kbp potentially encoding 154 genes [2]. To date, the functions of about 50% genes in AcMNPV genome have been determined and according to their functions, the genes could be divided into genes essential for viral DNA replication, such as lef-1, lef-2, lef-3, p143, dnapol, and auxiliary genes that can regulate the transcription of viral genes in vivo.

Ac68 is a member of the 29 baculovirus core set genes [3], locating at 58,720–59,298 nt in AcMNPV genome and encoding a putative protein of 192 amino acids with a predicted molecular mass of 22.3 kD. The amino acid identity between Ac68 protein and the homolog in Rachiplusia ou NPV is 95%; between Ac68 protein and the homolog in Bombyx mori NPV is 65%; in Epiphyas postvittana NPV is 44%; in Hyphantria cunea NPV is 44%; in Choristoneura fumiferana NPV is 43%; in Choristoneura fumiferana defective NPV is 42%. Sequence-based queries performed with interProScan program showed that ac68 was a protein of unknown function.

The putative promoter region of ac68, as that of other early genes, contains a consensus early promoter motif ACATT, −15 nt upstream of ATG which might initiate the transcription during the early phase of viral replication. In addition, ac68 gene also contains conserved sequences of TCATT and GATA, which are typical of most early genes, though locating at different sites, at +5 nt and +13 nt downstream of ATG, respectively [4]. Lulin Li et al. [5] reported that in a transient-expression assay system, ac68 did not appear to affect late gene expression.

In this study, an ac68 gene knockout bacmid and a repair bacmid were generated to examine the role of this gene. The mutant was constructed from AcMNPV bacmid by homologous recombination in Escherichia coli (E. coli). Green fluorescent protein (GFP) and polyhedrin were introduced into the mutant as markers. We found that the ac68 deletion mutant had no striking phenotype in Sf-9 cells, including BV production, occlusion body formation, and viral morphogenesis.

Materials and methods

Virus, cells, and insects

Spodoptera frugiperda (Sf) 9 cells were grown at 27°C in Grace’s medium supplemented with 10% fetal calf serum, penicillin, (50 U/ml) and streptomycin (50 μg/ml). AcMNPV strain accompanying the Bac-to-Bac® system from Invitrogen was used in this study. The wild-type AcMNPV bacmid (vAcwt-GP) containing GFP and polyhedrin genes was constructed via introducing polyhedrin and GFP genes into the polyhedrin locus of AcMNPV genome through transposition in E. coli. Larvae of T. ni were reared on an artificial diet at 28°C. AcMNPV BV stocks were prepared by infecting third instar T. ni larvae with polyhedra and extracting hemolymph from infected insects 3 days p.i. as previously described [6].

Expression of Ac68 in E. coli and preparation of Ac68-specfic antibody

Two primers 68P1: 5′-ATGGATCCATGGTATTCATTATCGAT-3′ (BamHI site was underlined) and 68P2: 5′-GCAAGCTTTTTAATTTTTGCTGCAA-3′ (HindIII site was underlined) were designed to amplify the ORF of ac68 from AcMNPV genomic DNA. The PCR product was cloned first into pMD18-T (TaKaRa) and then into the expression vector pET28a (Promega), which generated plasmid pET28a-ac68 with 6× His-tag sequence at the N terminus. E. coli DE3 cells containing pET28a-ac68 were grown to an optical density at about 0.6 of OD600 and induced by addition of 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After incubation for 12 h at 37°C, cells were harvested by centrifugation at 7,000g for 15 min at 4°C. The fusion protein present in the pellet was separated in 15% SDS-polyacrylamide gels and stained with Coomassie brilliant blue. The induced ac68 band was excised directly and the antiserum was raised in rats according to the method of Sambrook et al. using Freund’s adjuvant [7].

Total RNA isolation, reverse transcriptase (RT)-PCR

Total RNA was isolated from 1.6 × 106 mock-infected and AcMNPV-infected Sf-9 cells (multiplicity of infection (m.o.i) of 5 plaque-formins unit (PFU) per cell) at 0, 0.5, 1, 3, 6, 12, 24, 48, 72, and 96 h p.i., respectively, using the TRIZOL® Reagent from Invitrogen.

