Expression of the Autographa californica Nuclear Polyhedrosis Virus ...

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Virus Apoptotic Suppressor Gene p35 in Nonpermissive. Spodoptera littoralis ... tated the isolation of genetically engineered AcMNPVs with novel insecticidal ...
JOURNAL OF VIROLOGY, Oct. 1997, p. 7593–7599 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 71, No. 10

Expression of the Autographa californica Nuclear Polyhedrosis Virus Apoptotic Suppressor Gene p35 in Nonpermissive Spodoptera littoralis Cells† EDUARD GERSHBURG,1,2 HADASSAH RIVKIN,1

AND

NOR CHEJANOVSKY1*

Entomology Department, Institute for Plant Protection, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250,1 and Department of Botany, Tel Aviv University, Tel Aviv 69978,2 Israel Received 13 March 1997/Accepted 8 July 1997

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519–525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transienttransfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system. A recombinant AcMNPV bearing a small fragment of the helicase gene of Bombyx mori nuclear polyhedrosisvirus (NPV) within AcMNPV’s p143 coding region (the helicase gene of AcMNPV) replicated in both permissive S. frugiperda SF21 and nonpermissive B. mori BmN4 cell lines (14, 28). (iii) The hrf-1 gene of the Lymantria dispar NPV was shown to allow the multiplication of AcMNPV in nonpermissive Ld652Y cells (16, 37). (iv) The hcf-1 gene of AcMNPV was necessary for successful infection of TN368 cells and to some extent for improvement of the infectivity of the virus in Trichoplusia ni larvae but was not necessary for replication or infectivity in S. frugiperda cells and larvae, respectively (27). In addition, 18 baculovirus genes (lef genes) were required for expression of a late baculovirus promoter in SF21 cells (26, 38); three of them, ie-2, lef-7, and p35, were not required for expression in TN368 cells, suggesting that they could be involved in determining host range. AcMNPV does not infect Spodoptera littoralis (the Egyptian cotton worm), an important Mediterranean pest. Recently, we reported that infection of S. littoralis SL2 cells with wild-type AcMNPV results in apoptosis and concomitantly low yields of viral progeny (9). Since AcMNPV mutants with alterations in the p35 gene induce apoptosis of the permissive cell line of S. frugiperda, SF21, we hypothesized that p35 either is expressed poorly or is not functional, or both, in AcMNPV-infected SL2 cells (9). p35 has been shown to suppress apoptosis in various heterologous systems (18, 20, 29, 33, 35) which are evolutionarily more distant from SF9 cells than the SL2 system (which is closely related to the S. frugiperda lepidopterous cells). This led us to hypothesize that p35 may be functional but not sufficiently expressed in AcMNPV-infected SL2 cells. Thus, augmenting the expression of p35 in SL2 cells could allow us to determine its functionality in terms of suppression of apoptosis. In the present work, we show that by overexpressing p35 we were indeed able to reduce apoptosis of SL2 cells induced by either actinomycin D or AcMNPV. However, increasing p35

The Autographa californica nuclear polyhedrosis virus (AcMNPV) is considered the prototype of subgroup A of the Baculoviridae family of viruses which infect invertebrates and primarily insects. The complete genome of AcMNPV has been sequenced (1), and its replication in permissive cells has been extensively studied (for reviews, see references 4 and 23). The ability of AcMNPV to infect a wide number of hosts, 39 species of lepidopteran larvae belonging to 13 families (5, 17), and the availability of appropriate tissue culture systems have facilitated the isolation of genetically engineered AcMNPVs with novel insecticidal properties. Some of these are being evaluated for agricultural application (reviewed in references 6 and 30). The mechanisms that control the ability of baculoviruses to infect specific insect hosts are not clear. Their elucidation is important for better assessment of potential risks associated with the utilization of recombinant-baculovirus insecticides. Moreover, this knowledge could help expand the range of applications of specific AcMNPV recombinants to target economically important lepidopteran pests. Four baculovirus genes have been implicated in facilitating AcMNPV replication in a species-specific manner. (i) p35, characterized as an antiapoptotic gene, inhibitor of cysteine proteases of the CED-3/ICE family, caspases (2, 7, 24, 39), is required for efficient AcMNPV infection of Spodoptera frugiperda cells and larvae. AcMNPV p35 gene null mutants yielded low titers of budded virus (BV) (12, 21). Moreover, the absence of p35 function was complemented by apoptosis inhibitor genes (iap genes) derived from other baculoviruses (3, 15). (ii)

