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Short Communication |
1 Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford OX1 3SR, UK
2 School of Biological and Molecular Sciences, Oxford Brookes University, The Headington Campus, Oxford OX3 0BP, UK
Correspondence
Robert D. Possee
rdpo{at}ceh.ac.uk
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ABSTRACT |
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Present address: Department of Microbiology, The Moyne Institute of Preventative Medicine, Trinity College, Dublin 2, Ireland. 
Present address: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Infection of Spodoptera frugiperda (Sf21) cells with wild-type AcMNPV causes transient plasma-membrane blebbing at 12 h p.i. (Clem et al., 1991). In Sf21 cells infected with an AcMNPV p35 mutant, blebbing intensifies, resulting in disintegration of cells into apoptotic bodies. Cell blebbing is not observed in Trichoplusia ni (TN-368) cells infected with either wild-type virus or AcMNPV p35 mutants (Clem et al., 1991). We isolated a spontaneous AcMNPV mutant from a plaque assay conducted in TN-368 cells, during construction of a virus deficient in p35 function. The virus was supposed to have a 488 bp deletion from positions 116395 to 116883 of the AcMNPV C6 genome (Ayres et al., 1994). Thus, it lacked the p35 basal promoter region and the first 391 bases of the p35 coding region.
After infection of TN-368 cells (uninfected; Fig. 1a) with the p35-negative virus, an aberrant phenotype was observed at 3 days p.i. Instead of normal polyhedra production, as seen in AcMNPV-infected TN-368 cultures (Fig. 1b), cells contained few occlusion bodies (Fig. 1c). Furthermore, virus-infected cells exhibited extensive blebbing of vesicles (Fig. 1c). The virus was designated AcdefrT, as it was considered to be defective for replication in TN-368 cells, presumably owing to a spontaneous mutation.
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), was digested with EcoRI. This was mixed with AcdefrT DNA and used to transfect Sf21 cells (King & Possee, 1992). Medium containing virus was harvested at 4 days post-transfection and screened for cell blebbing and polyhedra production. Virus from a homogeneous polyhedra-producing recombinant plaque of wild-type appearance was amplified in Sf21 cells. It was designated AcdefrTp35r and exhibited a plaque phenotype indistinguishable from that of AcMNPV in Sf21 cells (Fig. 1f, g
). This suggested that the spontaneous mutation did not produce the same blebbing effect in Sf21 cells as observed in TN-368 cells.
Cultures of TN-368 cells were infected with AcdefrTp35r to determine the phenotype of the modified virus. As with AcdefrT, TN-368 cells infected with AcdefrTp35r showed a reduction in the number of polyhedra within each cell compared with wild-type infection, as judged by visual inspection of the cultures. However, the extensive cell blebbing seen with AcdefrT infection was not observed in cells infected with AcdefrTp35r (Fig. 1d), suggesting that this feature of the mutant phenotype was due to a combination of the unknown mutation and the non-functional p35 gene.
Induction of apoptosis in Sf21 cells by AcMNPV p35-negative viruses is associated with reduced BV formation (Hershberger et al., 1992; Clem & Miller, 1993). Therefore, BV production in AcdefrT-infected Sf21 or TN-368 cells was examined. Either cell type was infected with AcMNPV, AcdefrT or AcdefrTp35r (10 p.f.u. per cell). Medium was harvested at 72 h p.i. and viruses were titrated three times by plaque assay in the appropriate cell line. The mean titre for AcMNPV in Sf21 cells was 2·93x108 p.f.u. ml–1. As expected, AcdefrT showed greatly reduced levels of BV production in Sf21 cells compared with AcMNPV, owing to the absence of p35. The AcdefrT titres only reached a maximum of 1·24x104 p.f.u. ml–1. AcdefrTp35r, however, showed similar BV titres to the wild type, reaching 6·63x108 p.f.u. ml–1 at 72 h p.i. Similar experiments were done using TN-368 cells. In these cells, whilst the mean BV titre for the wild-type virus at 72 h p.i. was 4·8x106 p.f.u. ml–1, titres of both AcdefrT and AcdefrTp35r were found to reach 2·9x108 and 2·7x108 p.f.u. ml–1, respectively.
