1 Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
2 Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Correspondence
Michihiro Kobayashi
michihir{at}agr.nagoya-u.ac.jp
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ABSTRACT |
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Present address: Nikken Foods Co., Ltd, Haruoka 723-1, Hukuroi, Shizuoka 437-0122, Japan.
Present address: Insect Pathology Laboratory, Department of Entomology, College of Agriculture, University of the Philippines Los Baños College, Laguna 4031, Philippines.
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INTRODUCTION |
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One of the distinctive features of non-productive infection is observed in the cell line Ld652Y (Goodwin et al., 1978), derived from the gypsy moth, Lymantria dispar, which has been shown to be permissive for L. dispar multinucleocapsid NPV (LdMNPV) (Slavicek et al., 1992
) and Orgyia pseudotsugata MNPV (OpMNPV) (Bradford et al., 1990
). Infection of Ld652Y cells with Autographa californica MNPV (AcMNPV) results in a total shutdown of not only cellular but also viral protein synthesis at the level of translation (Guzo et al., 1991
, 1992
; Du & Thiem, 1997
; Mazzacano et al., 1999
). The suppressed protein synthesis in AcMNPV-infected Ld652Y cells is restored to the level of LdMNPV-infected Ld652Y cells when AcMNPV experimentally acquires the LdMNPV host range factor 1 (hrf-1) gene (Thiem et al., 1996
). The recombinant AcMNPV with the hrf-1 gene successfully replicates and produces high titres of progeny virions in both Ld652Y cells and L. dispar larvae (Thiem et al., 1996
; Chen et al., 1998
), indicating that hrf-1 is directly related to the AcMNPV productive infection in Ld652Y cells.
Another distinctive feature of non-productive infection is observed in those cells undergoing virus-induced apoptosis. Baculovirus-induced apoptosis was first demonstrated in Spodoptera frugiperda Sf21 cells infected with an AcMNPV mutant lacking a functional p35 gene (Clem et al., 1991). Subsequent studies have shown that wild-type (wt) AcMNPV with the intact p35 gene also induces apoptosis in cell lines from Spodoptera littoralis and Choristoneura fumiferana (CF-203) (Chejanovsky & Gershburg, 1995
; Palli et al., 1996a
) and that the apoptosis induced in AcMNPV-infected CF-203 cells is blocked by prior inoculation with C. fumiferana MNPV (Palli et al., 1996a
). In addition to AcMNPV, Spodoptera exigua MNPV (SeMNPV) and Heliothis armigera single nucleocapsid NPV (HaSNPV), which cause productive infection in cell lines from S. exigua and Trichoplusia ni (Hi5), have also been shown to induce apoptosis in S. littoralis and Heliothis zea cells, respectively (Yanase et al., 1998
; Dai et al., 1999
). In addition, it has been shown that T. ni (TN368) cells infected with the p35-defective AcMNPV mutant resist apoptosis and yield a high titre of progeny virions (Clem & Miller, 1993
). These results imply that baculoviruses generally encode a factor(s) that triggers apoptosis in the infected cells, and that whether the baculovirus-infected cells undergo apoptosis relies on an intricate relationship between cellular and viral functions that are involved in the induction and suppression of apoptosis.
The molecular mechanisms underlying the induction and suppression of apoptosis in cells infected with baculoviruses are largely unknown. Careful analysis of the timing of apoptotic events in p35-defective AcMNPV-infected S. frugiperda cells has suggested that NPV-induced apoptosis could be triggered by both early and late events in virus infection (LaCount & Friesen, 1997). Previous studies have also shown that AcMNPV-induced apoptosis of Sf21 cells is triggered by IE1, the product of the immediate-early viral gene ie1 (Prikhod'ko & Miller, 1996
), and suppressed by P35 and inhibitor of apoptosis (IAP) proteins, which are encoded by the genomes of certain NPVs (Clem, 1997
, 2001
). It has also been shown that IE1-induced apoptosis of Sf21 cells is augmented by the AcMNPV early gene product PE38 (Prikhod'ko & Miller, 1999
). In addition, apoptosis induced by baculovirus infection has been shown to be associated with the activation of caspases (Bertin et al., 1996
; Ahmad et al., 1997
; LaCount & Friesen, 1997
; Seshagiri & Miller, 1997
; LaCount et al., 2000
; Manji & Friesen, 2001
).
We have previously suggested that Ld652Y cells exhibit apoptosis following infection with SeMNPV and Spodoptera litura MNPV (SpltMNPV) (Wu et al., 2000; Laviña et al., 2001
). In the present study, we have demonstrated that Ld652Y cells readily undergo apoptosis following infection with a variety of NPVs, including Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HycuNPV), OpMNPV, SeMNPV and SpltMNPV. In addition, we have characterized the HycuNPV-induced apoptosis of Ld652Y cells, which exhibit severe apoptosis, and found that a substantial amount of the HycuNPV iap3 gene, whose product has been shown to play a role in blocking apoptosis, is expressed in HycuNPV-infected Ld652Y cells.