* Corresponding author. Mailing address: Entomology Department, The Institute for Plant Protection, The Volcani Center, POB 6, Bet Dagan 50-250, Israel. Phone: (972)-3-968-3694. Fax: (972)-3-960-4180. E-mail: [email protected]. † Contribution 2093 from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 7593

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FIG. 1. P35 synthesis in AcMNPV-infected S. littoralis and S. frugiperda cells. Extracts from SL2 (A) and SF9 (B) cells (4 3 105) infected with AcMNPV were harvested at various times p.i. (indicated below the lanes) and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using a-P35NF antiserum.

expression only slightly improved the yields of AcMNPV BV in infected SL2 cells and did not improve the 50% lethal doses (LD50s) of AcMNPV in S. littoralis fourth-instar larvae.

viously (10). Control larvae were injected with the same volume of TNM-FH complete medium. Mortality of larvae was recorded daily.

RESULTS MATERIALS AND METHODS Cell lines and viruses. S. littoralis SL2 and S. frugiperda SF9 cells were maintained and propagated in TNM-FH medium supplemented with 10% heatinactivated fetal bovine serum (36). Infection of the cells with wild-type (wt) AcMNPV strain E2 (34), 50% tissue culture infectious dose assays, and plaque titration of virus stocks were done as described previously (31). Plasmids and transfections. Plasmids pBB/BSst and pHSP35VI1 contain the p35 coding region under the control of the p35 and Drosophila melanogaster hsp70 promoters, respectively (13, 21). pHSP35VI1 also possesses an intact polyhedrin gene located back-to-back to the hsp70-p35 transcription unit and additional viral sequences which allow the transfer of the p35 and polyhedrin genes to the polyhedrin locus of AcMNPV by homologous recombination (13). pIE1 contains an intact ie-1 gene (19). DNA transfections were performed by a modified calcium phosphate method as previously described (10). Construction of the recombinant vHSP-P35. vHSP-P35 was constructed by cotransfection of SF9 cells with plasmid pHSP35VI1 and linearized polyhedrinnegative AcMNPV DNA by the calcium phosphate method. Polyhedrin-positive recombinant viruses were isolated by three rounds of plaque purification (10) and verified by restriction enzyme and PCR analyses of the viral DNA. Apoptosis assays. (i) DNA fragmentation. DNA oligonucleosomes were extracted from the virus-infected SL2 cells by using a 10 mM Tris (pH 8.0)–1 mM EDTA–1% sodium dodecyl sulfate (SDS) buffer containing 70 mg of proteinase K per ml (2 h at 37°C) followed by the addition of NaCl (final concentration, 1 M). The extracts were treated with phenol-chloroform and ethanol precipitated, and resuspended DNA was analyzed by agarose gel electrophoresis as described previously (9). Densitometric scanning of the gels was performed basically as described below for Western blot scanning. (ii) Actinomycin D-induced apoptosis. SL2 cells (105) were transfected with 10 mg of pHSP35VI1 or pBluescript. Twelve hours posttransfection, 10 mM bromodeoxyuridine (BrdU) was added and the mixture was incubated for 12 h to label the cellular DNA. The cells were heat shocked at 24 h posttransfection (30 min, 42°C, in a water bath), and 4 h later, 250 ng of actinomycin D per ml was added to induce apoptosis. The time course of BrdU-labeled oligonucleosome release to the cell medium was monitored by incubating aliquots of the cell supernatants (in triplicate), taken at various times, in an enzyme-linked immunosorbent assay (ELISA) plate coated with anti-DNA antibody. Bound BrdUlabeled oligonucleosomal DNA was detected by using anti-BrdU monoclonal antibody conjugated to peroxidase in an ELISA according to the recommendation of the manufacturer (Boehringer, Mannheim, Germany). Western blot analysis. AcMNPV-infected or plasmid-transfected cells were washed in phosphate saline buffer, harvested in 150 mM Tris (pH 8.0)–1 mM EDTA–1% aprotinin, and subjected to three cycles of freezing and thawing. After clearing by centrifugation (14,000 3 g at 4°C), 1.5 3 106 cell equivalents were subjected to SDS-polyacrylamide gel electrophoresis (25), and the polypeptides were transferred to a nitrocellulose membrane. Immunodetection of P35 was performed by using a-P35NF antiserum (22) as previously described (9). Relative levels of expressed P35 were determined by computerized densitometry scanning with a Bio-Image System 202D Apparatus (Rhenium, Jerusalem, Israel). Bioassays. Various doses of BVs whose titers in SF9 cells had been previously determined were injected into fourth-instar S. littoralis larvae as described pre-

