©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Baculovirus p35 Protein Inhibits Fas- and Tumor Necrosis Factor-induced Apoptosis (*)

David R. Beidler , Muneesh Tewari , Paul D. Friesen (1), Guy Poirier (2), Vishva M. Dixit (§)

From the (1)Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin 53706, and (2)Poly(ADP-ribose) Metabolism Group, Department of Molecular Endocrinology, Centre Hospitalier de l'Université Laval Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The baculovirus p35 gene product inhibits virally induced apoptosis, developmental cell death in Caenorhabditis elegans and Drosophila, and neuronal cell death in mammalian systems. Therefore, p35 likely inhibits a component of the death machinery that is both ubiquitous and highly conserved in evolution. We now show for the first time that p35 also inhibits Fas- and tumor necrosis factor (TNF)-induced apoptosis. Additionally, p35 blocks TNF- and Fas-induced proteolytic cleavage of the death substrate poly(ADP-ribose) polymerase from its native 116-kDa form to the characteristic 85-kDa form. This cleavage is thought to be catalyzed by an aspartate-specific protease of the interleukin 1-converting enzyme family designated prICE (Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346 -347). Our data suggest that p35 must directly or indirectly inhibit prICE. Given that p35 inhibits both TNF and Fas killing, along with previous reports of its ability to block developmental, viral, and x-irradiation-induced cell death, the present results indicate that TNF- and Fas-mediated apoptotic pathways must have components in common with these highly conserved death programs.


INTRODUCTION

Two members of the TNF()receptor family, TNFR-1 and Fas, are known mediators of host antiviral defense mechanisms(1, 2) . Activation of the TNF receptor in virally infected host cells results in their elimination from the host organism. Similarly, cytotoxic T-cells have been shown to kill infected host cells by activating the Fas pathway(3) . Activated cytotoxic T-cells express the Fas ligand, which upon engaging and activating the Fas receptor on the surface of the virally infected cells, delivers a death signal. Both elimination events proceed through a process of active, programmed cell death termed apoptosis(4) , which results in the death of virus-bearing host cells in order to attenuate viral spread. Apoptosis is characterized by a series of orchestrated intracellular events that lead to the selective removal of target cells without an accompanying inflammatory response. Apoptotic events are morphologically distinguishable by cellular shrinkage, nuclear condensation, cytoplasmic vacuolization, and membrane blebbing. Biochemical events include the activation of specific proteases resulting in the cleavage of certain recently identified protein substrates(5, 6) . There is now overwhelming genetic and biochemical evidence implicating interleukin 1-converting enzyme (ICE)-like proteases as components of the cell death pathway(7, 8) . This unusual class of cysteine proteases cleaves only following certain specific aspartic acid residues, and until of late, the only known substrate was prointerleukin-1, which is converted to the active cytokine by ICE. Now, however, the 116-kDa DNA repair enzyme poly(ADP-ribose) polymerase (PARP) has been shown to be proteolytically processed to a signature 85-kDa fragment by an aspartate-specific cleavage mediated by an ICE-like protease (termed prICE) that appears to be distinct from ICE itself(6) . Importantly, PARP cleavage has been observed in a variety of apoptotic responses(5) , including TNF- and Fas-mediated cell death (9).

