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ExpressionoftheAutographacalifornicanuclearpolyhedrosisvirusapoptoticsuppressorgenep35innonpermissiveSpodopteralittoraliscells

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EdwardGershburg

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NorChejanovsky

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JOURNAL OF VIROLOGY,0022-538X/97/$04.0010

Oct. 1997, p. 7593–7599 Vol. 71, No. 10

Copyright © 1997, American Society for Microbiology

Expression of the Autographa californica Nuclear PolyhedrosisVirus 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 polyhe-drosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology209:519–525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorlyexpressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determin-ing the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-inducedapoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viabilityof SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did notimprove significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showedsimilar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replicationin these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only hostrange determinant in this system.

The Autographa californica nuclear polyhedrosis virus(AcMNPV) is considered the prototype of subgroup A of theBaculoviridae family of viruses which infect invertebrates andprimarily insects. The complete genome of AcMNPV has beensequenced (1), and its replication in permissive cells has beenextensively studied (for reviews, see references 4 and 23). Theability of AcMNPV to infect a wide number of hosts, 39 speciesof lepidopteran larvae belonging to 13 families (5, 17), and theavailability of appropriate tissue culture systems have facili-tated the isolation of genetically engineered AcMNPVs withnovel insecticidal properties. Some of these are being evalu-ated for agricultural application (reviewed in references 6 and30).

The mechanisms that control the ability of baculoviruses toinfect specific insect hosts are not clear. Their elucidation isimportant for better assessment of potential risks associatedwith the utilization of recombinant-baculovirus insecticides.Moreover, this knowledge could help expand the range ofapplications of specific AcMNPV recombinants to target eco-nomically important lepidopteran pests.

Four baculovirus genes have been implicated in facilitatingAcMNPV replication in a species-specific manner. (i) p35,characterized as an antiapoptotic gene, inhibitor of cysteineproteases of the CED-3/ICE family, caspases (2, 7, 24, 39), isrequired for efficient AcMNPV infection of Spodoptera frugi-perda cells and larvae. AcMNPV p35 gene null mutants yieldedlow titers of budded virus (BV) (12, 21). Moreover, the ab-sence of p35 function was complemented by apoptosis inhibitorgenes (iap genes) derived from other baculoviruses (3, 15). (ii)

A recombinant AcMNPV bearing a small fragment of thehelicase gene of Bombyx mori nuclear polyhedrosisvirus (NPV)within AcMNPV’s p143 coding region (the helicase gene ofAcMNPV) replicated in both permissive S. frugiperda SF21 andnonpermissive B. mori BmN4 cell lines (14, 28). (iii) The hrf-1gene of the Lymantria dispar NPV was shown to allow themultiplication of AcMNPV in nonpermissive Ld652Y cells (16,37). (iv) The hcf-1 gene of AcMNPV was necessary for suc-cessful infection of TN368 cells and to some extent for im-provement of the infectivity of the virus in Trichoplusia nilarvae but was not necessary for replication or infectivity in S.frugiperda cells and larvae, respectively (27).

In addition, 18 baculovirus genes (lef genes) were requiredfor expression of a late baculovirus promoter in SF21 cells (26,38); three of them, ie-2, lef-7, and p35, were not required forexpression in TN368 cells, suggesting that they could be in-volved in determining host range.

AcMNPV does not infect Spodoptera littoralis (the Egyptiancotton worm), an important Mediterranean pest. Recently, wereported that infection of S. littoralis SL2 cells with wild-typeAcMNPV results in apoptosis and concomitantly low yields ofviral progeny (9). Since AcMNPV mutants with alterations inthe p35 gene induce apoptosis of the permissive cell line of S.frugiperda, SF21, we hypothesized that p35 either is expressedpoorly or is not functional, or both, in AcMNPV-infected SL2cells (9). p35 has been shown to suppress apoptosis in variousheterologous systems (18, 20, 29, 33, 35) which are evolution-arily more distant from SF9 cells than the SL2 system (which isclosely related to the S. frugiperda lepidopterous cells). This ledus to hypothesize that p35 may be functional but not suffi-ciently expressed in AcMNPV-infected SL2 cells. Thus, aug-menting the expression of p35 in SL2 cells could allow us todetermine its functionality in terms of suppression of apopto-sis. In the present work, we show that by overexpressing p35 wewere indeed able to reduce apoptosis of SL2 cells induced byeither actinomycin D or AcMNPV. However, increasing p35

* Corresponding author. Mailing address: Entomology Department,The Institute for Plant Protection, The Volcani Center, POB 6, BetDagan 50-250, Israel. Phone: (972)-3-968-3694. Fax: (972)-3-960-4180.E-mail: vpnjc@volcani.agri.gov.il.