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METHODS |
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Insect cell lines used in these experiments were IPLB-Ld652Y from the gypsy moth, L. dispar (Ld652Y; Goodwin et al., 1978) and FRI-SpIm1229 from the mulberry tiger moth, Spilosoma imparilis (SpIm; Mitsuhashi & Inoue, 1988
). Ld652Y cells were grown at 28 °C in TC100 medium (Invitrogen) supplemented with 10 % foetal bovine serum (FBS), whereas SpIm cells were maintained at 28 °C in MM medium (Mitsuhashi & Maramorosch, 1964
) supplemented with 3 % FBS.
DNA fragmentation assay.
Culture cells with apoptotic bodies in 25 cm2 culture flasks (Nunc 163371) were scraped into culture medium with a rubber policeman and collected by centrifugation at 3000 r.p.m. for 5 min at 4 °C. The precipitate was washed twice with PBS and stored frozen until used. DNA for the fragmentation assay was isolated as described previously (Palli et al., 1996b). Briefly, the thawed cells with apoptotic bodies were suspended in 200 µl lysis buffer (100 mM NaCl, 10 mM Tris/HCl, pH 7·9, 25 mM EDTA, 0·5 % SDS) containing 0·3 mg proteinase K ml-1, incubated at 55 °C for 12 h and digested with 1 mg RNase A ml-1 for 1 h at 37 °C. The DNA was extracted twice with an equal volume of phenol (saturated with 100 mM Tris/HCl, pH 8)/chloroform/isoamyl alcohol (24 : 1) and then once with chloroform alone. The extracted DNA was ethanol-precipitated and dissolved in TE-8 (10 mM Tris/HCl, pH 8, 1 mM EDTA).
In certain experiments, an NP-40 extraction procedure was used to yield preferentially the fragmented DNAs. In this procedure, cells washed with PBS were immediately lysed in 100 ml lysis buffer (1 % NP-40, 50 mM Tris/HCl, pH 7·5, 20 mM EDTA) and centrifuged at 4500 r.p.m. for 5 min at 4 °C in a microcentrifuge. The supernatant containing fragmented DNA was removed and incubated at 50 °C for 3 h after mixing well with 20 µl 10 % SDS and 5 µl RNase A (10 mg ml-1). After incubation, 5 µl proteinase K (15·6 mg ml-1) was added and the mixture was further incubated at 37 °C for 3 h. The DNA was ethanol-precipitated and dissolved in TE-8.
Caspase activity assay.
The caspase activity assay was performed using the caspase-3 fluorescent assay kit ApoProbe-3 (BioDynamics Laboratory), which allows quantitative detection of caspase-3-like protease activity. Monolayer cultures were infected with seven different NPVs. At different times post-infection (p.i.), cells were scraped into culture medium with a rubber policeman and collected by centrifugation at 3000 r.p.m. for 10 min at 4 °C. The cells were suspended in cell lysis buffer (included in the kit) and incubated on ice for 10 min and the cell lysates centrifuged at 12 000 r.p.m. for 3 min at 4 °C. The resultant supernatants were analysed for caspase-3-like protease activity using vAc-DEVD-AMC as the substrate. Accumulation of fluorescent product was monitored using a spectrofluorophotometer, model RF-5300PC (Shimadzu), with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Slot-blot hybridization analysis.
Slot-blot hybridization analysis was performed as described previously (Ikeda & Kobayashi, 1999). Virus-infected cells were scraped into the culture medium at different times p.i., precipitated at 10 000 r.p.m. for 15 min at 4 °C and suspended in distilled water. The cells in distilled water were treated with heated supersaturated sodium iodide and boiled for 10 min. After chilling on ice, the mixtures were blotted on to Hybond-N+ nylon membranes and hybridized with the DNA probe labelled with fluorescein according to the protocol of the Gene Images CDP-Star detection module (Amersham Pharmacia Biotech). The probe used for the detection of viral DNA was the HycuNPV iap3 (hycu-iap3) gene, which was amplified by PCR using 5'-ACGCACACGGCGGAGTTAAC-3' and 5'-AGTAGTGCGACACGTGGGAC-3' as the paired primers and HycuNPV genomic DNA as the template.
Northern blot analysis.