P35 synthesis in AcMNPV-infected SL2 cells. To compare the steady-state levels of P35 synthesized during the infectious cycle, both nonpermissive SL2 and permissive SF9 cells were infected with AcMNPV. At various times postinfection (p.i.), cell extracts were subjected to immunoblot analysis using aP35NF antiserum (Fig. 1). P35 was detected in virus-infected SF9 cells at 4 h p.i. and reached its maximal expression at about 18 to 34 h p.i. (Fig. 1B). In SL2 cells, very small amounts of P35 were observed at 4 h p.i.; maximal steady-state levels were detected at 18 h p.i. (Fig. 1A). However, P35 steady-state levels in SL2 cells dropped from 18 to 34 h p.i., probably due to cell death (compare Fig. 1A and B). Overall, P35 levels in those cells were extremely low compared to those in infected SF9 cells. These results suggested that a lack of sufficient amounts of P35 allows the progression of apoptosis initiated by AcMNPV infection of SL2 cells (9). However, this hypothesis did not rule out the possibility of p35’s nonfunctionality in SL2 cells. Transient expression of p35 in SL2 cells. We assumed that increasing the amount of P35 was a prerequisite to addressing the question of whether p35 is functional in SL2 cells. Previous studies have shown that high P35 levels are obtained in SF21 cells by expressing p35 under the control of the hsp70 heat shock promoter from Drosophila (13). We compared the levels of P35 obtained during transfection of two plasmids, pBB/BSst and pHSP35VI1, in which p35 was placed under the control of its own promoter or the hsp70 promoter, respectively (see Materials and Methods). A third plasmid, pIE1 (19), bearing the intact ie-1 gene coding for IE1, a transactivator of p35, was cotransfected with pBB/BSst. Immunoblot analysis detected higher levels of P35 in the pHSP35VI1-transfected cells than in cells transfected with pBB/BSst plus pIE1, and the levels were undetectable in pBB/BSst-transfected cells (Fig. 2A). On the basis of these results, we selected the pHSP35VI1 construct for our expression studies (see below). p35-mediated suppression of apoptosis. Actinomycin D treatment has been shown to induce apoptosis of lepidopteran SF21 cells, which was suppressed by p35 expression (8, 13). SL2 cells undergo apoptosis upon incubation with actinomycin D at concentrations above 100 ng/ml. The ability of p35 to block apoptosis in actinomycin D-

