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Infection of a Spodoptera frugiperda cell line with Bombyx mori nucleopolyhedrovirus
Virus Research 47 (1997) 179 – 185
Infection of a Spodoptera frugiperda cell line with Bombyx mori nucleopolyhedrovirus O. Martin, G. Croizier* Unite´ de Ge´ne´tique des Virus, Station de Recherches de Pathologie Compare´e, INRA-URA CNRS 2209, Saint Christol-Le`s-Ale`s, France Received 30 April 1996; accepted 30 October 1996
Abstract The interactions of Bombyx mori nucleopolyhedrovirus (BmNPV) with Spodoptera frugiperda cells (Sf9) were investigated. S. frugiperda cells are usually considered nonpermissive for BmNPV. However, in the present study, BmNPV DNA replication was observed and an increasing infectious titre, reaching 104 TCID50/ml on B. mori permissive cells by 6 days post-transfection, developed in the supernatant of infected Sf9 cells. Infection of Sf9 cells by BmNPV did not induce a discernible shutoff of cellular protein synthesis and no overt cytopathic effects were observed. These data indicate that the low permissivity of Sf9 cells for BmNPV replication is associated with an inapparent infection. © 1997 Elsevier Science B.V. Keywords: Baculovirus; Bombyx mori; Cellular permissivity; Nucleopolyhedrovirus; Sf9 cells
1. Introduction Baculoviruses are characterized by enveloped rod-shaped virions containing circular doublestranded DNA (Murphy et al., 1995). They are infectious to insects and in particular to a variety of economically important lepidopteran species (Gro¨ner, 1986) which gives them a potential role as insecticides (Carter, 1984). Two different * Corresponding author. Tel.: +33 0466783714; fax: + 33 0466524699; e-mail: [email protected]
approaches have been applied to overcome the problem of slow speed of kill characteristic of wild-type baculovirus insecticides. Insertion of non-baculovirus genes such as insect-selective toxins into the baculovirus genome, or deletion of the viral gene encoding ecdysteroid UDP-glucosyltransferase, has yielded improved biopesticides (Bonning and Hammock, 1996; O’Reilly, 1995). On the other hand, combining genome elements from viruses possessing different host ranges offers a method to produce recombinant viruses with extended host range (Kondo and Maeda,
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1991). A greater understanding at the molecular level of viral specificity and virulence, by analysis of the genes which determine baculovirus replication in given hosts, is another important component of strategies to improve their efficacy as biopesticides. Comparisons of Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV) replication in cells possessing different degrees of permissivity offer an attractive model to investigate the genetic determinants of specificity (Maeda et al., 1993). Spodoptera frugiperda cells are considered nonpermissive for BmNPV and permissive for AcMNPV, while B. mori cells are considered nonpermissive for AcMNPV and permissive for BmNPV (Kondo and Maeda, 1991; Croizier et al., 1994). Recently it was shown that the blockage of AcMNPV replication in Bombyx mori cells (Bm5) could be overcome by homologous recombination between AcMNPV genomic DNA and a 133 base pair DNA fragment of the BmNPV helicase gene (Croizier et al., 1994). AcMNPV replication in Bm5 cells could thus be achieved by a minor modification of its DNA helicase gene, which has been shown to be an essential gene for DNA replication (Kool et al., 1994). Further analyses of the determinant(s) of AcMNPV and BmNPV specificity might include the identification of AcMNPV DNA fragments capable of extending the host range of BmNPV. Previous results showed that coinfection of B. mori (BmN) cells with AcMNPV and BmNPV resulted in the inhibition of BmNPV replication (Kamita and Maeda, 1993). The deleterious effect of AcMNPV helicase during the coinfection (Kamita and Maeda, 1993) makes it difficult to obtain recombinant viruses directly in B. mori cells. Since coinfection of Sf21 cells with AcMNPV and BmNPV virus particles induces BmNPV replication and recombinant formation (Kondo and Maeda, 1991), recombinants are expected to arise from cotransfection with a mixture of AcMNPV and BmNPV DNAs. Analysis of the behaviour of BmNPV in S. frugiperda cells is a prerequisite to studying the formation of AcMNPV-BmNPV recombinants in S. frugiperda cells by rescue of BmNPV with AcMNPV DNA sequences.
In this study, we report the results of BmNPV infection of Sf9 cells. Analyses of viral DNA replication, appearance of viral proteins by immunofluorescence analysis, and extracellular virus production are described.
2. Materials and methods
2.1. Cells and 6iruses S. frugiperda (fall armyworm) IPLB SF 21-AE (Vaughn et al., 1977) clone 9 (Sf9) and B. mori (silkworm) (Grace, 1967) clone 5 (Bm5) were maintained at 27°C in TC100 medium supplemented with 10% fetal bovine serum (FBS) (heat inactivated at 56° for 30 min). AcMNPV strain 1.2 (Croizier and Quiot, 1981) and BmNPV strain SC7 (Croizier et al., 1994) were used in this study.