Transcripts of hycu-iap3 were examined by Northern blot analysis, essentially as described previously (Ikeda et al., 2001). Monolayer cultures of Ld652Y cells (8x106) were prepared in 80 cm2 culture flasks (Nunc 147589) and infected with HycuNPV at an input m.o.i. of 10. At different times p.i., total RNA was isolated by TRIzol reagent (Invitrogen) from the virus-infected cells, resolved on a 1·2 % agarose gel (SeaKem GTG, FMC BioProducts) and blotted on to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) under alkaline conditions. The RNA on the membrane was probed with 32P-labelled hycu-iap3 and analysed by imaging analyser (BAS 2000, Fuji Photo Film). The probe used was amplified by PCR as described for slot-blot analysis and labelled with [
-32P]dCTP (NEN Research Products) using the Rediprime II random prime labelling system (Amersham Pharmacia Biotech).
Immunoblot analysis.
Immunoblot analysis was carried out as described previously (Ikeda et al., 2001; Katou et al., 2001
). Briefly, polypeptides from infected cells were resolved by SDS-PAGE and blotted on to nitrocellulose membranes (Advantec Toyo) or Immobilon transfer membranes (Millipore). Antibodies against BmNPV polyhedrin (Shirata et al., 1999
), BmNPV occluded virions (Kobayashi et al., 1990
) and Hycu-IAP3 were used as primary antibodies. The antibodies against BmNPV polyhedrin and BmNPV occluded virions were raised in rabbits and the antibody against Hycu-IAP3 in mice for the present study. The immunopositive polypeptides were detected using HRP-conjugated goat anti-rabbit or anti-mouse IgG (Zymed). The BmNPV structural polypeptides and polyhedrin were visualized by Konica immunostaining HRP-1000, and Hycu-IAP3 by ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
Preparation of anti-Hycu-IAP3 antiserum.
For the preparation of anti-Hycu-IAP3 antiserum, a portion of the Hycu-IAP3 protein was generated using the pET-32b(+) expression vector (Novagen). A PCR product encoding 126 amino acid residues (Gly6Thr131) of the Hycu-IAP3 protein (accession no. AB088850) was amplified using the 3·3 kbp HindIII fragment of HycuNPV DNA as the template and paired primers 5'-CGGGATCCCGGAGTTAACATGGAA-3' and 5'-CCGCTCGAGCGGGTAATAAAACCC-3' containing HindIII and XhoI restriction sites (underlined), respectively. The PCR product was subcloned into the HindIIIXhoI site of the pET-32b(+) expression vector and introduced into Escherichia coli BL21-DE3-pLysS. Bacteria were grown at 37 °C and the portion of Hycu-IAP3 protein was expressed by induction with IPTG at a final concentration of 10 mM. The Hycu-IAP3 protein produced was purified by His Trap (Amersham Pharmacia) and the His Trap-purified Hycu-IAP3 protein was resolved by SDS-PAGE. The Hycu-IAP3 protein band was cut out from the gel after Coomassie brilliant blue staining and the gel slices containing the Hycu-IAP3 protein were homogenized and injected into mice for immunization.
Budded virion titration.
HycuNPV BVs in the medium of virus-infected cells were titrated by plaque assay on SpIm cells, as described previously (Shirata et al., 1999).
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RESULTS |
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Caspase-3-like protease activity was determined in NPV-infected Ld652Y cells using the ApoProbe-3 kit. At 60 h p.i., Ld652Y cells infected with NPVs were subjected to a caspase-3-like protease activity assay. The results showed that caspase-3-like protease activity increased significantly in Ld652Y cells infected with HycuNPV, SeMNPV, SpltMNPV, BmNPV and OpMNPV, as well as with AcMNPVp35 (Fig. 1C
). The caspase-3-like protease activity was higher in cells infected with HycuNPV, SeMNPV and SpltMNPV than in the cells infected with BmNPV and OpMNPV. In mock-infected Ld652Y cells and Ld652Y cells infected with AcMNPV and LdMNPV, no significant increase in caspase-3-like protease activity was observed.
Cytopathology, DNA fragmentation and caspase activation in HycuNPV-infected Ld652Y cells
To characterize further the NPV-induced apoptosis, HycuNPV-infected Ld652Y cells, which exhibited severe apoptosis, were analysed in some detail. Ld652Y cells were infected with HycuNPV at an input m.o.i. of 1, 5, 10 or 20 and examined at intervals for apoptosis induction. Microscopic observation showed that the severity of apoptosis induced in HycuNPV-infected Ld652Y cells was dependent on the input m.o.i. between 1 and 20. The Ld652Y cells infected at 20 p.f.u. per cell showed blebbing by 24 h p.i. and the number of cells exhibiting characteristics of apoptosis increased up to 72 h p.i.