To circumvent host defense mechanisms, viruses have evolved mechanisms to antagonize host death signals so that viral propagation can continue unabated in infected cells(10) . The baculovirus Autographa californica and Bombyx mori nuclear polyhedrosis viruses encode a 35-kDa protein (p35) that alleviates apoptosis in virally infected insect cells(11, 12) . p35-mediated inhibition of apoptosis correlates with increased viral replication and enhanced yields of viral progeny(11, 13) . Viral mutants lacking p35 induce premature death of the host cells with a resultant decline in viral yields. Surprisingly, p35 can inhibit not only virally induced apoptosis but also developmentally programmed cell death in the nematode Caenorhabditis elegans(14) . p35 expression in transgenic worms, for example, can rescue embryonic lethality resulting from a mutation in ced-9, an endogenous suppressor of cell death. Additionally, p35 has been found to suppress developmental cell death in Drosophila(15) . Ectopic targeted p35 expression to the eyes of transgenic flies eliminates all normally occurring cell death in this tissue and, furthermore, also inhibits x-irradiation-induced death. Remarkably, p35 can also inhibit apoptosis in mammalian neural cells deprived of neurotropic stimulation (16, 17). These studies suggest that p35 must interrupt a highly conserved and ubiquitous component of the death machinery. We now show for the first time that p35 inhibits TNF- and Fas-induced apoptosis and, in addition, it blocks the cleavage of PARP, a death substrate in the apoptotic pathway.


MATERIALS AND METHODS

Cell Lines and Plasmids

The TNF-sensitive MCF7 breast carcinoma cell line was provided by Dr. David R. Spriggs (University of Wisconsin). The Fas-sensitive MCF-Fas subclone was obtained by transfecting a Fas receptor expression construct into the MCF7 cells (18). The BJAB cell line was kindly provided by Dr. Fred Wang (Harvard Medical School, Cambridge, MA). The plasmid pPRM-35K-ORF containing the entire p35 open reading has been described previously (19). The entire p35 open reading frame obtained as a 0.9-kilobase pair EcoRI/XbaI fragment was cloned into the mammalian expression vector pcDNA3 (Invitrogen).

Transfections

Transfection by electroporation of p35/pcDNA3 and pcDNA3 (vector alone) into MCF7 cells and selection of stable lines was carried out as described previously(20) . For transient transfection of MCF-Fas lines, 10 cells were plated onto 6-well plates and incubated for 18 h. Transfections were by the Lipofectamine (Life Technologies, Inc.) procedure according to the manufacturer's instructions. Cells were exposed to agonist anti-Fas antibody (Upstate Biotechnology Inc.) 28 h following transfection with the indicated plasmids. Transient transfection of BJAB cells was by electroporation, followed by anti-Fas exposure 48 h later.

Assessment of TNF- and anti-Fas-induced Apoptosis/Toxicity

To quantitate TNF-induced apoptosis in stably transfected MCF7 lines, a previously described quantitative apoptosis assay using propidium iodide was used(20) . For measurement of TNF toxicity, 2 10 cells were plated in 24-well plates, incubated for 24 h, exposed to 20 ng/ml TNF (1.2 10 units/mg, Genentech) for 18 h, washed, and incubated for a further 48 h in TNF-free media. Wells were fixed and stained by adding 0.5% methylene blue in 50% ethanol for 30 min, and after washing, the incorporated methylene blue was solubilized in 1% Sarkosyl in phosphate buffered saline. The resulting color intensities were quantitated on a microplate reader (BioTek Instruments) at an absorbance of 630 nM.

For the transient apoptosis assays, cells transfected with a -galactosidase expression vector together with either a p35 expression construct or vector control were exposed to anti-Fas (250 ng/ml) for 16 h, fixed with 0.5% glutaraldehyde (EM Sciences) or methanol (for MCF-Fas and BJAB cells, respectively), and stained with 1 mg/ml 5-bromo-4-chloro-3-indoyl -D-galactoside (Boehringer Mannheim) to detect -galactosidase expression. The resulting -galactosidase positive (green) cells were examined for apoptotic morphology including cellular shrinkage, nuclear condensation, and cytoplasmic blebbing, by phase contrast microscopy (MCF-Fas), or by propidium iodide staining and fluorescence microscopy (BJAB).

RNA Isolation and Northern Analysis

Total RNA was isolated (21) and enriched for the poly(A) fraction using a kit (Qiagen) according to the manufacturer's instructions. RNA was transferred to nylon membranes and probed with a P-labeled 1-kilobase pair DNA fragment corresponding to the entire p35 open reading frame according to established procedures (22).