After the RNA samples were treated with RNase-Free DNase, RT-PCR was performed using RNA-PCR-M-MLV Kit (Promega) with 3 μg RNA as the template per time point. First-strand cDNA synthesis was performed using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase and the oligo(dT) according to the manufacturer’s instructions. The cDNA product was amplified by the gene-specific primers 68P1 and 68P2. AcMNPV ie-1 gene and vp39 gene were used as the control for early gene and late gene, respectively. The primer pair: ie-1U: 5′-ATGACGC- AAATTAATTTTAACGC-3′ and ie-1D: 5′-ACAATTTAGTTTTTGTTCCG-3′ were designed for ie-1; the primer pair: vp39U: 5′-GCGCTAGTGCCCGTGGGTAT-3′ and vp39D: 5′-TTAGACGGCTATTCCTCCACCTGC-3′ were designed for vp39. The obtained PCR products were analyzed in 1.0% agarose gel. Control experiments in which no reverse transcriptase was added prior to the PCR step were performed to detect any possible viral DNA contamination. ac68 RT-PCR products were gel purified and cloned into pMD18-T (TaKaRa) and sequenced with M13F(−47) or M13R(−48) primers.

In vitro expression of Ac68

For expression phase analysis of Ac68 protein, monolayer of Sf-9 cells were infected with mock or AcMNPV (m.o.i. of 5). Cells were harvested at 0, 6, 12, 24, 36, 48, 72, and 96 h p.i., respectively. To confirm that no Ac68 protein was expressed in ac68-KO virus infected cells, monolayer of Sf-9 cells were infected with ac68-wt or ac68-KO virus (m.o.i. of 5). Cells were harvested at 48 and 72 h p.i., respectively. Then the cells were pelleted and resuspended in phosphate-buffered saline (PBS, pH 7.4), lysed in SDS-polyacrylamide loading buffer and analyzed by Western blot. Ac68 antibodies and pre-serum were used at a dilution of 1:1,000. Immunoreactive proteins were visualized using goat anti-rat IgG and Horseradish Peroxidase.

Deletion of AcMNPV ac68

An ac68 knockout AcMNPV bacmid was generated by ET recombination system according to the manufacturer’s instructions. We first generated a transfer vector in which the ac68 locus region was replaced with Zeocin for antibiotic selection in E. coli. A 553-bp 5′ flank of ac68 was amplified by PCR from the AcMNPV bacmid using the primers 68US1: 5′-CGAAGCTTTTCTTCCATGTCTTTGAAAGATTGC-3′ (HindIII site was underlined) and 68US2: 5′-CGGAATTCATTAACATTGACCGTTTGATCGT-3′ (EcoRI site was underlined). The PCR product was digested with HindIII and EcoRI and then ligated into vector pBluescriptII-Zeocin (Li et al., unpublished) to generate the recombinant plasmid named pBluescriptII-US-Zeocin. With primers 68DS1: 5′-TGGGGATCCATGTTGCAGCAAAAATTAAAT-3′ (BamHI site was underlined) and 68DS2: 5′-AAGAGCTCGGCAAATTAAAATTAGCTG CGTC-3′ (SacI site was underlined), a 528-bp 3′ flank of ac68 was amplified by PCR from the AcMNPV bacmid. The PCR product was digested with BamHI and SacI and cloned into plasmid pBluescriptII-US-Zeocin that was digested with BamHI/SacI to generate a final ac68 knockout transfer vector named pBluescriptII-US-Zeocin-DS. This transfer vector was digested with HindIII and SacI, and the resulting linear 1.8 kbp fragment containing Zeocin gene cassette and ac68 flanking region was gel purified, resuspended in distilled water to a final concentration of 200 ng/μl.

An ac68 knockout AcMNPV bacmid was generated by ET recombination system according to the manufacturer’s instructions. DH10B cells which contain AcMNPV bacmid bMON14272 were transformed with plasmid pBAD-gbaA. Plasmid pBAD-gbaA can provide λ Red recombination function when it is induced by l-arabinose. Then DH10B electro-competent cells harboring plasmid pBAD-gbaA were made and electroporated with the 1.8 kbp fragments. The electroporated cells were incubated at 37°C for 1 h in 1 ml SOC medium, then 100 μl was spread onto low salt agar medium containing 25 μg/ml Zeocin, 100 μg/ml Amp, and 50 μg/ml Kanamycin. Plates were incubated at 37°C overnight, and colonies resistant to Zeocin and Kanamycin were selected, and target colony with ac68 knockout bacmid was confirmed by PCR.