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FIG. 2. Transient expression of p35 in transfected S. littoralis cells. (A) SL2 cells (1.5 3 106) were transfected with 10 mg of pHSP35VI1 (lane 1), 10 mg of pBB/BSst (lane 2), or 10 mg of pBB/BSst and 10 mg of pIE1 (lane 3). P35 synthesis was monitored by immunoblotting the cell extracts as described for Fig. 1. (B) Relative levels of P35 protein detected in lanes 1 (pHSP-P35 5 pHSP35VI1) and 3 (pP351pIE1 5 pBB/BSst plus pEI1) of panel A (10 mg of pBluescript was added to the single-plasmid transfections to obtain a total of 20 mg of DNA).

treated SL2 cells was studied by transfecting them with pHSP35VI1 and further incubating them with 250 ng of actinomycin D per ml. The time course of the apoptosis provoked by the addition of actinomycin D was monitored by using a semiquantitative ELISA-based assay which measures the amount of oligonucleosomal DNA released by the apoptotic cells (see Materials and Methods). A decrease of about 50% in the release of oligonucleosomal DNA was observed in SL2 cells transfected with pHSP35VI1 relative to that in control pBluescript-transfected cells (Fig. 3A). Untreated cells (no addition of actinomycin D) did not release oligonucleosomal DNA, and their optical densities were below 0.05. As expected, P35 was detected in the pHSP35VI1-transfected cells and was not detected in the pBluescript-transfected cells (Fig. 3B, lanes 2 to 5 and 6, respectively). pBB/BSst-transfected cells released amounts of oligonucleosomes equivalent to those released by pBluescript-transfected cells, and P35 was barely detectable (data not shown and Fig. 2A). A total of 60% of pHSP35VI1-transfected SL2 cells remained viable after exposure to actinomycin D, in contrast to 18% of pBluescript (or pBB/BSst)-transfected cells (Fig. 4 and data not shown), again indicating that high-level expression of p35 conferred resistance to apoptosis. p35 overexpression during viral infection. The experiments described above indicated that p35 expression in SL2 cells (i) could be elevated by placing its transcription under the control of the hsp70 promoter and (ii) resulted in a functional product, since it correlated with acquired resistance to actinomycin Dinduced apoptosis. Thus, to study the ability of P35 to block AcMNPV-induced apoptosis, we constructed a recombinant virus (vHSP-P35) in which the hsp70 promoter-p35 transcription unit was inserted at the polyhedrin locus of the AcMNPV genome (Fig. 5). SL2 cells were infected with vHSP-P35 at multiplicities of infection (MOIs) of 1 and 10, and p35 expression was detected as described above. Higher steady-state levels of P35 were produced in cells infected with the recombinant than in cells infected with wt AcMNPV (Fig. 6, lanes 2, 4, and 5 and lanes 3 and 6, respectively). Moreover, heat shocking the cells after infection resulted in a dramatic increase in P35 synthesis in the recombinant-virus-infected cells (Fig. 6, lanes 4 and 5). Apoptosis of the SL2 cells infected by wt and recombinant vHSPP35 viruses (MOI of 10) is shown in Fig. 7. Electrophoresis of DNA extracted from AcMNPV-infected cells shows the typical oligonucleosomal ladder characteristic of apoptosis (Fig. 7A,

lane 1), corresponding to the extensive cell death observed (Fig. 7C). Partial suppression of apoptosis was consistently observed in vHSP-P35-infected cells, as evidenced by the reduced extent of DNA fragmentation (Fig. 7A, lane 2 and 7B).

FIG. 3. Suppression of actinomycin D-induced apoptosis by p35 expression. SL2 cells (105) transfected with 10 mg of pHSP35VI1 (pHSP-P35) or pBluescript (pBSK) were labeled with 10 mM BrdU and heat shocked 24 h later, and at 28 h posttransfection 250 ng of actinomycin D per ml was added to induce apoptosis. At the indicated times after actinomycin D addition, cell supernatants were collected and aliquoted, and the release of BrdU-labeled oligonucleosomes was monitored by using a monoclonal antibody and ELISA (see Materials and Methods). (A) P35 steady-state levels were monitored by immunoblotting (B) (lanes 1 to 6 [times are indicated below the lanes]; lane M, molecular weight markers). Relative levels of P35 at the various times indicated in panel B are also shown (C); total optical density units are expressed as percentages of the highest P35 peak. Generally, a variation of about 15% was observed in the detection of P35 by Western blotting, which was attributed to differences in transfection efficiencies.