2.2. Antisera A rabbit received intramuscular injection of 2–3 mg of purified BmNPV virions (after thioglycolate digestion of occlusion bodies and sucrose gradient centrifugation) mixed with Freund’s adjuvant. One booster injection was delivered 3 weeks later. The rabbit was bled after a further 3 weeks. This immune serum was used for immunofluorescence microscopy. Dr L.K. Miller and colleagues kindly provided rabbit polyclonal antisera against the AcMNPV major capsid protein VP39. Dr G.W. Blissard’s laboratory kindly provided monoclonal antibody AcV5 that recognizes the denatured form of AcMNPV GP64 envelope fusion protein (Monsma and Blissard, 1995).
2.3. DNA purification AcMNPV and BmNPV were multiplied in Galleria mellonella and B. mori larvae, respectively. Genomic DNAs used for transfection were extracted from occlusion bodies obtained from dead larvae by standard methods (O’Reilly et al., 1992). Briefly, occlusion bodies were incubated in 0.1 M Na2CO3 for 10 min and the alkaline suspension
O. Martin, G. Croizier / Virus Research 47 (1997) 179–185
was clarified at 2000× g for 5 min. Virions were pelleted at 45 000 × g for 1 h in a SW 28 rotor (Beckman L7-55 Ultracentrifuge). DNA was phenol-extracted after digestion with proteinase K (500 mg/ml) in the presence of 1% SDS at 60°C for 30 min. DNA concentrations were estimated by UV spectrophotometry.
2.4. Lipofection Monolayers of Sf9 cells (5× 105 cells/35 mm dish) were transfected with DNA by liposome-mediated transfection using DOTAP (Boehringer, Germany): AcMNPV or BmNPV DNA (250 ng in 10 ml of TE buffer) was mixed with 8 ml of DOTAP and 600 ml of TC100 medium, and added to Sf9 cells. After 5 h, the medium was replaced with TC 100 supplemented with 10% FBS.
2.5. Dot blot analysis Monolayers of Sf9 cells (1.0 ×106 cells/35 mm dish) were infected with BmNPV at a multiplicity of infection (m.o.i.) of 20. At the designated time postinfection (p.i.) the cells were harvested, pelleted and washed once with 0.5 ml phosphatebuffered saline (PBS) pH 7.4. Extraction of total DNA from Sf9 cells was performed as described by O’Reilly et al. (1992). DNA was resuspended in 50 ml of TE buffer. 2 ml of the DNA solution was dot blotted onto a Hybond-N + membrane (Amersham, France). Nylon membrane was laid on a Whatman paper saturated with denaturation solution (1 M NaOH, 1.5 M NaCl). The membrane was air dried, and the DNA was UV-crosslinked to the membrane. Hybridization was carried out overnight at 68°C with a probe obtained by random priming of the PstI O fragment of BmNPV DNA cloned in pUC 19 (random priming DNA labeling kit, Boehringer, Germany).
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Laemmli sample buffer (Laemmli, 1970), followed by SDS-PAGE. After transfer to a nitrocellulose membrane (Trans-blot transfer medium, Bio-Rad, USA), the blots were blocked with non-fat dried milk (10% in a Tris/NaCl buffer 50 mM Tris HCl, 200 mM NaCl, pH7.4) and incubated with rabbit antiserum directed against VP39 protein diluted 1:2000 or monoclonal antibody directed against GP64 protein diluted 1:4000. They were then incubated with peroxidase conjugated anti-rabbit or anti-mouse IgG diluted 1:2000. Peroxidase activity was revealed using the ‘ECL kit’ (Amersham, France).
2.7. Immunofluorescence Sterile coverslips were deposited in tissue culture dishes and overlaid with 3 × 105 Sf9 cells in 2 ml medium. Cells were allowed to adhere for 1 h and were then infected at a m.o.i. of 10 with supernatant from BmNPV-infected Bm5 cells or supernatant from AcMNPV-infected Sf9 cells. After 3 h the supernatant was removed and replaced with fresh medium. At various time p.i. the cells were fixed in a 70% acetone, 30% methanol solution for 10 min at − 20°C. The cells were airdried and then overlaid with PBS for 15 min. Rabbit immune serum directed against BmNPV purified virions (after thioglycolate digestion of occlusion bodies and sucrose gradient centrifugation), diluted 1:100 in PBS, was added. After a 1 h incubation at 37°C, the coverslip was washed twice for 15 min in PBS and then incubated with fluorescein-labeled goat antibody directed against rabbit globulin (diluted 1:1000 in PBS) for 1 h. The cells were rinsed twice for 15 min in PBS, and the coverslips were placed face down on a drop of buffered glycerin on a microscope slide and examined on a Nikon epifluorescence microscope.