Deletion of ac68 from its locus in AcMNPV and correct insertion of Zeocin at the ac68 locus were confirmed by PCR. The relative positions of the primer pairs are shown in Fig. 3b. Primer pair 68US1 and 68DS2 will amplify a 1,200 bp wild type fragment and an 1,873 bp knockout fragment. Primer pair Zeo-U and Zeo-D were used to detect the correct insertion of the Zeocin gene cassette. Primer pair 68P1 and 68P2 was used to confirm the deletion of the ac68 gene. Primer pairs 68US1/Zeo-D, Zeo-U/68DS2, and 68P1/Zeo-D were used to examine the junction between the upstream or downstream flanking region and Zeocin.

Construction of knockout, repair, and wt AcMNPV bacmids containing polyhedrin and GFP

To construct repair bacmid, a 892-bp fragment containing ac68 gene with native promoter and polyadenylation signal was amplified by PCR using primers 68REP-U: 5′-CGTCTAGAAACAACTTTGTACAAACC-3′ (XbaI site was underlined) and 68REP-D: 5′-CGCTCGAGTTATTTAATTTTTGCT-3′ (XhoI site was underlined). The PCR product was digested with XbaI/XhoI and ligated with pFB-ieGP [8], which was also digested with the same enzymes, to generate pFB-ieGP-ac68. To prepare bacmid for transposition, the helper plasmid pMON7124 conferring resistance to tetracycline and encoding a transposase was transformed into DH10B harboring the ac68 knockout bacmid. Then these cells were transformed with donor plasmid pFB-ieGP or pFB-ieGP-ac68 to generate ac68 bacmid vAcac68KO-GP or ac68 repair bacmid vAcac68REP-GP. In addition, electro-competent DH10B cells containing pMON7124 helper plasmid and bacmid bMON14272 were transformed with pFB-ieGP to generate a control virus named vAcwt-GP. The transformed cells were incubated at 37°C for 4 h in 1 ml SOC medium with moderate shaking, spread onto LB agar medium containing 25 μg/ml Zeocin, 50 μg/ml Kanamycin, 7 μg/ml Gentamicin, 10 μg/ml Tetracycline, 100 μg/ml X-Gal, and 40 μg/ml IPTG plates, and then incubated at 37°C for 48 h. The typical white colonies resistant to Zeocin, Kanamycin, Gentamicin, and Tetracycline were selected and confirmed by PCR analysis.

Transfections and fluorescence microscopy

Sf-9 cells (106 cells/well) were seeded into six-well culture plates and were incubated at 27°C for 16–24 h before transfection. DNA of three bacmid constructs was isolated from each resulting E. coli which had discarded the helper plasmid pMON7124 by resistance crossing. Equimolar amounts of bacmid DNA were transfected into Sf-9 cells using Cellfectin Reagent (Invitrogen Life Technology). Bacmid DNA was mixed with 6 μl cellfectin in 200 μl non-serum Grace’s medium and incubated at 27°C for 45 min, then the mixed DNA-cellfectin solution was added to 800 μl non-serum Grace’s medium and overlaid onto the seeded Sf-9 cells. After incubation for 4 h, the supernatant was removed and the cells were washed twice with non-serum Grace’s medium. Finally the cells were incubated at 27°C with 2 ml Grace’s medium containing 10% fetal bovine serum. GFP expression and polyhedra formation were examined through fluorescence microscopy to study the role of vAcac68KO-GP, vAcac68REP-GP, and vAcwt-GP on Sf-9 cells.

Analysis of viral growth curve

To assess whether ac68 is required for virus production and determine the replication kinetics of the virus constructed, a virus growth curve analysis was performed as described previously [9]. For this experiment, Sf-9 cells were transfected in triplicate with each bacmid DNA (~2 μg/well), and the culture supernatants were collected from various time points. BV titers were determined using a TCID50 end-point dilution assay described previously [8]. Virus infection was determined by monitoring GFP expression with fluorescence microscopy. A well of the culture plate was identified as positive if there were one or more cells expressing GFP.