† Contribution 2093 from the Agricultural Research Organization,The Volcani Center, Bet Dagan, Israel

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

MATERIALS AND METHODS

Cell lines and viruses. S. littoralis SL2 and S. frugiperda SF9 cells were main-tained and propagated in TNM-FH medium supplemented with 10% heat-inactivated fetal bovine serum (36). Infection of the cells with wild-type (wt)AcMNPV strain E2 (34), 50% tissue culture infectious dose assays, and plaquetitration of virus stocks were done as described previously (31).

Plasmids and transfections. Plasmids pBB/BSst and pHSP35VI1 contain thep35 coding region under the control of the p35 and Drosophila melanogasterhsp70 promoters, respectively (13, 21). pHSP35VI1 also possesses an intactpolyhedrin gene located back-to-back to the hsp70-p35 transcription unit andadditional viral sequences which allow the transfer of the p35 and polyhedringenes 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 methodas previously described (10).

Construction of the recombinant vHSP-P35. vHSP-P35 was constructed bycotransfection of SF9 cells with plasmid pHSP35VI1 and linearized polyhedrin-negative AcMNPV DNA by the calcium phosphate method. Polyhedrin-positiverecombinant 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 ex-tracted from the virus-infected SL2 cells by using a 10 mM Tris (pH 8.0)–1 mMEDTA–1% sodium dodecyl sulfate (SDS) buffer containing 70 mg of proteinaseK per ml (2 h at 37°C) followed by the addition of NaCl (final concentration, 1M). The extracts were treated with phenol-chloroform and ethanol precipitated,and resuspended DNA was analyzed by agarose gel electrophoresis as describedpreviously (9). Densitometric scanning of the gels was performed basically asdescribed below for Western blot scanning.

(ii) Actinomycin D-induced apoptosis. SL2 cells (105) were transfected with 10mg of pHSP35VI1 or pBluescript. Twelve hours posttransfection, 10 mM bro-modeoxyuridine (BrdU) was added and the mixture was incubated for 12 h tolabel the cellular DNA. The cells were heat shocked at 24 h posttransfection (30min, 42°C, in a water bath), and 4 h later, 250 ng of actinomycin D per ml wasadded to induce apoptosis. The time course of BrdU-labeled oligonucleosomerelease to the cell medium was monitored by incubating aliquots of the cellsupernatants (in triplicate), taken at various times, in an enzyme-linked immu-nosorbent assay (ELISA) plate coated with anti-DNA antibody. Bound BrdU-labeled oligonucleosomal DNA was detected by using anti-BrdU monoclonalantibody conjugated to peroxidase in an ELISA according to the recommenda-tion of the manufacturer (Boehringer, Mannheim, Germany).

Western blot analysis. AcMNPV-infected or plasmid-transfected cells werewashed in phosphate saline buffer, harvested in 150 mM Tris (pH 8.0)–1 mMEDTA–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 equivalentswere subjected to SDS-polyacrylamide gel electrophoresis (25), and the polypep-tides were transferred to a nitrocellulose membrane. Immunodetection of P35was performed by using a-P35NF antiserum (22) as previously described (9).Relative levels of expressed P35 were determined by computerized densitometryscanning with a Bio-Image System 202D Apparatus (Rhenium, Jerusalem, Isra-el).