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FIG. 4. Protection of SL2 cells from actinomycin D-induced apoptosis by p35 expression. SL2 cells (10 5) were transfected with 10 mg of pHSP35VI1 (pHSPP35) or pBluescript (pBSR). Heat shock was performed at 24 h posttransfection, and actinomycin D (250 ng/ml) was added at 28 h posttransfection. Cell viability was determined by trypan blue exclusion. The viability of heat-shocked plasmidtransfected cells (as described above) prior to addition of actinomycin D was considered 100%. The results shown are means of three replicates, with standard deviations (error bars).

We estimated a reduction of about 50% apoptosis in vHSPP35-versus wt-infected SL2 cells by densitometric scanning of the gel and, independently, by counting intact versus blebbing vHSP-P35- and wt-infected SL2 cells (Fig. 7). Inhibition of apoptosis and viral yields. Apoptosis has been implicated in the significant reduction of BV yields observed in SL2 cells relative to virus yields measured in SF9 cells (9). Since overexpression of p35 reduced the extent of virus-induced apoptosis of SL2 cells, we determined the amount of progeny BVs released through one cycle of replication by infecting the cells with wt (AcMNPV) and recombinant (vHSPP35) viruses at an MOI of 10 PFU per cell. As can be seen in Fig. 8, vHSP-P35 yields increased slightly compared to those of AcMNPV (vHSP-P35 titers of 5.17 3 104 6 4.33 3 103 and 10.1 3 104 6 8.4 3 103 and AcMNPV titers of 1.55 3 104 6 1.29 3 103 and 2.48 3 104 6 2.06 3 103 PFU/ml at 24 and 48 h p.i., respectively). Also, a slight increase in BV yields was noted with cells infected at a MOI of 1 (not shown). vHSP-P35 titers from permissive SF9 cells infected at 48 h p.i. (same MOI as above) were 1.01 3 107 6 3.2 3 106 and 2.00 3 107 6 1.1 3 106 PFU/ml, with or without the cells being heat shocked, respectively. AcMNPV titers under the same conditions were 3.05 3 107 6 2.40 3 106 and 6.10 3 107 6 3.50 3 106 PFU/ml, with or without the cells being heat shocked, respectively. Routine determination of BV released from S. littoralis NPV (SlNPV)-infected SL2 cells yielded titers of about 108 PFU/ml (9). In order to determine if p35 overexpression improved the ability of AcMNPV to replicate at the organism level, we determined the approximate LD50s of vHSP-P35 and wt AcMNPV for fourth-instar S. littoralis larvae. That was accomplished by injecting vHSP-P35 and AcMNPV BV doses ranging from 102 to 105 PFU per larva. As can be seen in Fig. 9, approximate LD50s of 103 PFU/larvae were determined for vHSP-P35 and wt Ac-

FIG. 5. Structure of recombinant virus vHSP-P35. The recombinant virus possesses an additional p35 gene inserted in the polyhedrin gene locus back-toback with the polyhedrin gene. p35 and polh viral gene are indicated (black boxes). The direction of transcription (arrows) of these genes under the control of the different promoters (open boxes) and the original p35 gene resident in the AcMNPV genome are also indicated.

FIG. 6. Expression of p35 by recombinant virus vHSP-P35. SL2 cells were infected with recombinant virus (vHSP-P35) or wt AcMNPV (lanes 1, 2, 4, and 5 and lanes 3 and 6, respectively) at an MOI of 1 or 10. Infected cells were heat shocked at 1 h after infection or were not heat shocked (1HS or 2HS, respectively). Cell extracts prepared at 24 h p.i. were immunoblotted as described in the legend to Fig. 1 and in Materials and Methods. Lane M, prestained markers with molecular masses of 98, 64, 50, 30 and 16 kDa (bovine serum albumin, glutamic dehydrogenase, alcohol dehydrogenase, myoglobin, and lysozyme, respectively).