2.6. Protein analysis
3. Results
Sf9 cells were infected with supernatant from BmNPV-infected Bm5 and AcMNPV-infected Sf9. Bm5 cells were infected only with supernatant from BmNPV infected Bm5. Infected cells were harvested at 0, 6, 12, 24, 48, 96 h p.i. and lysed in
3.1. Increase of BmNPV titre in the supernatant of Sf 9 cells transfected with BmNPV DNA After liposome-mediated transfection of Sf9 cells with BmNPV SC7 DNA, cells were observed
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regularly and the supernatant was harvested at different time post infection. No occlusion bodies and no cytopathic effects were detected in the BmNPV SC7-infected Sf9 cells at any time postinfection. Moreover, no significant differences in cell growth curves between BmNPV SC7-infected and mock-infected Sf9 cells were detectable (data not shown). However, the infectious titre of the Sf9 supernatant harvested at 0, 72, 120, and 168 h p.i., assayed on Bm5 cells (Reed and Muench, 1938) increased with time from 0 to 104 TCID50/ ml (Fig. 1).
3.2. BmNPV DNA replication After infection of Sf9 cells with BmNPV SC7infected Bm5 cells supernatant at m.o.i. of 10, we examined the amount of BmNPV DNA present in Sf9 cells at various time postinfection BmNPV DNA replication in Sf9 cells was observed by dot blot hybridization (Fig. 2). Detectable levels of BmNPV DNA were present from 24 h p.i. and increased significantly up to 96 h p.i.
3.3. Detection of 6iral antigens Contrary to AcMNPV-infected Sf9 cells or to BmNPV-infected Bm5 cells, where host protein
Fig. 1. Titration curve of extracellular BmNPV virus present after transfection of Sf9 cells with BmNPV DNA. The titre was determined on Bm5 cells by end-point dilution in a 60-well plate.
Fig. 2. Kinetics of BmNPV DNA synthesis in BmNPV-infected Sf9 cells. Total cell DNA was isolated from BmNPV-infected Sf9 cells at the indicated time postinfection. DNA was blotted and hybridized with labeled BmNPV DNA. Upper line from left to right: uninfected cell, BmNPV-infectedSf9 cell at respectively 0, 12, 24, 48, and 96 h p.i. Lower line BmNPV genomic DNA used as standard at 10, 1, 10 − 1, 10 − 2, 10 − 3, 10 − 4 ng.
synthesis was progressively inhibited, in BmNPVinfected Sf9 cells the pattern of 35S-labeled proteins did not vary from 0 to 96 h. No detectable change, and particularly no shutoff of cellular protein synthesis, was observed. The protein pattern resembled that of mock infected Sf9 cells (data not shown). However, using a rabbit polyclonal immune serum directed against BmNPV antigens, it was possible to detect the appearance of viral proteins. Compared to infected cells at 0 h p.i., which showed no immunofluorescence signal (data not shown), viral antigens appeared at 24 h p.i. and 5% of cells showed an immunofluorescence signal (Fig. 3A). This percentage remained stable up to 5 days p.i. (data not shown). In mock infected cells no immunofluorescence signal was detected (Fig. 3C), and in AcMNPV-infected Sf9 cells more than 90% of the cells were detected by the antiserum (Fig. 3B). Using the same immune serum, it was not possible to detect a signal in the immunoblot experiment. However, using a polyvalent immune serum directed against alkali-solubilized occlusion bodies, it was possible to detect at 96 h p.i. a specific signal from infected cells (data not shown). BmNPV-infected Sf9 cells were also analysed by western blotting with antisera to AcMNPV VP39 and to AcMNPV GP64. Both antisera cross-reacted with BmNPV antigen, since VP 39 and GP64 were revealed in BmNPV infected Bm5 cells (data not shown). Moreover, a
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Fig. 3. Immunofluorescence microscopy of Sf9 cells infected with BmNPV and AcMNPV at 24 h p.i. (A) Cells infected with BmNPV. (B) Cells infected with AcMNPV. (C) Mock-infected cells. Immune serum directed against BmNPV virus particle proteins. Bar =100 mm.
comparison of AcMNPV and BmNPV gp64 sequences shows that the AcMNPV epitope recognized by the AcV5 antibody was identical to the BmNPV epitope. In BmNPV-infected Sf9 cells neither the early VP39 protein nor the early and late GP64 protein were detected by ECL chemiluminescence, suggesting that no significant accumulation of these early proteins occurs during the cross-infection.