Bioassays. Various doses of BVs whose titers in SF9 cells had been previouslydetermined were injected into fourth-instar S. littoralis larvae as described pre-

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

RESULTS

P35 synthesis in AcMNPV-infected SL2 cells. To comparethe steady-state levels of P35 synthesized during the infectiouscycle, both nonpermissive SL2 and permissive SF9 cells wereinfected with AcMNPV. At various times postinfection (p.i.),cell extracts were subjected to immunoblot analysis using a-P35NF antiserum (Fig. 1). P35 was detected in virus-infectedSF9 cells at 4 h p.i. and reached its maximal expression atabout 18 to 34 h p.i. (Fig. 1B). In SL2 cells, very small amountsof P35 were observed at 4 h p.i.; maximal steady-state levelswere detected at 18 h p.i. (Fig. 1A). However, P35 steady-statelevels in SL2 cells dropped from 18 to 34 h p.i., probably dueto cell death (compare Fig. 1A and B). Overall, P35 levels inthose cells were extremely low compared to those in infectedSF9 cells.

These results suggested that a lack of sufficient amounts ofP35 allows the progression of apoptosis initiated by AcMNPVinfection of SL2 cells (9). However, this hypothesis did not ruleout the possibility of p35’s nonfunctionality in SL2 cells.

Transient expression of p35 in SL2 cells. We assumed thatincreasing the amount of P35 was a prerequisite to addressingthe question of whether p35 is functional in SL2 cells. Previousstudies have shown that high P35 levels are obtained in SF21cells by expressing p35 under the control of the hsp70 heatshock promoter from Drosophila (13). We compared the levelsof P35 obtained during transfection of two plasmids, pBB/BSstand pHSP35VI1, in which p35 was placed under the control ofits own promoter or the hsp70 promoter, respectively (seeMaterials and Methods). A third plasmid, pIE1 (19), bearingthe intact ie-1 gene coding for IE1, a transactivator of p35, wascotransfected with pBB/BSst. Immunoblot analysis detectedhigher levels of P35 in the pHSP35VI1-transfected cells thanin cells transfected with pBB/BSst plus pIE1, and the levelswere undetectable in pBB/BSst-transfected cells (Fig. 2A). Onthe basis of these results, we selected the pHSP35VI1 con-struct for our expression studies (see below).

p35-mediated suppression of apoptosis. Actinomycin Dtreatment has been shown to induce apoptosis of lepidopteranSF21 cells, which was suppressed by p35 expression (8, 13). SL2cells undergo apoptosis upon incubation with actinomycin D atconcentrations above 100 ng/ml.

The ability of p35 to block apoptosis in actinomycin D-

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 wereharvested at various times p.i. (indicated below the lanes) and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using a-P35NF antiserum.

7594 GERSHBURG ET AL. J. VIROL.

treated SL2 cells was studied by transfecting them withpHSP35VI1 and further incubating them with 250 ng of acti-nomycin D per ml. The time course of the apoptosis provokedby the addition of actinomycin D was monitored by using asemiquantitative ELISA-based assay which measures theamount of oligonucleosomal DNA released by the apoptoticcells (see Materials and Methods). A decrease of about 50% inthe release of oligonucleosomal DNA was observed in SL2cells transfected with pHSP35VI1 relative to that in controlpBluescript-transfected cells (Fig. 3A). Untreated cells (noaddition of actinomycin D) did not release oligonucleosomalDNA, and their optical densities were below 0.05. As expected,P35 was detected in the pHSP35VI1-transfected cells and wasnot detected in the pBluescript-transfected cells (Fig. 3B, lanes2 to 5 and 6, respectively). pBB/BSst-transfected cells releasedamounts of oligonucleosomes equivalent to those released bypBluescript-transfected cells, and P35 was barely detectable(data not shown and Fig. 2A).

A total of 60% of pHSP35VI1-transfected SL2 cells re-mained viable after exposure to actinomycin D, in contrast to18% of pBluescript (or pBB/BSst)-transfected cells (Fig. 4 anddata not shown), again indicating that high-level expression ofp35 conferred resistance to apoptosis.

p35 overexpression during viral infection. The experimentsdescribed above indicated that p35 expression in SL2 cells (i)could be elevated by placing its transcription under the controlof the hsp70 promoter and (ii) resulted in a functional product,since it correlated with acquired resistance to actinomycin D-induced apoptosis. Thus, to study the ability of P35 to blockAcMNPV-induced apoptosis, we constructed a recombinantvirus (vHSP-P35) in which the hsp70 promoter-p35 transcrip-tion unit was inserted at the polyhedrin locus of the AcMNPVgenome (Fig. 5).

