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1 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
2 Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 2E5, Canada
Correspondence
Peter J. Krell
pkrell{at}uoguelph.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Details of primers and oligonucleotides are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, since baculoviruses are generally slow-acting, synthetic chemical insecticides are often preferred. Baculoviruses can be enhanced by insertions of exogenous genes expressing, for example, insect-specific neurotoxins (e.g. AaIT; Maeda, 1989). However, release of such genetically modified viruses carrying foreign gene inserts into the environment might not be approved (Black et al. 1997; Inceoglu et al. 2006). Baculoviruses with enhanced lethality, but lacking such exogenous genes, should be more environmentally benign and acceptable, as suggested by Saville et al. (2004).
Most baculoviruses encode a chitinase (chiA) and a viral protease (v-cath) which are retained in cells and released upon virus-induced lysis to liquefy host carcasses at the end of the infection (Hawtin et al., 1997). Relative to infection with wild-type Autographa californica multiple nucleopolyhedrovirus (AcMNPV), insect survival times are shortened by 25 % with a mutant of AcMNPV overexpressing (polh promoter) a secreted chitinase (Gopalakrishnan et al., 1995; Saville et al., 2002). Consequently, the natural baculovirus host liquefaction process can be exploited to enhance virulence by mere misexpression of chitinase. However, the viruses generated by Gopalakrishnan et al. (1995) and Saville et al. (2002) were non-occluded. Occluded baculoviruses are more amenable to field use because of both their environmental stability and their obligate per os infectivity. Upon per os infection, an occlusion-positive AcMNPV mutant expressing a secreted form of its native chiA from its native locus and promoter was more lethal to insects (Saville et al., 2004) than a control AcMNPV, but less so than a polh promoter-driven chiA in an occlusion-negative virus (Saville et al., 2002). Therefore, earlier release (secretion) of chitinase enhances lethality, but overexpression of chitinase considerably increases the potential for reducing infected insect survival times. In another study, insects were killed much more quickly upon infection with recombinant AcMNPV expressing the Sarcophaga peregrina cathepsin L (ScathL) from the strong late p6.9 promoter than with a recombinant virus expressing the same gene from the weak immediate-early ie-1 promoter (Harrison & Bonning, 2001). Moreover, ScathL expression from the p6.9 promoter killed insects faster than viruses expressing either the AaIT or the LqhIT2 neurotoxin from the p6.9 promoter (Harrison & Bonning, 2000, 2001). Together, these findings indicate that reprogramming (for overexpression) of the native baculovirus chiA and/or v-cath expression may enhance the effectiveness of such baculoviruses against pest insects.
The genomic organization of chiA and v-cath is conserved in many baculoviruses. They are located on opposite genomic strands, with their respective promoters contained within a common intergenic region (summarized by Slack et al., 2004). Forty-five base pairs separate the AcMNPV chiA and v-cath translational start codons, and promoters for both chiA (Hawtin et al., 1995) and v-cath (Hill et al., 1995) have been mapped to this intergenic region.
Although the time-course of expression of chitinase as determined by enzyme activity, immunoblotting and immunolocalization has been reported (Thomas et al., 1998, 2000; Saville et al., 2002, 2004; Hawtin et al., 1995), there are no reports of temporal expression of RNA or a comparison of temporal expression of both RNA and enzyme synthesis simultaneously. While the 5' end of the chiA RNA has been mapped (Hawtin et al., 1995), the 3' end has not. Similarly, while the time-course of V-CATH expression has been reported by immunoblotting or enzyme activity (Hom & Volkman, 2000; Hom et al., 2002; Slack et al., 1995), little is known about the temporal expression of v-cath RNA. We report here on the temporal expression profiles of both AcMNPV chiA RNA and protein synthesis and mapping of both the chiA RNA 5' and 3' ends. We also followed the temporal expression of AcMNPV v-cath RNA, monitored the effects of altering the chiA promoter on cathepsin expression and mapped the 3' end of the v-cath RNA.