Electron microscopy

For electron microscopy, Sf-9 cells (5 × 106 per 100-mm-diameter tissue culture dish) were infected with BV (vAcac68KO-GP or vAcwt-GP) at a MOI of 5. At 24, 48, and 72 h p.i., cells were harvested, washed once with Grace’s medium lacking fetal serum, and centrifuged at 3,000g for 10 min. Then cells were fixed, dehydrated, embedded, sectioned, and stained as described previously [8]. Samples were observed under a JEM-100CXII transmission electron- microscope at an accelerating voltage of 80 KV.

Bioassays

In order to determine whether ac68 deletion has any effect on the infectivity, bioassays were performed as described previously [10]. BVs diluted in Grace’s medium at the concentration of 2 × 106 TCID50/ml were injected into the hemocoel of fourth-instar T. ni larvae. PIBs were harvested from dead larvae, purified by centrifugation, and resuspended in double distilled water as described previously [6]. To determine the LD50, 10 μl samples containing 0 (control), 2 × 102, 1 × 103, 2 × 103, 1 × 104, and 2 × 104 PIBs, respectively, were applied onto a small piece of artificial diet. Newly molted third-instar T. ni larvae were reared at 27°C individually in 12-well plates and were infected by allowing them to feed on a diet contaminated with above concentrations of ac68 wt or ac68 deleted PIBs. Fresh diet was added into the wells when all the virus-contaminated diet was consumed. Control experiment was made with uncontaminated diet and each experiment was made three times repeatedly. Forty five larvae per dosage were used in the experiment. Mortality was recorded at 12 h intervals until larvae died or pupated. The data were analyzed by probit analysis. The LT50 were determined in T. ni larvae by droplet feeding assays as previously described [11]. Newly molted third-instar T. ni larvae were starved for 6 h, then were fed a solution containing 1% sucrose (w/v), 0.01% blue food coloring (FD and C Blue NO.1), as well as PIBs (1 × 108 PIBs/ml). Orally infected larvae were maintained individually in the dark at 27°C and mortality was recorded every 8 h until larvae died or pupated. Thirty six larvae were used per treatment group and the experiments were made three times repeatedly. The data were analyzed by Kaplan-Meier estimator in the bioassays.

Results

RT-PCR analysis of ac68 transcripts

To determine the temporal expression of ac68 transcripts at early, late, and very late phases of virus infection, RT-PCR was performed using total RNA isolated from AcMNPV-infected Sf-9 cells at different time points as template. We took advantage of AcMNPV ie-1 gene and vp39 gene as the controls for the early gene and late gene, respectively. As expected, a 1722 bp ie-1 transcript was amplified by ie-1 specific primers from 0.5 h p.i. to 72 h p.i. and a 1,041 bp vp39 transcript was detectable from 12 h p.i. to 96 h p.i. when amplifying by vp39 specific primers (Fig. 1). Using the gene-specific primers 68P1 and 68P2, a single band with an expected size of 592 bp was amplified at 3 h p.i., which remained detectable up to 96 h p.i. (Fig. 1), indicating that ac68 is an early gene according to the current view on early versus late transcription [12], in agreement with the sequence analysis of ac68, in which an early baculovirus transcription initiation motif ACATT was found at −15 nt upstream of ATG. The RT-PCR products obtained were cloned into pMD18-T and sequenced. The obtained sequence matched, as expected, the ac68 sequence. No RT-PCR product was detected in mock-infected Sf-9 cells.

Fig. 1
figure 1

RT-PCR analysis of ac68 transcription. Total RNA was extracted from AcMNPV-infected Sf-9 cells at different times post infection (p.i.). PCR products of different genes are indicated on the left. Times p.i. are indicated above the lanes. The sizes (in bp) of different PCR products are indicated on the right. IE1 and vp31 are positive controls for early and late genes

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Immunodetection of Ac68 in AcMNPV-infected cells

Antibody was prepared by immunization of rats with purified Ac68 produced in E. coli. Western blot analysis of extracts from AcMNPV-infected cells revealed a specific 25 kDa band when using the Ac68 antiserum (Fig. 2), a little larger than the predicted size of Ac68, which was 22.3 kDa, implying possible posttranslational modification of the protein.