SL2 cells were infected with vHSP-P35 at multiplicities ofinfection (MOIs) of 1 and 10, and p35 expression was detectedas described above. Higher steady-state levels of P35 wereproduced in cells infected with the recombinant than in cellsinfected with wt AcMNPV (Fig. 6, lanes 2, 4, and 5 and lanes3 and 6, respectively). Moreover, heat shocking the cells afterinfection resulted in a dramatic increase in P35 synthesis in therecombinant-virus-infected cells (Fig. 6, lanes 4 and 5). Apo-ptosis of the SL2 cells infected by wt and recombinant vHSP-P35 viruses (MOI of 10) is shown in Fig. 7. Electrophoresis ofDNA extracted from AcMNPV-infected cells shows the typicaloligonucleosomal ladder characteristic of apoptosis (Fig. 7A,

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

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 levelsof 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 thesingle-plasmid transfections to obtain a total of 20 mg of DNA).

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 hposttransfection 250 ng of actinomycin D per ml was added to induce apoptosis. Atthe indicated times after actinomycin D addition, cell supernatants were collectedand aliquoted, and the release of BrdU-labeled oligonucleosomes was monitored byusing a monoclonal antibody and ELISA (see Materials and Methods). (A) P35steady-state levels were monitored by immunoblotting (B) (lanes 1 to 6 [times areindicated below the lanes]; lane M, molecular weight markers). Relative levels ofP35 at the various times indicated in panel B are also shown (C); total opticaldensity units are expressed as percentages of the highest P35 peak. Generally, avariation of about 15% was observed in the detection of P35 by Western blotting,which was attributed to differences in transfection efficiencies.

VOL. 71, 1997 AcMNPV p35 EXPRESSION IN S. LITTORALIS CELLS 7595

We estimated a reduction of about 50% apoptosis in vHSP-P35-versus wt-infected SL2 cells by densitometric scanning ofthe gel and, independently, by counting intact versus blebbingvHSP-P35- and wt-infected SL2 cells (Fig. 7).

Inhibition of apoptosis and viral yields. Apoptosis has beenimplicated in the significant reduction of BV yields observed inSL2 cells relative to virus yields measured in SF9 cells (9).Since overexpression of p35 reduced the extent of virus-in-duced apoptosis of SL2 cells, we determined the amount ofprogeny BVs released through one cycle of replication by in-fecting the cells with wt (AcMNPV) and recombinant (vHSP-P35) viruses at an MOI of 10 PFU per cell. As can be seen inFig. 8, vHSP-P35 yields increased slightly compared to those ofAcMNPV (vHSP-P35 titers of 5.17 3 104 6 4.33 3 103 and10.1 3 104 6 8.4 3 103 and AcMNPV titers of 1.55 3 104 61.29 3 103 and 2.48 3 104 6 2.06 3 103 PFU/ml at 24 and 48 hp.i., respectively). Also, a slight increase in BV yields was notedwith cells infected at a MOI of 1 (not shown). vHSP-P35 titersfrom permissive SF9 cells infected at 48 h p.i. (same MOI asabove) were 1.01 3 107 6 3.2 3 106 and 2.00 3 107 6 1.1 3106 PFU/ml, with or without the cells being heat shocked,respectively. AcMNPV titers under the same conditions were3.05 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 abil-ity of AcMNPV to replicate at the organism level, we determinedthe approximate LD50s of vHSP-P35 and wt AcMNPV forfourth-instar S. littoralis larvae. That was accomplished by in-jecting vHSP-P35 and AcMNPV BV doses ranging from 102 to105 PFU per larva. As can be seen in Fig. 9, approximate LD50sof 103 PFU/larvae were determined for vHSP-P35 and wt Ac-

MNPV, by graphic extrapolation. Thus, no significant differ-ences in LD50s between recombinant and wt AcMNPVs innonpermissive S. littoralis larvae were observed.