We also report the expression of AcMNPV chiA under the control of native and alternate promoters (polh, p6.9) in the native chiA locus, demonstrating that chiA expression could be modulated by altering promoters. The temporal transcription profile of AcMNPV v-cath and the negative effect on v-cath transcription resulting from insertion of alternate chiA promoters were also noted.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were cultured at 27 °C in Grace's insect medium (Invitrogen) supplemented with 10 % FBS (Invitrogen), penicillin (1 U ml–1) and streptomycin (1 µg ml–1). AcMNPV strain E2 was used to generate the recombinants described herein. Viral plaque isolation and titration by end-point dilution were carried out as described by O'Reilly et al. (1992). For analysis of virus growth kinetics and RNA/protein expression, cells were infected at an m.o.i. of 0.01 or 10, respectively. For all viral time-course experiments, t=0 is the time when virus inoculum was removed after 1 h of rocking at ambient temperature (O'Reilly et al., 1992).
Construction of promoter-exchange donor vectors.
A pBluescript-based transfer vector was constructed for introducing the selectable EGFP cassette, and later replacing that with alternate chiA promoters, into the AcMNPV chiA/v-cath intergenic region through homologous recombination (Fig. 1). Primer and oligonucleotide sequences are listed in Supplementary Table S1. The left genomic-flanking portion of the donor cassette, amplified from AcMNPV DNA using the chiAKpnI/chiAXhoI primer set, contained the full chiA ORF and partial downstream lef-7. First, the lef-7/chiA-containing PCR fragment was cloned into the multiple cloning site (MCS) of pBluescript by utilizing the PCR primer-introduced KpnI and XhoI restriction sites to create the plasmid pBSK.lef-7/chiA. The right genomic-flanking fragment, amplified from AcMNPV DNA by a PCR with the cathXbaI/cathSstI primer set, contained the entire v-cath ORF and a partial downstream gp64 sequence. The v-cath/gp64-containing amplicon was then cloned into the remaining MCS of pBSK.lef-7/chiA by utilizing the PCR primer-introduced XbaI and SstI restriction sites. This plasmid, containing the right and left chiA/v-cath intergenic promoter-flanking regions, was designated pBSK.chiA/v-cath. The EGFP selection cassette containing the AcMNPV polh promoter, EGFP ORF and SV40 polyadenylation signal was amplified from pBlueGFP (Cheng et al., 2001) by a PCR using the polh.egfpF/SV40.egfpR primer set. The SpeI site-containing polh.EGFP.SV40 PCR amplicon was digested with SpeI and then blunt/SpeI-ligated into the remaining MCS of pBSK.chiA/v-cath that had been digested with SmaI and SpeI, thus generating pBSK.chiA/EGFP/v-cath (designated AcEGFP in Fig. 1a). The AcMNPV p6.9 promoter (86886–87061) was amplified by PCR from AcMNPV DNA with the p6.9chiAF/p6.9chiAR primer set. The p6.9 promoter PCR fragment containing primer-introduced XhoI and XbaI sites was cloned by digestion (with XhoI and XbaI) and ligated into a likewise-digested pBSK.chiA/v-cath plasmid, thus generating pBSK.p6.9.chiA/v-cath, so that the p6.9 promoter would drive chiA transcription (designated Acp6.9.chiA in Fig. 1a). The sequence representing the AcMNPV polh promoter (4461–4520) was designed as two complementary oligomers (polh.chiAF and polh.chiAR) that, when annealed, had compatible overhangs for ligation into an XhoI/XbaI-digested pBSK.chiA/v-cath plasmid, thus generating pBSK.polh.chiA/v-cath, so that the polh promoter would drive chiA transcription (designated Acpolh.chiA in Fig. 1a). The late baculovirus promoter sequence (TAAG) or the complement sequence (ATTC) in polh.chiAF and polh.chiAR oligomers and the p6.9.chiAR primer were designed so as to reconstruct most of the native v-cath promoter in the chiA-reprogrammed viruses. The integrity of all cloned sequences was verified by sequencing.
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). To generate AcEGFP, cells were transfected with 1 µg each of pBSK.chiA/EGFP/v-cath and AcMNPV genomic DNA. EGFP+ viral plaques were screened by UV microscopy and purified through five plaque isolations. One plaque isolate of CHIA–/V-CATH– and EGFP+ virus with a verified genomic organization was used for all subsequent analyses and for the generation of the Acpolh.chiA and Acp6.9.chiA viruses (Fig. 1a
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Recombinant viruses were generated by transfection of cells with 1 µg each of AcEGFP genomic DNA and pBSK.polh.chiA/v-cath or pBSK.p6.9.chiA/v-cath. Viruses with rescued chitinase activity were enriched by end-point dilution and identified using a fluorigenic assay (McCreath & Gooday, 1992). EGFP– viruses were plaque-purified through five cycles. One CHIA+/EGFP– Acpolh.chiA and one CHIA+/EGFP– Acp6.9.chiA isolate, with genomic structures verified by Southern blot hybridization of restriction enzyme-digested viral DNA, were used for all subsequent analyses. Sequencing of PCR amplicons spanning the AcEGFP, Acpolh.chiA and Acp6.9.chiA chiA/v-cath intergenic regions amplified using the chiAR/cathR primer set (Supplementary Table S1) verified the authenticity of the genomic modifications.