Fig. 2
figure 2

Time course of Ac68 expression in AcMNPV-infected Sf-9 cells. Times p.i. are indicated above the lanes. The pre-stained protein standards are indicated on the left and the corresponding band to Ac68 is indicated on the right

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Construction of ac68 knockout AcMNPV bacmid

To investigate the function of ac68 during the viral infection cycle, we generated a bacmid by ET recombination system according to the manufacturer’s instructions, in which a 120-bp fragment (nt 59,179–59,298) of the ac68 gene coding region was replaced by Zeocin gene (Fig. 3a). The region contains a strong hydrophobicity domain as predicted using the Bioedit 7.0 software.

Fig. 3
figure 3

Strategy for construction of ac68 knockout AcMNPV bacmid and confirmation by PCR analysis. (a) Schematic diagram showing the structure of ac68 locus in wild-type and ac68-deleted virus and the replacement of a 120 bp fragment by Zeocin resistance gene. (b) Positions of primer pairs used in the confirmation of the ac68 gene deletion and correct insertion of Zeocin resistance gene cassette. (c) PCR analysis of different primer pairs. Marker: DNA marker with sizes indicated. The virus templates are shown above each lane and the primer pairs used are shown below

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The deletion of ac68 was confirmed by PCR with several primer pairs (Fig. 3c). Primer pair 68US1 and 68DS2 produced an 1,873-bp product from the mutant genome, in comparison with a 1,200-bp product from the wild-type genome. Primer pair 68US1/Zeo-D produced no PCR product in vAcwt, but a fragment of 1,345-bp in vAcac68KO. Primers Zeo-U/Zeo-D produced no PCR product in vAcwt, but a fragment of 792-bp in vAcac68KO. Primers Zeo-U/68-DS2 produced no PCR product in vAcwt, but a fragment of 1,300-bp in vAcac68KO. Primers 68P1/68P2 produced a 592-bp fragment in vAcwt, but no PCR product in vAcac68KO. Primers 68P1/Zeo-D produced no PCR product, but a 1,259-bp fragment in vAcac68KO. These results demonstrated that Zeocin gene cassette had replaced ac68 gene in AcMNPV bacmid successfully and no intact ac68 gene cassette existed in the vAcac68KO genome.

Immunodetection of Ac68 in the extracts from ac68-wt or ac68-KO infected cells

To confirm that the truncated Ac68 is not expressed in the ac68-KO virus infected cells, antibodies against the Ac68 protein was used to perform western blot analysis. The result showed that a 25 kDa band was detected in the extracts from ac68-wt infected cells, while no similar band in the extracts from ac68-KO infected cells was detected, indicating that the truncation has successfully blocked ac68 gene expression (Fig. 4).

Fig. 4
figure 4

Western blot analysis of Ac68 in Sf-9 cells infected with Ac-wt or ac68-KO virus. Virus used for infection and times p.i. are indicated above the lanes. The pre-stained protein standards are indicated on the left

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Construction of knockout, repair, and wt AcMNPV bacmids containing polyhedrin and GFP (green fluorescent protein)

Transposition events were confirmed by PCR with primer set PUC/M13 just outside of the transposition locus, which produced PCR fragment with expected sizes from specific bacmid constructs (data not shown). In addition, GFP expression and occlusion body formation in bacmid DNA transfected Sf-9 cells as examined by fluorescence microscopy further confirmed the accomplishment of the transposition events.

Analysis of knockout, repair, and wt AcMNPV replication in infected Sf-9 cells

To further assess whether ac68 is required for virus production, the bacmids with the ac68 gene removed (vAcac68KO-GP), repaired (vAcac68REP-GP), or the wild-type control (vAcwt-GP) were transfected into Sf9 cells. Fluorescence could be detected at 24 h p.t., and at this time point all cultures exhibited isolated GFP expression. At 72 h p.t. or 96 h p.t., approximately 100% transfected cells were observed expressing GFP with no difference among the cells transfected with different bacmids (Fig. 5a). Supernatants from cultures transfected with vAcwt-GP, vAcac68KO-GP, or vAcac68REP-GP at 96 h p.t. were collected and the titers were determined by TCID50 (50% Tissue Culture Infective Dosage). The collected supernatants were then used to infect Sf-9 cells at a MOI of 5. Fluorescence was observed at 48 h p.i. in all cultures infected with the supernatants (data not shown).