MNPV, by graphic extrapolation. Thus, no significant differences in LD50s between recombinant and wt AcMNPVs in nonpermissive S. littoralis larvae were observed. DISCUSSION Virus-induced apoptosis fully develops in SL2 and is suppressed in SF21 (or SF9) cells infected with wt AcMNPV (7, 9, 11, 22). To determine whether AcMNPV’s failure to suppress apoptosis in infected SL2 cells is due to poor expression of the apoptotic suppressor gene p35, we compared the steady-state levels of P35 in SL2 and SF9 cells infected with AcMNPV. P35 levels were indeed much lower in infected SL2 cells than in their SF9 counterparts (Fig. 1A and B, respectively). Studies of P35’s mode of action have indicated that it binds stoichiometrically to CED-3/ICE-like death proteases of invertebrate and vertebrate origin, inhibiting their activity (2, 7, 24, 39). Thus, the above results suggest that apoptosis in infected SL2 cells could occur due to insufficient amounts of P35 available to bind to and inhibit a putative SL2 CED-3/ICE-related protease activated by AcMNPV. Alternatively, P35 levels in nonpermissive SL2 cells could be equivalent to those in permissive cells, but p35 could be nonfunctional, unable to suppress apoptosis, i.e., P35 could either not bind or bind with lower affinity to the SL2 caspase (2). A prerequisite to examination of these alternatives was overexpression of p35. Expression of p35 directed by the Drosophila hsp70 promoter resulted in remarkably higher steady-state levels of P35 in both plasmid-transfected and recombinant-virus-infected

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FIG. 7. Overexpression of p35 reduces apoptosis of SL2 cells. (A) Intracellular DNA fragmentation. DNA was extracted from SL2 cells infected with wt virus or vHSP-P35 virus (lanes 1 and 2, respectively). Lane M, l DNA digested with BstEII. (B and C) SL2 cells infected with vHSP-P35 and the wt, respectively. Light micrographs (magnification 3200) were prepared as previously described (9). Intact and blebbing cells were counted (250 to 350 cells per field, three replicates). The extent of apoptosis of vHSP-P35-infected SL2 cells relative to that of wt-infected cells was estimated.

SL2 cells (Fig. 2 and 6). p35 expression was further enhanced by heat shocking the cells (Fig. 6 and data not shown). Does p35 overexpression protect SL2 cells from apoptosis? Apoptosis of SL2 cells was induced by actinomycin D and AcMNPV infection. In the case of actinomycin D-treated cells, p35 expression correlated with inhibition of apoptosis, estimated directly by measuring the amount of oligonucleosomal

DNA released by the cells (Fig. 3), and as evidenced by higher cell viability (Fig. 4). Overexpression of p35 has been shown to augment viability of SF21 cells, protecting them from apoptosis induced by either actinomycin D or expression of the p32 form of the human ICE protease gene (7, 13). Recombinant vHSP-P35 infection also induced apoptosis of SL2 cells, albeit to a lower extent than wt AcMNPV (Fig. 7A).

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FIG. 8. Growth curve of recombinant (vHSP-P35) and wt AcMNPVs. BVs collected from SL2 cells infected vHSP-P35 (squares) and the wt (circles) at various times p.i. were titrated in SF9 cells by the 50% tissue culture infective dose method (see Materials and Methods). The results are the averages of three independent experiments.