RNA isolation, electrophoresis and blotting.
Total RNA was isolated from infections (m.o.i. of 10) at 0–48 h p.i. using the RNeasy-TRIzol method (Bowtell & Sambrook, 2003). RNA from mock-infected cells was isolated at t=0. High Range RNA molecular weight ladder (Fermentas) and 5 µg of each denatured RNA sample were electrophoresed in denaturing (2.2 M formaldehyde, 1x MOPS) 1.3 % agarose gels. Following electrophoresis, RNA was transferred to positively charged nylon membrane according to the instructions of the manufacturer (Schleicher and Schuell). To determine the chiA transcript sizes more accurately, 1 µg Acp6.9.chiA 24 h p.i. poly(A)+ RNA was electrophoresed alongside a DIG-labelled High Range RNA size marker (Roche) in denaturing (2.2 M formaldehyde, 1x MOPS) 1.0 % agarose gels, blotted and hybridized as described above.
Northern blot hybridization and analysis.
Gel-purified (Qiagen) chiA and v-cath PCR amplicons served as templates in PCRs incorporating only one (nested) primer for amplification of DIG-labelled ssDNA complementary to either chiA or v-cath RNA. The chiA template amplicon (106132–106940) was amplified with the chiAXhoI/chiART primer set. The v-cath template amplicon (106962–107598) was amplified with the cathXbaI/cathRT primer set. DIG-labelled antisense chiA ssDNA (470 nt; 106471–106940) or v-cath ssDNA (563 nt; 106962–107507) probes were generated from the chiAXhoI/chiART or cathXbaI/cathRT template amplicons with the chiAcDNA or cathcDNA primers according to the manufacturer's instructions (Roche). Hybridization solutions comprised 2 µl labelled probe added to 10 ml pre-warmed (42 °C) DIG Easy Hyb (Roche). RNA blots of individual viruses were pre-hybridized (30 min, 42 °C, 20 ml DIG Easy Hyb) and then hybridized with 10 ml hybridization solution (15 h, 42 °C). Probes were detected with the DIG nucleic acid detection kit and protocol (Roche) using 1 ml CSPD substrate (Roche) per blot. Chemiluminescence was detected by using X-ray film (Kodak X-Omat).
Densitometric analysis of chiA autoradiograms.
RNA densitometry and quantification of the 2.6 kb chiA bands was performed using a Bio-Rad GS-800 calibrated densitometer and Quantity One software (contour method). Relative intensities of each band were expressed as relative adjusted densities, and were plotted against time in h p.i. Values of 0 were assigned for lanes lacking detectable 2.6 kb chiA transcripts.
RACE and RT-PCR analysis.
RACE analysis was performed according to Sambrook & Russell (2001). One microgram of 24 h p.i. total RNA from AcMNPV-, Acpolh.chiA- and Acp6.9.chiA-infected cells was reverse-transcribed using the Superscript II enzyme and protocol (Invitrogen). Reverse transcription for chiA 5' RACE employed the chiART primer. The 5' cDNA, purified using the QIAquick PCR purification kit (Qiagen), was ethanol-precipitated, resuspended in 10 µl water and polyadenylated using terminal transferase (Roche). PCRs (50 µl) contained 16 pmol oligoT17 adaptor primer, 32 pmol adaptor primer, 32 pmol nested chiA-specific primer (chiART2), 2.6 U Taq-Pwo polymerase and 1/20 volume of the A-tailed cDNA. After 30 cycles, PCR amplicons were ligated into pGEM-T Easy vector (Promega). Five plasmid clones arising from each viral chiA 5' RACE preparation were sequenced using M13 primers.