4. Discussion Infection or transfection of Sf9 cells by BmNPV SC7 genomic DNA does not produce any cytopathic effect and no plaques could be obtained in test assays, leading to the current idea of Sf9 being nonpermissive for BmNPV. However, a low production of infectious BmNPV SC7 virus particles was observed in the present work by titration of the supernatant of BmNPV-transfected Sf9 cells on permissive Bm5 cells. In contrast to previous unsuccessful attempts to detect BmNPV replication in S. frugiperda cells following classical infection (Kondo and Maeda, 1991), the transfection approach revealed a low level of replication of BmNPV in Sf9 cells. Transfection eliminated the background of input virus present in classical infection. The infection curve obtained by transfection progresses from zero at 0 h p.i. to reach 104 TCID50/ml 4 days later. Whether
the effects of transfection and infection on BmNPV replication in Sf9 are different is unknown. However, examination of titration curves of BmNPV-infected S. frugiperda cells shows a positive slope consistent with limited replication (Kondo and Maeda, 1991; our unpublished data). Transfection may be used to monitor the permissivity of S. frugiperda cells, as virus entry into the cell is not in many cases a host range-restrictive step (Carbonell and Miller, 1987; Morris and Miller, 1992) and blockage of replication must occur after the entry of virions into the cell (Boyce and Bucher, 1996). The kinetics of BmNPV DNA replication in Sf9 cells observed by dot blot indicated that viral DNA synthesis takes place, albeit at a much lower level than in cells infected with AcMNPV. This result indicates that all genes required for viral DNA replication in Sf9, as determined by Kool et al. (1994), are present in the BmNPV genome. It is likely that BmNPV replication genes are homologous with those of AcMNPV, since AcMNPV and BmNPV are very closely related (Zanotto et al., 1993; Barrett et al., 1995). Moreover, the limited production of infectious virus particles and the fact that protein patterns do not change during infection of Sf9 cells by BmNPV suggest that early BmNPV gene(s) are not well adapted to the Sf9 cell environment. However, in immunofluorescence experiments the presence of any detectable virion antigen is limited to 5% of
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the cells infected by BmNPV. This suggests that infection of Sf9 cells with BmNPV results in a small number of cells producing reasonably high levels of virus, rather than most of cells producing very low levels. The viral particles budding from these infected cells appear unable to infect neighboring cells as observed with cells infected with gp64 − virus by Monsma et al. (1996). This result is distinct from the infection of Lymantria dispar cells with AcMNPV, where a shutoff of cellular and viral protein synthesis occurs in the absence of virion production (Guzo et al., 1992). Here, a symptomless basic infection occurred in the absence of detectable cellular protein synthesis inhibition. It is possible that the inability to shut down host cell protein synthesis could explain the low production of virions. The occurrence of DNA replication and the appearance of an increasing infectious titre in the supernatant of BmNPV-transfected Sf9 are consistent with Sf9 cells being semipermissive for BmNPV replication. The failure to detect early proteins such as GP64 or VP39 on Western blots, using polyclonal or monoclonal antibodies directed against AcMNPV proteins, indicates that no notable accumulation of these proteins occurred. Both antibodies detected heterologous BmNPV antigens VP39 and GP64 in BmNPV-infected Bm5 cells used as controls. The limit of detection of the western technique was too high to reveal infection levels which are three to four orders of magnitude lower than in classical productive infection. The low replication rate of BmNPV in Sf9 cells might explain why Sf9 has so far been considered a nonpermissive cell line for BmNPV replication. Whether the interaction described here for BmNPV SC7 and Sf9 cells can be extended to other BmNPV genotypes remains to be determined. Further analyses are in progress to uncover, at the gene level, at which step(s) of the virus cycle productive replication of BmNPV in Sf9 is impeded. Initial investigations are aimed at explaining the failure of progeny BmNPV virus particles to cause secondary infections in Sf9 cells.
Acknowledgements We are grateful to Ian Smith (Central Science Laboratory, UK) for valuable help in preparing the manuscript. This work was supported in part by EEC contract Bio2-CT94-3069.
References Barrett, J.W., Krell, P.J. and Arif, B.M. (1995) Characterization, sequencing and phylogeny of the ecdysteroid UDPglucosyltransferase gene from two distinct nuclear polyhedrosis viruses isolated from Choristoneura fumiferana. J. Gen. Virol. 76, 2447 – 2456. Bonning, B.C. and Hammock, B.D. (1996) Development of recombinant baculoviruses for insect control. Annu. Rev. Entomol. 41, 191 – 210. Boyce, F.M. and Bucher, N.L.R. (1996) Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl. Acad. Sci. USA 93, 2348 – 2352. Carbonell, L.F. and Miller, L.K. (1987) Baculovirus interaction with nontarget organism: a virus-borne reporter is not expressed in two mammalian cell lines. Appl. Environ. Microbiol. 53, 1412 – 1417. Carter, J. (1984) Viruses as pest-control agents. Biotechnol. Genet. Eng. Biol. 1, 375 – 419. Croizier, G. and Quiot, J.M. (1981) Obtention and analysis of two genetic recombinants of baculoviruses of Lepidoptera, Autographa californica SPEYER and Galleria mellonella L. Ann. Virol. (Inst. Pasteur), 132 E, 3 – 18. Croizier, G., Croizier, L., Argaud, O. and Poudevigne, D. (1994) Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene. Proc. Natl. Acad. Sci. USA 91, 48 – 52. Grace, T.D.C. (1967) Establishment of a line of cells from silkworm Bombyx mori. Nature 216, 613. Gro¨ner, A. (1986) Specificity and safety of baculoviruses. In: R.R. Granados and B.A. Federici (Eds.) The Biology of Baculoviruses. Biological Properties and Molecular Biology Vol. 1. CRC Press, Boca Raton Florida, pp. 177 – 202. Guzo, D., Rathburn, H., Guthrie, K. and Dougherty, E. (1992) Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines. J. Virol. 66, 2966 – 2972. Kamita, G.S. and Maeda, S. (1993) Inhibition of Bombyx mori nuclear polyhedrosis virus (NPV) replication by the putative DNA helicase gene of Autographa californica NPV. J. Virol. 67, 6239 – 6245. Kondo, A. and Maeda, S. (1991) Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus. J. Virol. 65, 3625 – 3632.