DISCUSSION

Virus-induced apoptosis fully develops in SL2 and is sup-pressed in SF21 (or SF9) cells infected with wt AcMNPV (7, 9,11, 22). To determine whether AcMNPV’s failure to suppressapoptosis in infected SL2 cells is due to poor expression of theapoptotic suppressor gene p35, we compared the steady-statelevels of P35 in SL2 and SF9 cells infected with AcMNPV. P35levels were indeed much lower in infected SL2 cells than intheir SF9 counterparts (Fig. 1A and B, respectively). Studies ofP35’s mode of action have indicated that it binds stoichiomet-rically to CED-3/ICE-like death proteases of invertebrate andvertebrate origin, inhibiting their activity (2, 7, 24, 39). Thus,the above results suggest that apoptosis in infected SL2 cellscould occur due to insufficient amounts of P35 available tobind to and inhibit a putative SL2 CED-3/ICE-related proteaseactivated by AcMNPV. Alternatively, P35 levels in nonpermis-sive 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 theSL2 caspase (2). A prerequisite to examination of these alter-natives was overexpression of p35.

Expression of p35 directed by the Drosophila hsp70 pro-moter resulted in remarkably higher steady-state levels of P35in both plasmid-transfected and recombinant-virus-infected

FIG. 4. Protection of SL2 cells from actinomycin D-induced apoptosis by p35expression. SL2 cells (10 5) were transfected with 10 mg of pHSP35VI1 (pHSP-P35) or pBluescript (pBSR). Heat shock was performed at 24 h posttransfection,and actinomycin D (250 ng/ml) was added at 28 h posttransfection. Cell viabilitywas determined by trypan blue exclusion. The viability of heat-shocked plasmid-transfected cells (as described above) prior to addition of actinomycin D wasconsidered 100%. The results shown are means of three replicates, with standarddeviations (error bars).

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

FIG. 6. Expression of p35 by recombinant virus vHSP-P35. SL2 cells wereinfected with recombinant virus (vHSP-P35) or wt AcMNPV (lanes 1, 2, 4, and5 and lanes 3 and 6, respectively) at an MOI of 1 or 10. Infected cells were heatshocked at 1 h after infection or were not heat shocked (1HS or 2HS, respec-tively). Cell extracts prepared at 24 h p.i. were immunoblotted as described in thelegend to Fig. 1 and in Materials and Methods. Lane M, prestained markers withmolecular masses of 98, 64, 50, 30 and 16 kDa (bovine serum albumin, glutamicdehydrogenase, alcohol dehydrogenase, myoglobin, and lysozyme, respectively).

7596 GERSHBURG ET AL. J. VIROL.

SL2 cells (Fig. 2 and 6). p35 expression was further enhancedby 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 andAcMNPV infection. In the case of actinomycin D-treated cells,p35 expression correlated with inhibition of apoptosis, esti-mated directly by measuring the amount of oligonucleosomal

DNA released by the cells (Fig. 3), and as evidenced by highercell viability (Fig. 4). Overexpression of p35 has been shown toaugment viability of SF21 cells, protecting them from apoptosisinduced by either actinomycin D or expression of the p32 formof the human ICE protease gene (7, 13).

Recombinant vHSP-P35 infection also induced apoptosis ofSL2 cells, albeit to a lower extent than wt AcMNPV (Fig. 7A).

FIG. 7. Overexpression of p35 reduces apoptosis of SL2 cells. (A) Intracellular DNA fragmentation. DNA was extracted from SL2 cells infected with wt virus orvHSP-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. Lightmicrographs (magnification 3200) were prepared as previously described (9). Intact and blebbing cells were counted (250 to 350 cells per field, three replicates). Theextent of apoptosis of vHSP-P35-infected SL2 cells relative to that of wt-infected cells was estimated.

VOL. 71, 1997 AcMNPV p35 EXPRESSION IN S. LITTORALIS CELLS 7597

The infected cells needed to be heat shocked in order to obtainsignificant p35 expression and exhibit reduced cellular DNAfragmentation, 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 correlatedwith the ability of p35 to prevent actinomycin D-induced apo-ptosis of SF21 cells (13). Another study has shown that SF21cells stably transformed to express p35 are resistant to ap-optosis induced by actinomycin D (8). Taken together, theseand our results lead to the conclusion that a functional P35,product of p35 expression, protects the SL2 cell from apo-ptotic death induced by actinomycin D or AcMNPV infec-tion.