For 3' RACE, 1 µg of 24 h p.i. AcMNPV, Acpolh.chiA or Acp6.9.chiA total RNA was reverse-transcribed (Superscript II; Invitrogen) using the oligoT17 adaptor primer. PCR amplification of chiA and v-cath 3' RACE products utilized 32 pmol nested oligodT adaptor primer (noligoT) and 32 pmol of either a positive-sense chiA-specific primer (chiA3RACE) or a positive-sense v-cath-specific primer (cath3RACE). Amplicons were cloned as for 5' RACE, and three plasmids arising from AcMNPV chiA and v-cath 3' RACE were sequenced using M13 primers. Acpolh.chiA and Acp6.9.chiA chiA 3' RACE was done in an identical manner, and a single clone from each virus was sequenced. To characterize chiA transcripts further, polyadenylated RNA extracted using a PolyAtract kit (Promega) from 18 h p.i. RNA was reverse-transcribed using the oligoT17 adaptor primer. The cDNA was used for PCR using different primer pairs (see Fig. 3b).
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). AcMNPV virus grown in wells for 1 week served as a positive control for chitinase activity.
Colorimetric chitinase assay.
Chitinase production was monitored during in vitro replication (0–48 h p.i.) for three independent experiments. Infected cells were dislodged from plates by triturating with a Pasteur pipette, pelleted (600 g, 5 min), washed (PBS, pH 6.2), resuspended in 100 µl lysis buffer (50 mM Na2HPO4, pH 6.0) and stored at –70 °C. Protein concentrations of lysate supernatants (12 000 g, 15 min, 4 °C) were determined using the Bio-Rad protein concentration kit. Fifty microlitre aliquots of total protein samples (1 µg µl–1) in 50 mM Na2HPO4 (pH 6.0) were added to pre-warmed reaction tubes containing 750 µl 0.5 mg CM-chitin-RBV ml–1 (Loewe) in 50 mM Na2HPO4, pH 7.0. Reactions were incubated with agitation at 37 °C for 2 h before addition of 300 µl 0.1 M HCl, brief vortexing, chilling on ice (10 min) and centrifugation (10 000 g, 5 min, 4 °C) to pellet undigested substrate. The A550 of room temperature supernatants was measured against reaction blanks with 50 µl buffer only. Amounts of enzyme activity (units enzyme per A550 unit) were expressed relative to Serratia marcescens chitinase (Sigma) and expressed as mU chitinase (mg lysate protein)–1.
Cysteine protease assay.
Mock- and virus-infected cells (48 h p.i.) were assessed for cysteine protease activity, in the presence and absence of a cysteine protease inhibitor (E-64), based on the assay described by Ohkawa et al. (1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was verified by PCR and sequencing. The chiA of our AcMNPV E2 virus had one nucleotide difference (C106346T; C592T chiA coding sequence) from the C6 strain (Ayres et al., 1994), which conferred a novel EcoRV site for E2 and a predicted amino acid shift (P198S). The commercial BEV (Bac-to-Bac; Invitrogen) chiA also had the novel EcoRV site.
Analysis of virus growth kinetics
Virus growth curves from two independent experiments were compared for AcMNPV and modified viruses. The kinetics of virus growth for all three viruses were similar through 72 h p.i. (Fig. 2). Differences between final titres of AcMNPV and the Acpolh.chiA and Acp6.9.chiA viruses were not significant (P=0.13 for Acpolh.chiA, P=0.12 for Acp6.9.chiA) (Fig. 2).
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) and at 24 h p.i. for Northern blots (Fig. 3a) and total RNA from 24 h p.i. for RACE analyses to determine the nature of the chiA transcript (Fig. 3c). Patterns of chiA transcription through 48 h p.i. of AcMNPV and reprogrammed (Acpolh.chiA, Acp6.9.chiA) viruses using a strand-specific chiA ssDNA probe were compared (Fig. 3a). No detectable hybridization to mock-infected cellular RNA was found. A major 2.6 kb RNA band, a minor 1.9 kb and several minor large (>5 kb) RNA bands were detected for all three viruses. Based on densitometric analysis, the AcMNPV and Acpolh.chiA chiA major 2.6 kb RNA was first detectable by 9 h p.i., whereas the Acp6.9.chiA chiA transcript was detectable earlier, by 6 h p.i. At 9 h p.i., there was much less Acpolh.chiA than AcMNPV chiA RNA detectable, but more than was detectable for Acp6.9.chiA at 6 h p.i. Peak transcription of the 2.6 kb RNA occurred at 12 h p.i. for AcMNPV, 18 h p.i. for Acp6.9.chiA and 24 h p.i. for Acpolh.chiA (Fig. 4a). Transcription from the polh or p6.9 promoters was about twice that from the native AcMNPV chiA promoter. Levels of the AcMNPV chiA 2.6 kb RNA declined beyond 15 h p.i., with only a small decline for the Acp6.9.chiA promoter and none for Acpolh.chiA. Although each lane was loaded with what was calculated to be 5 µg RNA, the greater intensity of the rRNA band for AcMNPV at 6, 9 and 12 h p.i. (Fig. 3a) suggested that a relatively larger amount was loaded in these lanes. Thus, the relative amounts of 2.6 kb chiA RNA reported for these times might be slightly overestimated. The levels of expression of the 1.9 kb and the >5.0 kb RNAs were higher for Acp6.9.chiA and Acpolh.chiA than for AcMNPV.