The AcdefrT-infected TN-368 cells exhibited extensive plasma-membrane blebbing, suggesting that apoptosis was induced. Apoptosis is associated with caspase activation from procaspases in cells. Therefore, caspase activation was monitored in AcMNPV-, AcdefrT- and AcdefrTp35r-infected Sf21 and TN-368 cells (Fig. 2). Cell cytosolic extracts were assayed by using a caspase-3 colorimetric activity assay kit (Chemicon International, Inc.). The kit provides a means of assaying the activity of caspases that recognize the DEVD motif. It is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labelled substrate DEVD-pNA. Thus, levels of free pNA reflect levels of caspase activity within the cell. In Sf21 cells, levels of caspase activity were five times higher in AcdefrT-infected cultures than with AcMNPV or AcdefrTp35r (Fig. 2a). This was consistent with the absence of p35 in AcdefrT. In TN-368 cells infected with the same three viruses, extracts were prepared at 24, 36 and 48 h p.i. (Fig. 2b). At 24 h p.i., caspase activity in AcdefrT-infected cells was marginally higher than in extracts from mock-infected cells or those infected with AcMNPV or AcdefrTp35r. By 36 h p.i., the levels of caspase activity in AcdefrT-infected TN-368 cells were nearly double those observed in any of the other samples. At 48 h p.i., caspase activity in AcdefrT-infected cells was over sixfold higher than that in mock- or AcdefrTp35r-infected cells. Levels of caspase activity in AcMNPV-infected cells were also higher at 48 h p.i., but a two-tailed Student's t-test confirmed the difference in activity between AcdefrT and AcMNPV samples to be statistically significant.
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a). A partial digest was carried out on AcdefrT genomic DNA by using the restriction enzyme MboI. Digests producing fragments of approximately 30 kbp were pooled and ligated into the SuperCos 1 vector (Stratagene). The identity of cloned AcdefrT genomic fragments was ascertained by sequencing the ends of each insert and alignment with the complete AcMNPV sequence (Ayres et al., 1994). Clones were selected spanning most of the AcMNPV genome (Fig. 3a).
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), containing the Escherichia coli lacZ gene inserted into the p35 locus, and used to transfect TN-368 cells. The progeny virus was titrated in the same cell type after 5 days. Cell blebbing in AcdefrT-infected TN-368 cells was due to a combination of the unknown mutation and the absence of p35. Thus, by using AcMNPVp35– DNA in this experiment, mutant plaques could be identified on the basis of both a reduction in number of polyhedra and the occurrence of plasma-membrane blebbing.
Plaque screening revealed possible AcdefrT-like plaques in those dishes infected with medium harvested from cells transfected with AcMNPVp35– genomic DNA and cosdef 4 (Fig. 3a). In the same experiment, none of the other cosmids resulted in the production of AcdefrT-like plaques. The AcMNPV gene complement within cosdef 4 was analysed to identify genes that might be responsible for the phenotype of AcdefrTp35r-infected TN-368 cells. The reduction in polyhedra and increase in BV production are characteristics of the Few Polyhedra (FP) phenotype, which occurs upon serial passage of nucleopolyhedroviruses in cell culture due to mutations in the FP-25 gene locus (Hink & Vail, 1973; Potter et al., 1976; Fraser et al., 1983; Beames & Summers, 1988, 1989). As FP-25 was found to be located on cosdef 4, this gene locus was analysed in both AcdefrT and AcdefrTp35r.
The FP-25 locus of AcMNPV, AcdefrT and AcdefrTp35r was amplified by using PCR with primers (20 nt; MWG Biotech) designed outside the coding region (Fig. 3b). Amplified fragments (1·2 kbp) were found to be the same size in all three viruses (data not shown), thus confirming the absence of any major insertions or deletions in this region of the AcdefrT genome.
The PCR-amplified fragments from AcMNPV, AcdefrT and AcdefrTp35r were sequenced. Analysis revealed that both AcdefrT and AcdefrTp35r had an A insertion within a run of 7 adenines between positions +181 and +188 of FP-25K (Fig. 3c). The insertion of this base produced a frameshift mutation resulting in the premature occurrence of a stop codon and subsequent truncation of the FP-25 protein from the wild-type 214 aa to 63 aa.
Confirmation that this mutation was responsible for the observed phenotype of each virus and that no other mutation present in the viral genome was responsible was provided by reconstructing both AcdefrT and AcdefrTp35r. A 366 bp region of the AcdefrT FP-25 locus containing the point mutation was PCR-amplified by using Pfu polymerase and purified by using a Qiagen PCR purification kit. This fragment was mixed with either AcMNPV or AcMNPVp35– DNA and used to cotransfect TN-368 cells. Medium was harvested and screened for AcdefrT- and AcdefrTp35r-type plaques. Mutant plaques were identified successfully and the reconstructed viruses (recAcdefrT and recAcdefrTp35r) were amplified.
Both recAcdefrT (Fig. 1h) and recAcdefrTp35r (data not shown) produced the same plaque phenotypes as the original AcdefrT and AcdefrTp35r viruses, respectively, in TN-368 cells and Sf21 cells (data not shown). Sequencing FP-25 from each of the two recreated viruses confirmed the presence of only the inserted adenine in the same position as in AcdefrT and AcdefrTp35r. An analysis of BV titres, similar to that described above, was carried out to confirm that recAcdefrT produced levels of BV similar to AcdefrT in TN-368 cells. As expected, both AcdefrT and recAcdefrT produced higher levels of BV than did AcMNPV in this cell line. The mean BV titre for the wild-type virus was found to be 4·6x106 p.f.u. ml–1 at 72 h p.i. Consistent with earlier analyses of BV titres, AcdefrT BV levels were higher than those observed for the wild type, reaching 2·6x108 p.f.u. ml–1 at 72 h p.i. Titres of recAcdefrT were also found to be greater than those of AcMNPV, as well as exceeding those of AcdefrT, reaching 1·2x109 p.f.u. ml–1 by 72 h p.i. Together, these results confirmed that the reduction in polyhedra and increase in BV production observed in AcdefrTp35r-infected TN-368 cells was due to a mutation in the FP-25 gene alone.