O. Martin, G. Croizier / Virus Research 47 (1997) 179–185 Kool, M., Ahrens, C.H., Goldbach, R.W., Rohrmann, G.F. and Vlak, J.M. (1994) Identification of genes involved in DNA replication of the Autographa californica baculovirus. Proc. Natl. Acad. Sci. USA 91, 11212–11216. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Maeda, S., Kamita, S.G. and Kondo, A. (1993) Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-Kilobase-pair DNA fragment originating from Bombyx mori NPV. J. Virol. 67, 6239 –6245. Monsma, S.A. and Blissard, G.W. (1995) Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. J. Virol. 69, 2583 – 2595. Monsma, S.A., Oomens, A.G.P. and Blissard, G.W. (1996) The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J. Virol. 70, 4607 –4616. Morris, T.D. and Miller, L.K. (1992) Promoter influence on
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baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines. J. Virol. 66, 7397 – 7405. Murphy, F.A., Fauquet, C.M., Bishop, D.H.L., Ghabrial, S.A., Jarvis, A.W., Martelli, G.P., Mayo, M.A. and Summers, M.D. (1995) Virus Taxonomy. Springer Verlag, Vienna. O’Reilly, D.R., Miller, L.K. and Luckow, V.A. (1992) Baculovirus Expression Vectors: A Laboratory Manual. W.H. Freeman, New York. O’Reilly, D.R. (1995) Baculovirus-encoded ecdysteroid UDPglucosyltransferases. Insect. Biochem. Molec. Biol. 25, 541 – 550. Reed, L.J. and Muench, H. (1938) A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493 – 494. Vaughn, J.L., Goodwin, R.H., Tompkins, G.L. and McCawley, P. (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro Cell. Dev. Biol. 13, 213 – 217. Zanotto, P.M.A., Kessing, B.D. and Maruniak, J.E. (1993) Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J. Invertebr. Pathol. 62, 147 – 164.
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Infection of a Spodoptera frugiperda cell line with Bombyx mori nucleopolyhedrovirus O. Martin, G. Croizier* Unite´ de Ge´ne´tique des Virus, Station de Recherches de Pathologie Compare´e, INRA-URA CNRS 2209, Saint Christol-Le`s-Ale`s, France Received 30 April 1996; accepted 30 October 1996
Abstract The interactions of Bombyx mori nucleopolyhedrovirus (BmNPV) with Spodoptera frugiperda cells (Sf9) were investigated. S. frugiperda cells are usually considered nonpermissive for BmNPV. However, in the present study, BmNPV DNA replication was observed and an increasing infectious titre, reaching 104 TCID50/ml on B. mori permissive cells by 6 days post-transfection, developed in the supernatant of infected Sf9 cells. Infection of Sf9 cells by BmNPV did not induce a discernible shutoff of cellular protein synthesis and no overt cytopathic effects were observed. These data indicate that the low permissivity of Sf9 cells for BmNPV replication is associated with an inapparent infection. © 1997 Elsevier Science B.V. Keywords: Baculovirus; Bombyx mori; Cellular permissivity; Nucleopolyhedrovirus; Sf9 cells
1. Introduction Baculoviruses are characterized by enveloped rod-shaped virions containing circular doublestranded DNA (Murphy et al., 1995). They are infectious to insects and in particular to a variety of economically important lepidopteran species (Gro¨ner, 1986) which gives them a potential role as insecticides (Carter, 1984). Two different * Corresponding author. Tel.: +33 0466783714; fax: + 33 0466524699; e-mail: [email protected]
approaches have been applied to overcome the problem of slow speed of kill characteristic of wild-type baculovirus insecticides. Insertion of non-baculovirus genes such as insect-selective toxins into the baculovirus genome, or deletion of the viral gene encoding ecdysteroid UDP-glucosyltransferase, has yielded improved biopesticides (Bonning and Hammock, 1996; O’Reilly, 1995). On the other hand, combining genome elements from viruses possessing different host ranges offers a method to produce recombinant viruses with extended host range (Kondo and Maeda,
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1991). A greater understanding at the molecular level of viral specificity and virulence, by analysis of the genes which determine baculovirus replication in given hosts, is another important component of strategies to improve their efficacy as biopesticides. Comparisons of Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV) replication in cells possessing different degrees of permissivity offer an attractive model to investigate the genetic determinants of specificity (Maeda et al., 1993). Spodoptera frugiperda cells are considered nonpermissive for BmNPV and permissive for AcMNPV, while B. mori cells are considered nonpermissive for AcMNPV and permissive for BmNPV (Kondo and Maeda, 1991; Croizier et al., 1994). Recently it was shown that the blockage of AcMNPV replication in Bombyx mori cells (Bm5) could be overcome by homologous recombination between AcMNPV genomic DNA and a 133 base pair DNA fragment of the BmNPV helicase gene (Croizier et al., 1994). AcMNPV replication in Bm5 cells could thus be achieved by a minor modification of its DNA helicase gene, which has been shown to be an essential gene for DNA replication (Kool et al., 1994). Further analyses of the determinant(s) of AcMNPV and BmNPV specificity might include the identification of AcMNPV DNA fragments capable of extending the host range of BmNPV. Previous results showed that coinfection of B. mori (BmN) cells with AcMNPV and BmNPV resulted in the inhibition of BmNPV replication (Kamita and Maeda, 1993). The deleterious effect of AcMNPV helicase during the coinfection (Kamita and Maeda, 1993) makes it difficult to obtain recombinant viruses directly in B. mori cells. Since coinfection of Sf21 cells with AcMNPV and BmNPV virus particles induces BmNPV replication and recombinant formation (Kondo and Maeda, 1991), recombinants are expected to arise from cotransfection with a mixture of AcMNPV and BmNPV DNAs. Analysis of the behaviour of BmNPV in S. frugiperda cells is a prerequisite to studying the formation of AcMNPV-BmNPV recombinants in S. frugiperda cells by rescue of BmNPV with AcMNPV DNA sequences.