Our inability to achieve 100% suppression of apoptosis byoverexpressing p35 could be due to the lack of synchronizationof the infected SL2 cells. Thus, different cells at differentphases of the cell cycle may possess differential sensitivities toinduction, and suppression, of apoptosis (24). One way toovercome this problem could be the isolation of SL2 cellsstably transformed to express p35 (reference 8 and see be-low).

P35 levels and AcMNPV progeny yields. Apoptosis, con-ceived as an antiviral response of the host, blocks viral repli-cation and reduces virus yields (8, 9, 11, 12, 21). Thus, sup-pression of apoptosis by p35 overexpression was expected toenhance the yield of BV progeny. We consistently measured afivefold increase in BV yield of vHSP-P35, which overex-pressed p35, compared to wt AcMNPV progeny yields. Thisreflects only a slight improvement in the ability of AcMNPV tocomplete a productive infection. The above result could beattributed to an unexpected second mutation in the genome ofthe recombinant virus vHSP-P35; however, BV yields fromrecombinant- and wt-infected SF9 permissive cells were simi-lar. Another possible explanation is the low level of compe-tence of the SL2 cells to sustain NPV replication, but we havepreviously reported that SlNPV replicates to high titers in this

cell line (9). Moreover, we report here that vHSP-P35 and wtLD50s for S. littoralis larvae were found to be not much differ-ent (Fig. 9).

A 100- to 1,000-fold increase in BV yields has been reportedfor revertants of p35 null mutants (12, 21). Also, p35 mutantsshowed about 1,000-fold-higher LD50s than their revertants orwt AcMNPV in S. frugiperda BV-infected fourth-instar larvae(12). Recently, we have isolated a stably transfected SL2-de-rived cell line that expresses p35 constitutively and is com-pletely resistant to AcMNPV-induced apoptosis. Under theseconditions, no dramatic improvement in BV titers of wtAcMNPV compared to those of infected neomycin-resistantcontrol cells was observed (17a).

Thus, taken together, these data lead to the conclusion thatapoptosis is not the only block to AcMNPV replication in SL2cells, meaning that p35 is probably not the only AcMNPV hostrange determinant in this system (and probably at the organ-ism level). Study of the expression of other early viral genes,such as those required for efficient late-gene expression andDNA replication (26, 38), may provide further clues to un-derstanding the deficient replication of AcMNPV in SL2cells.

Finally, recent studies have indicated that Choristoneurafumiferana MNPV (CfMNPV) is able to suppress AcMNPV-induced apoptosis of C. fumiferana CF-203 cells and rescue theinfectivity of AcMNPV for T. ni larvae (32). SlNPV infection ofSL2 cells is permissive and does not induce apoptosis (9).Coinfection experiments with AcMNPV and S. littoralis NPVmay yield a permissive infection and help elucidate the mech-anism of abortion of the AcMNPV infection in SL2 cells.Moreover, the availability of in vitro AcMNPV DNA replica-tion assays (23, 26) may help define the minimal set of genesrequired to obtain efficient amplification of the viral genome inSL2 cells (9).

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

FIG. 8. Growth curve of recombinant (vHSP-P35) and wt AcMNPVs. BVscollected from SL2 cells infected vHSP-P35 (squares) and the wt (circles) atvarious times p.i. were titrated in SF9 cells by the 50% tissue culture infectivedose method (see Materials and Methods). The results are the averages of threeindependent experiments.

7598 GERSHBURG ET AL. J. VIROL.

ACKNOWLEDGMENTS

We thank Paul D. Friesen for providing plasmid pBB/BSst, thea-P35NF antiserum, and the vD35K/lacZ virus; Lois K. Miller for plas-mid pHSP35VI1; and Linda A. Guarino for the pIE1 plasmid.

We acknowledge support for this research by the Israel ScienceFoundation under grant 398/96-1 to N.C.

ADDENDUM

SF9 (subclone of SF21) cells are as susceptible to apoptosisby the AcMNPV null mutant vD35K/lacZ as SF21 cells.

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