Mutations in the AcMNPV FP-25 would be expected to result in the production of FP mutants in both Sf21 and TN-368 cells. This was not the case, however, with the FP-25 mutation found here. Although truncation of the FP-25 protein was predicted to be severe, reducing the 214 aa protein to only 63 residues, the difference in phenotype observed in these two cell types suggests that it did not affect its ability to function normally in Sf21 cells.
The work described in this report indicates that infection of TN-368 cells with an AcMNPV mutant possessing mutations in FP-25 and p35 induces plasma-membrane blebbing in TN-368 cells. An assay of caspase-3-like activity in insect cells showed that elevated levels of caspase activity were present in AcdefrT-infected TN-368 cells. This indicated that an apoptotic response had been generated in these cells after infection with a virus lacking functional p35 and 25K. The TN-368 cells have been found to be more resistant to apoptosis than Sf21 cells. Whilst infection with AcMNPV p35 mutants or treatment with the RNA-synthesis inhibitor actinomycin D induced apoptosis in Sf21 cells, cell death did not occur in TN-368 cells exposed to these same stimuli (Clem et al., 1994). The observation of a unique phenotype associated with mutations in FP-25 and p35 was unexpected. The characteristics associated most commonly with the FP genotype are a reduced number of polyhedra per cell compared with the wild type, occlusions containing no virions or virions of altered morphology, altered intranuclear envelopment and the production of more BV than by the wild type (Ramoska & Hink, 1974; Potter et al., 1976; Wood, 1980). Fraser et al. (1983) found a common feature of many AcMNPV and Galleria mellonella (Gm) MNPV mutants to be insertion of moderately repetitive host-DNA sequences into a region of the genome encoding a 25 kDa protein. These were later correlated with large insertions of host-cell DNA or deletions of viral DNA, detectable by restriction-endonuclease analysis, in this region of the genome (Beames & Summers, 1988, 1989). Targeted mutation of AcMNPV 25K confirmed alterations to this gene to be sufficient to result in these complex characteristics of the FP phenotype (Harrison & Summers, 1995). Although FP mutants of Lymantria dispar (Ld) MNPV and Helicoverpa armigera (Ha) SNPV have been identified (Slavicek et al., 1995; Bischoff & Slavicek, 1997; Chakraborty & Reid, 1999; Lua et al., 2002), these mutations were the result of point changes or small insertions or deletions in the FP-25K coding region (Bischoff & Slavicek, 1997; Lua et al., 2002). This suggests that LdMNPV and HaSNPV mutants may arise through a mechanism different from that involved in the formation of most AcMNPV and GmMNPV FP mutants. However, the frameshift mutation within AcdefrT is clearly more like the minor changes observed to occur within the LdMNPV and HaSNPV genomes. This feature, linked with the absence of p35 in AcdefrT-infected cells, must be responsible for the apparent induction of apoptosis in TN-368 cells infected with this virus. Whether or not such dual mutations accumulate in the genomes of baculoviruses in insect larvae is unclear. However, we did note an increase in caspase-3-like activity in AcMNPV-infected TN-368 cells, which might indicate accumulation of these mutations in a proportion of the wild-type virus population.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Clem, R. J. & Miller, L. K. (1993). Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. J Virol 67, 3730–3738.
Clem, R. J., Fechheimer, M. & Miller, L. K. (1991). Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390.
Clem, R. J., Robson, M. & Miller, L. K. (1994). Influence of infection route on the infectivity of baculovirus mutants lacking the apoptosis-inhibiting gene p35 and the adjacent gene p94. J Virol 68, 6759–6762.
Fraser, M. J., Smith, G. E. & Summers, M. D. (1983). Acquisition of host cell DNA sequences by baculoviruses: relationship between host DNA insertions and FP mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses. J Virol 47, 287–300.
Funk, C. J., Braunagel, S. & Rohrmann, G. F. (1997). Baculovirus structure. In The Baculoviruses, pp. 7–32. Edited by L. K. Miller. New York: Plenum.
Griffiths, C. M., Barnett, A. L., Ayres, M. D., Windass, J., King, L. A. & Possee, R. D. (1999). In vitro host range of Autographa californica nucleopolyhedrovirus recombinants lacking functional p35, iap1 or iap2. J Gen Virol 80, 1055–1066.[Abstract]
Harrison, R. L. & Summers, M. D. (1995). Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. J Gen Virol 76, 1451–1459.
Hershberger, P. A., Dickson, J. A. & Friesen, P. D. (1992). Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication. J Virol 66, 5525–5533.
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Received 1 July 2005;
accepted 18 November 2005.
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