HycuNPV-infected Ld652Y cells were also examined for the oligomeric fragmentation of cellular DNA (Fig. 2). Ld652Y cells were infected with HycuNPV at an m.o.i. of 1, 5, 10 or 20, and cellular DNA was extracted from infected cells at 96 h p.i. Analysis of the DNA on agarose gels showed that oligomeric DNA laddering was observed, even in cells infected at an m.o.i. of 1 (Fig. 2A
). In Ld652Y cells infected at an m.o.i. of 20, the oligomeric DNA ladder was detectable by 24 h p.i., became clearly observed at 48 h p.i. and decreased gradually from 72 to 96 h p.i. (Fig. 2B
).
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DISCUSSION |
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The suggestion that inherent cellular properties are closely related to the prompted apoptosis induction in NPV-infected Ld652Y cells is supported by circumstantial evidence derived from studies on virus-encoded apoptosis-suppressing factors. Previous studies in our laboratory have shown that HycuNPV does not possess the homologue of p35 but encodes three species of iap homologue (hycu-iap1, -2 and -3). Functional analyses with Sf9 cells transiently expressing Hycu-IAPs have further shown that hycu-iap3 exhibits clear apoptosis-suppressing activity, which depends on the amount of Hycu-IAP3 protein accumulated in the cells (M. Ikeda and others, unpublished data). Immunoblot analysis in the present study showed that the amount of Hycu-IAP3 protein accumulated in HycuNPV-infected Ld652Y cells was higher than or comparable with that in HycuNPV-infected SpIm cells, which showed no apoptotic response and generated a high titre of progeny virions. In addition, there was no apparent difference between HycuNPV-infected Ld652Y and SpIm cells in the time course of Hycu-IAP3 expression. It is thus probable that not only viral IAPs but also cellular IAPs or related factors play an important role in the suppression of apoptosis in insect cells triggered by NPV infection. Alternatively, it is possible that Hycu-IAP3 is non-functional or is required at higher levels for the suppression of apoptosis in this particular system relating to Ld652Y cells and NPVs. It may also be possible that the induction of apoptosis of NPV-infected Ld652Y cells is specifically associated with some unidentified apoptotic pathway that is insensitive to suppression by IAPs.
In S. frugiperda cells in which apoptosis is induced by infection with the mutant AcMNPVp35, production of viral progeny is severely reduced due to delay or lack of viral gene expression in both cultured cells (Hershberger et al., 1992
; Clem & Miller, 1993
) and insect larvae (Clem & Miller, 1993
). In HycuNPV-infected Ld652Y cells, on the other hand, our data demonstrated that neither BVs nor viral structural proteins and polyhedrin are yielded at detectable levels. Our results in HycuNPV-infected Ld652Y cells agree with those observed in S. littoralis and C. fumiferana cells undergoing apoptosis following infection with wt AcMNPV (Chejanovsky & Gershburg, 1995
; Palli et al., 1996a
). Using a caspase inhibitor, zVAD-FMK, we have also found that suppression of apoptosis in HycuNPV-infected Ld652Y cells does not result in the restoration of progeny virion production (H. Ishikawa, unpublished data), suggesting that the defects in progeny virion production are not merely due to the apoptosis induced in the infected cells. This result agrees with previous results in wt AcMNPV-infected S. littoralis cells (Gershburg et al., 1997
) and HaSNPV-infected T. ni cells (Dai et al., 1999
).
The mechanisms underlying apoptosis induction in Ld652Y cells observed in the present study are not known. In the HycuNPV-infected Ld652Y cells, our data demonstrated that the virus replication cycle is restricted at a step prior to viral late gene expression, suggesting that HycuNPV-induced apoptosis of Ld652Y cells is an event triggered in the early phase of viral infection. Previous studies have demonstrated that apoptosis of AcMNPV-infected S. frugiperda cells is triggered by an immediate-early viral protein, IE1 (Prikhod'ko & Miller, 1996) and suppressed by P35 and IAPs encoded by the viral genome (Clem, 1997
, 2001
). Recent studies from our laboratory with the transient expression assay have shown that the HycuNPV ie1 gene is sufficient to induce apoptosis in Ld652Y cells (H. Ishikawa, unpublished data).
Consistent with the previous results (Slavicek et al., 1992; Guzo et al., 1992
; Du & Thiem, 1997
; Mazzacano et al., 1999
), our results showed that Ld652Y cells were permissive for LdMNPV, while infection of Ld652Y cells with AcMNPV resulted in no appreciable apoptotic response. Since LdMNPV and AcMNPV have been characterized in Ld652Y cells to induce productive infection (Slavicek et al., 1992
) and total shut-down of protein synthesis, respectively (Guzo et al., 1992
; Du & Thiem, 1997
; Mazzacano et al., 1999
), our results indicate that Ld652Y cells display three distinct types of cytopathic response following infection with different NPVs. Thus, Ld652Y cells provide an excellent system for understanding the molecular mechanisms of NPVcell interactions.
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ACKNOWLEDGEMENTS |
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Received 6 September 2002;
accepted 6 November 2002.