In this study, we report the results of BmNPV infection of Sf9 cells. Analyses of viral DNA replication, appearance of viral proteins by immunofluorescence analysis, and extracellular virus production are described.
2. Materials and methods
2.1. Cells and 6iruses S. frugiperda (fall armyworm) IPLB SF 21-AE (Vaughn et al., 1977) clone 9 (Sf9) and B. mori (silkworm) (Grace, 1967) clone 5 (Bm5) were maintained at 27°C in TC100 medium supplemented with 10% fetal bovine serum (FBS) (heat inactivated at 56° for 30 min). AcMNPV strain 1.2 (Croizier and Quiot, 1981) and BmNPV strain SC7 (Croizier et al., 1994) were used in this study.
2.2. Antisera A rabbit received intramuscular injection of 2–3 mg of purified BmNPV virions (after thioglycolate digestion of occlusion bodies and sucrose gradient centrifugation) mixed with Freund’s adjuvant. One booster injection was delivered 3 weeks later. The rabbit was bled after a further 3 weeks. This immune serum was used for immunofluorescence microscopy. Dr L.K. Miller and colleagues kindly provided rabbit polyclonal antisera against the AcMNPV major capsid protein VP39. Dr G.W. Blissard’s laboratory kindly provided monoclonal antibody AcV5 that recognizes the denatured form of AcMNPV GP64 envelope fusion protein (Monsma and Blissard, 1995).
2.3. DNA purification AcMNPV and BmNPV were multiplied in Galleria mellonella and B. mori larvae, respectively. Genomic DNAs used for transfection were extracted from occlusion bodies obtained from dead larvae by standard methods (O’Reilly et al., 1992). Briefly, occlusion bodies were incubated in 0.1 M Na2CO3 for 10 min and the alkaline suspension
O. Martin, G. Croizier / Virus Research 47 (1997) 179–185
was clarified at 2000× g for 5 min. Virions were pelleted at 45 000 × g for 1 h in a SW 28 rotor (Beckman L7-55 Ultracentrifuge). DNA was phenol-extracted after digestion with proteinase K (500 mg/ml) in the presence of 1% SDS at 60°C for 30 min. DNA concentrations were estimated by UV spectrophotometry.
2.4. Lipofection Monolayers of Sf9 cells (5× 105 cells/35 mm dish) were transfected with DNA by liposome-mediated transfection using DOTAP (Boehringer, Germany): AcMNPV or BmNPV DNA (250 ng in 10 ml of TE buffer) was mixed with 8 ml of DOTAP and 600 ml of TC100 medium, and added to Sf9 cells. After 5 h, the medium was replaced with TC 100 supplemented with 10% FBS.
2.5. Dot blot analysis Monolayers of Sf9 cells (1.0 ×106 cells/35 mm dish) were infected with BmNPV at a multiplicity of infection (m.o.i.) of 20. At the designated time postinfection (p.i.) the cells were harvested, pelleted and washed once with 0.5 ml phosphatebuffered saline (PBS) pH 7.4. Extraction of total DNA from Sf9 cells was performed as described by O’Reilly et al. (1992). DNA was resuspended in 50 ml of TE buffer. 2 ml of the DNA solution was dot blotted onto a Hybond-N + membrane (Amersham, France). Nylon membrane was laid on a Whatman paper saturated with denaturation solution (1 M NaOH, 1.5 M NaCl). The membrane was air dried, and the DNA was UV-crosslinked to the membrane. Hybridization was carried out overnight at 68°C with a probe obtained by random priming of the PstI O fragment of BmNPV DNA cloned in pUC 19 (random priming DNA labeling kit, Boehringer, Germany).