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RACE analysis of chiA
By 5' RACE, the chiA transcripts initiated at –13 for AcMNPV, –60 for Acpolh.chiA chiA and –43 for Acp6.9.chiA, all equivalent to the first A of the AcMNPV TAAG at –13 bp, relative to the chiA ORF ATG (Figs 1b and 3c). chiA transcripts of AcMNPV, Acpolh.chiA and Acp6.9.chiA all terminated at the same relative genomic site (104537), 745 nucleotides downstream of the chiA ORF and 15 nucleotides beyond the AcMNPV polyadenylation signal (AATAAA) at 104552–7.
RT-PCR mapping of chiA RNA
Based on 5' and 3' RACE and the immediate downstream chiA sequence, the smallest transcript size from each of the native, polh or p6.9 promoters would be approximately 2.4 kb (Fig. 3c). Using oligoT17-primed cDNA, expected amplicons of 2.3, 2.2 and 1.4 kb were readily detected with the primer pairs noligoT/R1, F2/R1 and F3/R1, respectively. Although 2.9, 2.6, 2.4 and 1.6 kb amplicons were found for primer pairs F1/R2, F1/R1, F2/R2 and F3/R2, their relative levels were much lower. No amplicons were detected using the noligoT/R2 primer set, starting upstream of the 5' end of the RNA as determined by 5' RACE (Fig. 3b), or in the RT-negative control.
Temporal accumulation of chitinase in infected cells
The rate and/or amount of enzyme accumulation were monitored during virus replication (Fig. 4b). Chitinase-specific activities [(mU mg total lysate protein)–1] were relative to digestion of CM-chitin-RBV by S. marcescens chitinase. Chitinase activity increased exponentially in AcMNPV virus-infected cells between 15 (2.5±1.3 mU mg–1) and 18 h p.i. (25±1.9 mU mg–1) and then tapered off up to 48 h p.i. (62±6.4 mU mg–1) (Fig. 4b). Relative to AcMNPV, there was a marked increase in chitinase-specific activity for both Acp6.9.chiA (160±7.8 mU mg–1) and Acpolh.chiA (219±5.5 mU mg–1) at 48 h p.i. Chitinase activity was first detectable at 15 h p.i. for AcMNPV (5.7±5.3 mU mg–1), but not for Acpolh.chiA, as expected for a polh promoter-driven chiA. Acp6.9.chiA chitinase activity (21.3±10.4 mU mg–1) was first detectable at 12 h p.i. Chitinase activity increased exponentially in Acp6.9.chiA-infected cells between 12 (21.3±10.4 mU mg–1) and 18 h p.i. (95±5.3 mU mg–1), and more linearly from 18 to 48 h p.i. (160±7.8 mU mg–1). Moreover, the Acp6.9.chiA chitinase activity (26±4.6 mU mg–1) was about 5-fold higher at 18 h p.i. than that of either AcMNPV or Acpolh.chiA. The Acpolh.chiA chitinase activity was not detected until 18 h p.i. (17.7±1.4 mU mg–1) and then increased exponentially up to 36 h p.i. (178±25.1 mU mg–1) and more slowly thereafter to 48 h p.i. (219±15.9 mU mg–1). AcMNPV and Acp6.9.chiA chitinase enzyme activity was first detectable about 6 h after the corresponding chiA-specific RNA was first detectable in Northern blots. There was, however, a 9 h time lag between the first detection of Acpolh.chiA chiA RNA (at 9 h p.i.) and chitinase enzyme activity (at 18 h p.i.).