Fig. 5
figure 5

(a) Fluorescence micrographs of Sf-9 cells transfected with bacmids vAcac68KO-GP, vAcwt-GP, or ac68 repair vAcac68REP-GP. Bacmids used for transfection were indicated above and the times after transfection were indicated on the left. (b) Micrographs of Sf-9 cells infected with vAcac68KO-GP, vAcwt-GP, or vAc ac68REP-GP virus, showing polyhedra in the infected cells. Viruses used for infection were indicated above and the time post infection was indicated on the left. (c) Viral growth curves. Sf-9 cells were transfected with bacmids vAcac68KO-GP, vAcwt-GP, or vAcac68REP-GP, and the supernatants were collected at the indicated time points and the titers were determined by TCID50 assay. Each sample was performed in triplicate

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Occlusion bodies appeared in all cultures infected with vAcwt-GP, vAc ac68KO-GP, or vAcac68REP-GP viruses at around 48 h p.i. And occlusion bodies were observed in almost every single cell infected with vAcwt-GP, vAcac68KO-GP, or vAcac68REP-GP viruses at 72 h p.i. (Fig. 5b). The above results indicated that ac68 deletion did not affect infectious budded virus generation and polyhedra formation.

To further assess the effect of ac68 deletion on virus replication and determine the replication kinetics of the virus constructs, the time-course of budded virus production after transfection with the three bacmids was studied. At 24, 48, 72, and 96 h p.t. time points, the supernatants were collected and the BV titers were determined by TCID50 end-point dilution assay on Sf-9 cells. The result revealed steady increase in virus production and almost the same slope of the growth curves for the three viruses (Fig. 5c).

Electron microscopic analysis of mock, wt, knockout virus-infected Sf-9 cells

To further analyze whether the deletion of ac68 has any effect on virus morphogenesis, we examined the cells infected with BV (vAcwt-GP or vAcac68KO-GP) by electron microscopy. As expected, the sections of vAcwt-GP infected Sf-9 cells showed typical cytological changes and viral morphogenesis of NPV infection in the field of vision, and no morphological difference was found between cells infected with wild type and ac68 knockout viruses. Typical virogenic stroma, the putative site of nucleocapsids assembly with an electron-dense lattice-like structure, could be found easily within the nucleus of the ac68 knockout or wild type virus-infected cells (data not shown), with a lot of nucleocapsids at the edges of the electron-dense virogenic stroma, which did not exist in mock-infected cells (data not shown). At 48 h p.i., many enveloped virions containing multiple nucleocapsids could be observed in the sections, which formed in the ring zone and were embedded in the polyhedra. The number, as well as the sizes and shapes, of polyhedra in the ac68 knockout virus-infected cells were indistinguishable to that in wild type virus-infected cells (data not shown). These observations suggested that the deletion of ac68 gene had no effect on the occlusion body morphogenesis.

Effect of ac68 deletion on AcMNPV infectivity for Trichoplusia ni larvae

The infectivities of vAcwt-GP and vAcac68KO-GP were determined for newly molted fourth T. ni larvae in 50% lethal dose (LD50) and 50% lethal time (LT50) bioassays. The results of dose-mortality (LD50) assessing showed that there was no significant difference between vAcwt-GP and vAcac68KO-GP viruses (Table 1), but it took 16 h longer for ac68 deleted virus to kill the T. ni larvae than did for ac68 wt or ac68 repair virus in LT50 bioassay (Table 2). There is significant difference (P < 0.01) between the LT50 of vAcac68KO-GP and that of vAcwt-GP or vAcac68REP-GP by the analysis of Tukey HSD.