PARP Western Blot

The preparation of cell lysates and immunoblotting of TNF- and anti-Fas-treated MCF7 lines was performed as described previously(6) . The anti-PARP monoclonal antibody (clone C2-10, IgG1(23) ) was used at a dilution of 1:10,000, and the secondary antibody (anti-mouse Ig/horseradish peroxidase; Sigma) was used at a dilution of 1:1,000. The peroxidase signal was visualized by chemiluminescence (ECL, Amersham Corp.).


RESULTS AND DISCUSSION

To determine if p35 expression was capable of blocking TNF-induced apoptosis, MCF7, a TNF-sensitive human breast carcinoma cell line(20) , was stably transfected with a p35 expression construct, and clonal cell lines were derived. Fig. 1A shows that after an 18-h exposure to TNF (20 ng/ml), only 12% of a vector-transfected line (V-2) was not apoptotic as recognized by staining cellular DNA with propidium iodide and assessing nuclear fragmentation by fluorescence microscopy. However, in two p35-transfected lines, p35#4 and p35#5, approximately 90% of the cells were resistant to apoptosis. A third p35-expressing line, p35#2, had intermediate numbers of apoptotic cells. To confirm that assessing nuclear fragmentation by fluorescence microscopy corresponded to gross toxicity, cell survival following an 18-h TNF exposure was determined by incubating the cells for a further 48 h in TNF-free media. As shown in the inset to Fig. 1A, p35#4 and p35#5, and to a lesser extent, p35#2, displayed increased survival rates when compared with vector controls. The relative lower survival rates compared with that assessed by staining cellular DNA could either be due to reversible growth inhibition during the 18-h TNF incubation or the presence of additional apoptotic events even after the 18-h TNF exposure. Northern blots of poly(A)-enriched RNA from these lines verified p35 expression in the p35 stable transfectants (Fig. 1B). The p35#2 line had considerably less transcript when compared with p35#4 and p35#5, which would explain the intermediate levels of protection observed in p35#2. Therefore, there exists a dose-response relationship between the level of p35 expression and the degree of protection conferred from TNF-induced cell death.


Figure 1: Inhibition of TNF-induced apoptosis in MCF7 cells by p35. A, sensitivity of stable transfectants to TNF-induced apoptosis (20 ng/ml for 18 h) as determined by staining cellular DNA with propidium iodide and assessing nuclear fragmentation by fluorescence microscopy. V-2, control, vector-transfected clone; p35#2, p35#4, and p35#5, p35-transfected clones. Inset, TNF-induced cell death (20 ng/ml for 18 h) assessed by the methylene blue toxicity assay and expressed as the relative percentage of live cells compared with untreated control cultures. Data represent the mean ± S.D. from duplicate samples of two separate experiments. B, p35 transcript levels in the corresponding clones. C, photograph of ethidium bromide-stained gel to assess equivalency of loading as judged by the intensity of the 18 and 28 S rRNA bands.



It had previously been found that anti-Fas killing in the parental MCF7 line required the concomitant presence of cycloheximide, a protein synthesis inhibitor, to enhance Fas toxicity(20) . To allow a comparison of TNF- and Fas-induced killing in the same line without cycloheximide coincubation, thereby avoiding complications resulting from the toxicity of cycloheximide itself, the level of Fas expression was augmented by transfecting with a Fas expression construct and deriving a stable transfectant designated MCF-Fas(18) . This cell line was susceptible to Fas-induced death in the absence of a protein synthesis inhibitor. MCF-Fas cells were transiently cotransfected with a -galactosidase reporter and a p35 expression or vector control construct followed by Fas activation by cross-linking with an agonistic antibody. Assessment of nuclear morphology of -galactosidase-positive cells revealed that p35 markedly protected cells from Fas-induced apoptosis (Fig. 2A). Additional transient transfections performed in BJAB cells, a B cell line that is sensitive to Fas-induced apoptosis(20) , also demonstrated that p35 protected cells from Fas-induced apoptosis (Fig. 2B).