v-cath transcription, mapping and cysteine protease activities
As the v-cath promoter is in the same intergenic region as the chiA promoter, changing the chiA promoter might have influenced v-cath expression. AcMNPV v-cath transcription (0–48 h p.i.) was assessed by Northern blot analysis with a strand-specific DIG-labelled ssDNA probe as described in Methods (Fig. 5). A v-cath-specific 1.5 kb RNA was detected from 9 to 48 h p.i. (Fig. 5). Transcription of this RNA peaked by 15–18 h p.i., as judged by the relative intensities of the 1.5 kb bands. v-cath-specific RNA could not be detected at any time in Northern blots for Acpolh.chiA or Acp6.9.chiA (data not shown).
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have already reported it. Significant E-64-inhibitable cysteine protease was found in AcMNPV-infected cells, but the mock-, Acpolh.chiA- or Acp6.9.chiA-infected cells contained very little detectable cysteine protease activity at 48 h p.i. (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Our Northern blot data showed a major chiA RNA migrating at about 2.6 kb for all three viruses. However, the temporal expression patterns for reprogrammed chiA were in accordance with that expected for polh or p6.9 promoters. AcMNPV chiA RNA was expressed from 9 to 48 h p.i. followed by enzyme activity from 15 to 48 h p.i. The profile of AcMNPV chiA RNA transcription was similar to that of enzyme accumulation 6 h later (Fig. 4a, b). This is consistent with the intracellular detection of CHIA from 10 h p.i. by Hawtin et al. (1995) and from 12 h p.i. by Thomas et al. (2000) and Saville et al. (2004).
There were marked differences in both the timing of production and amounts of similarly sized chiA transcripts between AcMNPV, Acpolh.chiA and Acp6.9.chiA. The intensities of the major 2.6 kb chiA RNA bands of reprogrammed viruses were greater than for AcMNPV. The temporal Acpolh.chiA chiA transcription pattern was typical of native AcMNPV polyhedrin transcription (Friesen & Miller, 1985; Possee & Howard, 1987; Ooi et al., 1989), and was well below that of chiA from AcMNPV from 9 up to 15 h p.i. As expected, the chiA transcription profile of Acp6.9.chiA resembled that of native p6.9 transcription described by Wilson et al. (1987).
Altering the chiA promoter did not influence either the strand size or termination site of the chiA transcript. The mapping of the AcMNPV chiA transcript initiation site to the first A of the TAAG sequence, determined by 5' RACE, confirms that of Hawtin et al. (1995). The initiation of Acpolh.chiA and Acp6.9.chiA chiA-specific transcripts also mapped to the first A of the equivalent late promoter (TAAG) sequences just upstream (40–60 bp) of the chiA ORF (Fig. 1). None of the Acp6.9.chiA 5' sequence data mapped chiA transcriptional initiation from the second, further upstream (–154 to –157), TAAG element, suggesting that this one is not used. Based on 5' and 3' RACE and the 2.6 kb size of the major chiA transcript, these observations indicated that the transcripts initiated just upstream of the chiA ATG and terminated at the same site, about 0.75 kb beyond the 1653 bp chiA ORF, for all viruses.
Based on our RT-PCR mapping of chiA transcripts, a minor amount of a large polyadenylated RNA containing the chiA ORF is initiated and terminated beyond the sites we have mapped for chiA by 5' and 3' RACE. Since baculoviruses transcribe large, overlapping polycistronic yet functionally monocistronic RNAs (Lubbert & Doerfler, 1984; Friesen & Miller 1985; Wilson et al., 1987), some chiA transcripts might similarly be longer. In AcMNPV, the nearest upstream ORF in the same orientation as chiA is gp64, expressed as both an immediate-early (by 1 h p.i.) and late gene (Jarvis & Garcia, 1994; Whitford et al., 1989). Thus, the longer (>2.6 kb) chiA-specific RNA we detected by RT-PCR mapping and as early as 0 h p.i. (1 h after addition of virus) might have initiated from the gp64 promoter.