Table 1 Dose-mortality of vAcwt-GP and vAcac68KO-GP for third-instar T. ni larvae
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Table 2 Time-mortality of vAcac68KO-GP, vAcwt-GP and vAcac68REP-GP for third-instar T. ni larvae
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Discussion

Ac68 is a highly conserved gene in lepidopteron NPVs, but sequence-based queries performed with InterProScan program did not show significant sequence similarities to non-baculovirus proteins with known functions.

Ac68 locates downstream of ac67 (lef-3) gene with 159 overlap nucleotides at 5′ terminus; lef-3 is a single stranded DNA-binding protein which is necessary for viral DNA replication [13]. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG condon [14], i.e. 439 nucleotides in the lef-3 transcription overlap with the ac68 gene. In order to keep the lef-3 gene intact and its function, we disrupted only the putative 3′ terminal portion of ac68 gene, which represents a hydrophobic domain as predicted with the Bioedit 7.0 soft. A 792-bp DNA fragment containing the complete Zeocin gene was inserted into the deleted locus, which interrupted the conformation of Ac68 protein, making it extremely unlikely that the fusion protein could maintain the activity of Ac68 protein.

In this report, the mutant virus was examined for phenotype. Successful propagation, infectious progeny virus generation, formation of occlusion bodies, and polyhedra morphogenesis, and the virus growth curve of the ac68 knockout virus indicated little or no effect of ac68 deletion on virus replication in tissue culture and in T. ni larvae.

The ac68 gene had originally been identified in transient-expression assays as unnecessary for late gene expression [5], which was confirmed by our results. In this study, ac68 mRNA was detected from 3 to 96 h p.i., and was most abundant from 24 to 72 h p.i., identifying ac68 as an early gene that is expressed in both early and late phases, though late conserved motif in its 5′ flanking region could not be found. To date, most early genes that have been discovered in AcMNPV genome are essential to the production of BV such as ac38, EXON0, lef-3 [15–17]. Some early genes such as pk2, lef-6, and ac18 have little effect on the production of BV, which are regarded as auxiliary genes [18–20]. Auxiliary genes are not essential for viral replication but play a potential role in modulating or regulating late transcription in the life cycle of AcMNPV. It is accepted generally that auxiliary genes of baculovirus can function at the cellular level, i.e. protein kinase 2 (pk2), or at the organismal level, i.e. cathepsin (cath) [20]. Lef-6 is an early gene which is not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription [19]. Ac18 is also an early gene and is not essential for the propagation of AcMNPV, but it affects the efficient infectivity in T. ni larvae [20]. In this study, ac68 was identified as an early gene that was not required for viral replication in cell culture or in T. ni larvae, though ac68 accelerated the mortality of infected insect hosts by approximately 16 h, which might provide a selective advantage for the virus.

Western blot analysis confirmed that no Ac68 protein was expressed in the ac68-KO virus infected cells. In ac68-wt virus infected cells, a 25 kDa band was detected from 36 to 96 h after infection.

The deduced amino acid sequence analysis showed six phosphorylation sites in the Ac68 protein with NetPhos software (http://www.expasy.ch), speculating Ac68 protein relevant to phosphorylation, which plays a regulatory role in the life cycle of AcMNPV. Further experiments will help to clarify the posttranslational modification and the specific function of Ac68 protein in the life cycle of AcMNPV.

In addition, ac68 gene is adjacent to orf69 with 23 overlap nucleotides at C terminus. The AcMNPV orf69 gene is a late gene and encodes a Methyltransferase (MTase1). Early transcripts of ac68 gene contains at least the N-terminal part of the orf69 open reading frame, which could potentially direct the synthesis of MTase1 during the early phase of infection [21]. Although the disruption site was in the C-terminal coding region of ac68, it was still within 5′-TIR of ac69. Therefore the disruption affects the expression of both ac68 and ac69. Fortunately Wu and Guarino [21] had reported that the disruption of ac69 had no effect on the production of BV.

Our results, together with transient-expression assays [5] and the reports by Wu and Guarino [21], demonstrated that Ac68 protein may be an accessory factor or a minor component in regulating or modulating the transcription of late gene during the infection cycle. Therefore, it will be of great interest to more precisely elucidate the molecular mechanism of action of ac68 gene in the life cycle of AcMNPV.