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Laemmli sample buffer (Laemmli, 1970), followed by SDS-PAGE. After transfer to a nitrocellulose membrane (Trans-blot transfer medium, Bio-Rad, USA), the blots were blocked with non-fat dried milk (10% in a Tris/NaCl buffer 50 mM Tris HCl, 200 mM NaCl, pH7.4) and incubated with rabbit antiserum directed against VP39 protein diluted 1:2000 or monoclonal antibody directed against GP64 protein diluted 1:4000. They were then incubated with peroxidase conjugated anti-rabbit or anti-mouse IgG diluted 1:2000. Peroxidase activity was revealed using the ‘ECL kit’ (Amersham, France).
2.7. Immunofluorescence Sterile coverslips were deposited in tissue culture dishes and overlaid with 3 × 105 Sf9 cells in 2 ml medium. Cells were allowed to adhere for 1 h and were then infected at a m.o.i. of 10 with supernatant from BmNPV-infected Bm5 cells or supernatant from AcMNPV-infected Sf9 cells. After 3 h the supernatant was removed and replaced with fresh medium. At various time p.i. the cells were fixed in a 70% acetone, 30% methanol solution for 10 min at − 20°C. The cells were airdried and then overlaid with PBS for 15 min. Rabbit immune serum directed against BmNPV purified virions (after thioglycolate digestion of occlusion bodies and sucrose gradient centrifugation), diluted 1:100 in PBS, was added. After a 1 h incubation at 37°C, the coverslip was washed twice for 15 min in PBS and then incubated with fluorescein-labeled goat antibody directed against rabbit globulin (diluted 1:1000 in PBS) for 1 h. The cells were rinsed twice for 15 min in PBS, and the coverslips were placed face down on a drop of buffered glycerin on a microscope slide and examined on a Nikon epifluorescence microscope.
2.6. Protein analysis
3. Results
Sf9 cells were infected with supernatant from BmNPV-infected Bm5 and AcMNPV-infected Sf9. Bm5 cells were infected only with supernatant from BmNPV infected Bm5. Infected cells were harvested at 0, 6, 12, 24, 48, 96 h p.i. and lysed in
3.1. Increase of BmNPV titre in the supernatant of Sf 9 cells transfected with BmNPV DNA After liposome-mediated transfection of Sf9 cells with BmNPV SC7 DNA, cells were observed
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regularly and the supernatant was harvested at different time post infection. No occlusion bodies and no cytopathic effects were detected in the BmNPV SC7-infected Sf9 cells at any time postinfection. Moreover, no significant differences in cell growth curves between BmNPV SC7-infected and mock-infected Sf9 cells were detectable (data not shown). However, the infectious titre of the Sf9 supernatant harvested at 0, 72, 120, and 168 h p.i., assayed on Bm5 cells (Reed and Muench, 1938) increased with time from 0 to 104 TCID50/ ml (Fig. 1).
3.2. BmNPV DNA replication After infection of Sf9 cells with BmNPV SC7infected Bm5 cells supernatant at m.o.i. of 10, we examined the amount of BmNPV DNA present in Sf9 cells at various time postinfection BmNPV DNA replication in Sf9 cells was observed by dot blot hybridization (Fig. 2). Detectable levels of BmNPV DNA were present from 24 h p.i. and increased significantly up to 96 h p.i.
3.3. Detection of 6iral antigens Contrary to AcMNPV-infected Sf9 cells or to BmNPV-infected Bm5 cells, where host protein
Fig. 1. Titration curve of extracellular BmNPV virus present after transfection of Sf9 cells with BmNPV DNA. The titre was determined on Bm5 cells by end-point dilution in a 60-well plate.
Fig. 2. Kinetics of BmNPV DNA synthesis in BmNPV-infected Sf9 cells. Total cell DNA was isolated from BmNPV-infected Sf9 cells at the indicated time postinfection. DNA was blotted and hybridized with labeled BmNPV DNA. Upper line from left to right: uninfected cell, BmNPV-infectedSf9 cell at respectively 0, 12, 24, 48, and 96 h p.i. Lower line BmNPV genomic DNA used as standard at 10, 1, 10 − 1, 10 − 2, 10 − 3, 10 − 4 ng.
synthesis was progressively inhibited, in BmNPVinfected Sf9 cells the pattern of 35S-labeled proteins did not vary from 0 to 96 h. No detectable change, and particularly no shutoff of cellular protein synthesis, was observed. The protein pattern resembled that of mock infected Sf9 cells (data not shown). However, using a rabbit polyclonal immune serum directed against BmNPV antigens, it was possible to detect the appearance of viral proteins. Compared to infected cells at 0 h p.i., which showed no immunofluorescence signal (data not shown), viral antigens appeared at 24 h p.i. and 5% of cells showed an immunofluorescence signal (Fig. 3A). This percentage remained stable up to 5 days p.i. (data not shown). In mock infected cells no immunofluorescence signal was detected (Fig. 3C), and in AcMNPV-infected Sf9 cells more than 90% of the cells were detected by the antiserum (Fig. 3B). Using the same immune serum, it was not possible to detect a signal in the immunoblot experiment. However, using a polyvalent immune serum directed against alkali-solubilized occlusion bodies, it was possible to detect at 96 h p.i. a specific signal from infected cells (data not shown). BmNPV-infected Sf9 cells were also analysed by western blotting with antisera to AcMNPV VP39 and to AcMNPV GP64. Both antisera cross-reacted with BmNPV antigen, since VP 39 and GP64 were revealed in BmNPV infected Bm5 cells (data not shown). Moreover, a
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Fig. 3. Immunofluorescence microscopy of Sf9 cells infected with BmNPV and AcMNPV at 24 h p.i. (A) Cells infected with BmNPV. (B) Cells infected with AcMNPV. (C) Mock-infected cells. Immune serum directed against BmNPV virus particle proteins. Bar =100 mm.