The temporal chiA transcription profiles of Acpolh.chiA and Acp6.9.chiA relative to that of AcMNPV reflect the temporal profiles of the CHIA enzyme activity. The lag time between the first detection of Acpolh.chiA chiA RNA and the first detection of Acpolh.chiA chitinase activity was 3 h longer than the 6 h lag between RNA and enzyme for AcMNPV and Acp6.9.chiA. This difference in lag times could reflect the much lower level of chiA transcription of Acpolh.chiA at 9 to 12 h p.i., subsequently leading to undetectable levels of CHIA. Although Acpolh.chiA chiA transcripts were detectable at 9 and 12 h p.i., they were much less abundant than for either AcMNPV or Acp6.9.chiA at these times. Nevertheless, there might also have been unknown differences in the trafficking of nascent CHIA or the enzyme load of infected cells between AcMNPV and the two mutant viruses.
Much more chitinase activity was detected in cells infected with the chiA-reprogrammed viruses compared with AcMNPV. Fivefold more chitinase accumulated in Acp6.9.chiA virus-infected cells than in AcMNPV- or Acpolh.chiA-infected cells at 18 h p.i. Compared with AcMNPV infection, about 3- and 4-fold more chitinase activity accumulated at 48 h p.i. in Acp6.9.chiA- and Acpolh.chiA-infected cells, respectively, coincident with the differential chiA transcription rates of each virus.
We detected the 1.5 kb v-cath-specific RNA as early as 9 h p.i., much earlier than the 1 day reported by Hill et al. (1995). This was also much earlier than the detection of the enzyme at 22 h p.i. by immunoblot and activity assays (Slack et al.,1995) and 24 h p.i. by immunoblot (Hom et al., 2002). Since we did not do a time-course of cathepsin activity, we do not know whether the time-course of v-cath translation in our experiments reflects that of Slack et al. (1995) and Hom et al. (2002). The 1.5 kb size of v-cath transcripts determined from the Northern blot (Fig. 5) is slightly larger than the 1.2 kb size of transcript that was expected from transcriptional initiation from the TAAG late promoter sequence, 27 nucleotides upstream of the v-cath ATG (Hill et al., 1995), and termination at a site 173 nucleotides downstream of the translational termination sequence of the 966 bp ORF.
Although both the chiA and v-cath genes are transcribed at the same time, from about 9 h p.i., V-CATH is not translated until about 22 h p.i., as reported by Slack et al. (1995), which is about 10 h later than CHIA in our studies. The requirement for active CHIA for proper V-CATH folding and intracellular targeting (Hom & Volkman, 2000; Hom et al., 2002; Daimon et al., 2007) may explain the need for this delay in translation of v-cath relative to that of chiA RNA. Moreover, premature expression of V-CATH may damage infected cells too early, compromising the level of virus production.
Although we reconstituted the late promoter TAAG sequence and most of the native v-cath sequence up to the v-cath ATG in the chiA/v-cath intergenic regions of Acpolh.chiA and Acp6.9.chiA (Fig. 1b), no v-cath transcripts or virus-induced cysteine protease activity was detected in infected cells at 48 h p.i., as they were for AcMNPV. For Bombyx mori nucleopolyhedrovirus (BmNPV) in which much of the chiA ORF was deleted but the entire intergenic region remained intact, there was little effect on BmNPV v-cath expression (Daimon et al., 2007), suggesting that not more than the intergenic region was needed for v-cath expression. Our results suggest that the entire intergenic region upstream of the native v-cath TAAG promoter, which was modified in our alternate polh.chiA or p6.9.chiA promoters, might be needed for v-cath transcription. Nonetheless, our genomic alterations that modulated chiA and v-cath expression did not affect the in vitro replication kinetics of the modified viruses in comparison to AcMNPV.
In summary, we generated AcMNPV-derived viruses having chiA expression reprogrammed at its native locus using endogenous AcMNPV (polh or p6.9) promoters in the chiA/v-cath intergenic locus. Moreover, transcription of chiA in AcMNPV and two recombinant viruses occurred from about 6 h in advance of CHIA enzyme activity. We also demonstrated that, although we maintained most of the native putative promoter region from the TAAG to the v-cath ATG, the insertion of the alternate chiA promoters (polh, p6.9) adversely affected the transcription of v-cath. Further efforts to enable simultaneous reprogrammed expression of both chiA and v-cath could lead to the generation of an environmentally benign yet enhanced baculovirus insecticide.
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ACKNOWLEDGEMENTS |
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Received 19 January 2007;
accepted 9 May 2007.
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