The infected cells needed to be heat shocked in order to obtain significant p35 expression and exhibit reduced cellular DNA fragmentation, blebbing, and cell destruction (Fig. 6 and 7). Apoptosis of SF21 cells induced by a p35 null mutant virus, vAcAnh, was prevented by p35 expression, and this correlated with the ability of p35 to prevent actinomycin D-induced apoptosis of SF21 cells (13). Another study has shown that SF21 cells stably transformed to express p35 are resistant to apoptosis induced by actinomycin D (8). Taken together, these and our results lead to the conclusion that a functional P35, product of p35 expression, protects the SL2 cell from apoptotic death induced by actinomycin D or AcMNPV infection. Our inability to achieve 100% suppression of apoptosis by overexpressing p35 could be due to the lack of synchronization of the infected SL2 cells. Thus, different cells at different phases of the cell cycle may possess differential sensitivities to induction, and suppression, of apoptosis (24). One way to overcome this problem could be the isolation of SL2 cells stably transformed to express p35 (reference 8 and see below). P35 levels and AcMNPV progeny yields. Apoptosis, conceived as an antiviral response of the host, blocks viral replication and reduces virus yields (8, 9, 11, 12, 21). Thus, suppression of apoptosis by p35 overexpression was expected to enhance the yield of BV progeny. We consistently measured a fivefold increase in BV yield of vHSP-P35, which overexpressed p35, compared to wt AcMNPV progeny yields. This reflects only a slight improvement in the ability of AcMNPV to complete a productive infection. The above result could be attributed to an unexpected second mutation in the genome of the recombinant virus vHSP-P35; however, BV yields from recombinant- and wt-infected SF9 permissive cells were similar. Another possible explanation is the low level of competence of the SL2 cells to sustain NPV replication, but we have previously reported that SlNPV replicates to high titers in this

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cell line (9). Moreover, we report here that vHSP-P35 and wt LD50s for S. littoralis larvae were found to be not much different (Fig. 9). A 100- to 1,000-fold increase in BV yields has been reported for revertants of p35 null mutants (12, 21). Also, p35 mutants showed about 1,000-fold-higher LD50s than their revertants or wt AcMNPV in S. frugiperda BV-infected fourth-instar larvae (12). Recently, we have isolated a stably transfected SL2-derived cell line that expresses p35 constitutively and is completely resistant to AcMNPV-induced apoptosis. Under these conditions, no dramatic improvement in BV titers of wt AcMNPV compared to those of infected neomycin-resistant control cells was observed (17a). Thus, taken together, these data lead to the conclusion that apoptosis is not the only block to AcMNPV replication in SL2 cells, meaning that p35 is probably not the only AcMNPV host range determinant in this system (and probably at the organism level). Study of the expression of other early viral genes, such as those required for efficient late-gene expression and DNA replication (26, 38), may provide further clues to understanding the deficient replication of AcMNPV in SL2 cells. Finally, recent studies have indicated that Choristoneura fumiferana MNPV (CfMNPV) is able to suppress AcMNPVinduced apoptosis of C. fumiferana CF-203 cells and rescue the infectivity of AcMNPV for T. ni larvae (32). SlNPV infection of SL2 cells is permissive and does not induce apoptosis (9). Coinfection experiments with AcMNPV and S. littoralis NPV may yield a permissive infection and help elucidate the mechanism of abortion of the AcMNPV infection in SL2 cells. Moreover, the availability of in vitro AcMNPV DNA replication assays (23, 26) may help define the minimal set of genes required to obtain efficient amplification of the viral genome in SL2 cells (9).

FIG. 9. Mortality of virus-infected S. littoralis larvae. Twenty-four larvae per dose (fourth instar) were injected with vHSP-P35 or wt BVs. Percent mortality was calculated as the number of dead larvae (excluding larvae killed by the injection, normally one or two) divided by the number of larvae which survived the infection. No mortality was observed for mock-infected larvae

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AcMNPV p35 EXPRESSION IN S. LITTORALIS CELLS ACKNOWLEDGMENTS

We thank Paul D. Friesen for providing plasmid pBB/BSst, the a-P35NF antiserum, and the vD35K/lacZ virus; Lois K. Miller for plasmid pHSP35VI1; and Linda A. Guarino for the pIE1 plasmid. We acknowledge support for this research by the Israel Science Foundation under grant 398/96-1 to N.C.

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