Figure 2: Inhibition of Fas-induced apoptosis in MCF-Fas and BJAB cells by p35. Transient cotransfections with a -galactosidase reporter construct plus either vector control or a p35 expression construct were carried out in MCF-Fas (A) or BJAB (B) cells. Cultures were incubated in the presence (diagonalbars) or absence (solidbars) of an agonist anti-Fas antibody ((250 ng/ml) for 16 h). Morphology of -galactosidase-positive cells was assessed by phase contrast microscopy (MCF-Fas) or propidium iodide staining and fluorescence microscopy (BJAB). In each experiment, two separate counts of at least 200 cells were performed. Data represent the mean ± S.D. from three experiments (MCF-Fas) or two experiments (BJAB).



Analysis of the integrity of the death substrate PARP in the vector-transfected MCF7 line revealed complete cleavage of the native 116-kDa PARP to the signature 85-kDa proteolytic fragment within 16 h of exposure to TNF (Fig. 3A). However, in the p35#4 and p35#5 lines, only low levels of the 85-kDa proteolytic product were seen even after 24-48 h of TNF exposure. The underexpressing p35#2 line displayed a PARP cleavage rate that was only marginally slower than the vector-transfected control, V-2. Similar results were seen when MCF7 cells were treated with anti-Fas (Fig. 3B). While PARP cleavage was slower and not as complete as in TNF-treated cultures, p35 still significantly inhibited its cleavage in the p35#4 and p35#5 clones and to a lesser extent in the intermediate expressing p35#2 line. Therefore, as with TNF-induced killing, there exists a dose-response relationship between the level of p35 expression and the degree of inhibition of PARP cleavage.


Figure 3: p35 inhibits TNF- and Fas-induced PARP cleavage. MCF7 stable transfectants were exposed to TNF (A) or anti-Fas (B) for the indicated time periods after which cell lysates were prepared for immunoblotting with the anti-PARP monoclonal antibody as described under ``Materials and Methods.'' The 116-kDa band represents the intact protein, and the 85-kDa band denotes the signature cleavage product.



In summary, the baculovirus p35 protein was able to block not only Fas- and TNF-induced apoptosis but also Fas- and TNF-induced PARP cleavage. Given that p35 inhibits both TNF and Fas killing, along with previous reports of its ability to block developmental, viral, and x-irradiation-induced cell death, the present results indicate that TNF- and Fas-mediated apoptotic pathways must have components in common with these highly conserved death programs. Inhibition of cleavage of the death substrate PARP has also been observed with the anti-apoptotic cowpox virus CrmA gene product(9) . However, CrmA is a serpin capable of inhibiting ICE family members whereas p35 possesses no homology to any protease inhibitor. This suggests that p35 must inhibit cleavage of the death substrate PARP and ultimately cell death at a site unique in the cell death pathway. Thus, p35 protein will be an invaluable tool in helping identify components of this highly conserved cell death pathway.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 64803 (to V. M. D.), a grant from the National Cancer Institute of Canada (to G. P.), and United States Public Health Service Award HL 07517Y14 (to D. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel.: 313-747-0264; Fax: 313-764-4308; E-mail: vishva dixit@mailqm.pds.med.umich.edu.

The abbreviations used are: TNF, tumor necrosis factor; ICE, interleukin 1-converting enzyme; PARP, poly(ADP-ribose) polymerase.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. David R. Spriggs for providing the MCF7 cell line, Dr. Fred Wang for providing the BJAB line, and Marja Jäättelä for producing the MCF-Fas line. We would like to thank Karen O'Rourke and Arul Chinnaiyan for their helpful advice and technical assistance, and Ian M. Jones for help in preparation of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.