comparison of AcMNPV and BmNPV gp64 sequences shows that the AcMNPV epitope recognized by the AcV5 antibody was identical to the BmNPV epitope. In BmNPV-infected Sf9 cells neither the early VP39 protein nor the early and late GP64 protein were detected by ECL chemiluminescence, suggesting that no significant accumulation of these early proteins occurs during the cross-infection.
4. Discussion Infection or transfection of Sf9 cells by BmNPV SC7 genomic DNA does not produce any cytopathic effect and no plaques could be obtained in test assays, leading to the current idea of Sf9 being nonpermissive for BmNPV. However, a low production of infectious BmNPV SC7 virus particles was observed in the present work by titration of the supernatant of BmNPV-transfected Sf9 cells on permissive Bm5 cells. In contrast to previous unsuccessful attempts to detect BmNPV replication in S. frugiperda cells following classical infection (Kondo and Maeda, 1991), the transfection approach revealed a low level of replication of BmNPV in Sf9 cells. Transfection eliminated the background of input virus present in classical infection. The infection curve obtained by transfection progresses from zero at 0 h p.i. to reach 104 TCID50/ml 4 days later. Whether
the effects of transfection and infection on BmNPV replication in Sf9 are different is unknown. However, examination of titration curves of BmNPV-infected S. frugiperda cells shows a positive slope consistent with limited replication (Kondo and Maeda, 1991; our unpublished data). Transfection may be used to monitor the permissivity of S. frugiperda cells, as virus entry into the cell is not in many cases a host range-restrictive step (Carbonell and Miller, 1987; Morris and Miller, 1992) and blockage of replication must occur after the entry of virions into the cell (Boyce and Bucher, 1996). The kinetics of BmNPV DNA replication in Sf9 cells observed by dot blot indicated that viral DNA synthesis takes place, albeit at a much lower level than in cells infected with AcMNPV. This result indicates that all genes required for viral DNA replication in Sf9, as determined by Kool et al. (1994), are present in the BmNPV genome. It is likely that BmNPV replication genes are homologous with those of AcMNPV, since AcMNPV and BmNPV are very closely related (Zanotto et al., 1993; Barrett et al., 1995). Moreover, the limited production of infectious virus particles and the fact that protein patterns do not change during infection of Sf9 cells by BmNPV suggest that early BmNPV gene(s) are not well adapted to the Sf9 cell environment. However, in immunofluorescence experiments the presence of any detectable virion antigen is limited to 5% of
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the cells infected by BmNPV. This suggests that infection of Sf9 cells with BmNPV results in a small number of cells producing reasonably high levels of virus, rather than most of cells producing very low levels. The viral particles budding from these infected cells appear unable to infect neighboring cells as observed with cells infected with gp64 − virus by Monsma et al. (1996). This result is distinct from the infection of Lymantria dispar cells with AcMNPV, where a shutoff of cellular and viral protein synthesis occurs in the absence of virion production (Guzo et al., 1992). Here, a symptomless basic infection occurred in the absence of detectable cellular protein synthesis inhibition. It is possible that the inability to shut down host cell protein synthesis could explain the low production of virions. The occurrence of DNA replication and the appearance of an increasing infectious titre in the supernatant of BmNPV-transfected Sf9 are consistent with Sf9 cells being semipermissive for BmNPV replication. The failure to detect early proteins such as GP64 or VP39 on Western blots, using polyclonal or monoclonal antibodies directed against AcMNPV proteins, indicates that no notable accumulation of these proteins occurred. Both antibodies detected heterologous BmNPV antigens VP39 and GP64 in BmNPV-infected Bm5 cells used as controls. The limit of detection of the western technique was too high to reveal infection levels which are three to four orders of magnitude lower than in classical productive infection. The low replication rate of BmNPV in Sf9 cells might explain why Sf9 has so far been considered a nonpermissive cell line for BmNPV replication. Whether the interaction described here for BmNPV SC7 and Sf9 cells can be extended to other BmNPV genotypes remains to be determined. Further analyses are in progress to uncover, at the gene level, at which step(s) of the virus cycle productive replication of BmNPV in Sf9 is impeded. Initial investigations are aimed at explaining the failure of progeny BmNPV virus particles to cause secondary infections in Sf9 cells.
Acknowledgements We are grateful to Ian Smith (Central Science Laboratory, UK) for valuable help in preparing the manuscript. This work was supported in part by EEC contract Bio2-CT94-3069.
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