The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation

Yves Langelier1,2, Stéphane Bergeron1, Stéphane Chabaud1, Julie Lippensa,1, Claire Guilbault1,3, A. Marie-Josée Sasseville1, Stéphan Denis1, Dick D. Mosserb,3 and Bernard Massie2,3,4

Centre de recherche du Centre hospitalier de l'Université de Montréal, Hôpital Notre-Dame, 1560 Sherbrooke Est, Montréal, Québec, CanadaH2L 4M11
Département de microbiologie et immunologie de l’Université de Montréal, Montréal, Québec, Canada2
Institut de recherche en biotechnologie, 6100 ave Royalmount, Montréal, CanadaH4P 2R23
INRS-IAF Université du Québec, Laval, Québec, CanadaH7N 4Z34

Author for correspondence: Yves Langelier (at Centre de recherche du Centre hospitalier de l’Université de Montréal). Fax +1 514 412 7590. e-mail yves.langelier{at}umontreal.ca


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1({Delta}2–357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6{Delta} and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The mechanisms for establishment of latent infection by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in neurons and the subsequent reactivation are poorly understood. During latency, a single HSV gene, the latency-associated transcript (LAT) gene, is transcribed and is essential for efficient reactivation. Recently, it was shown that LAT promotes neuronal survival after HSV-1 infection by impairing apoptosis (Ahmed et al., 2002 ; Perng et al., 2000 ). During reactivation, the pattern of gene expression is different from that observed in the lytic cycle: the expression of early (E) genes, notably the gene for the R1 subunit of ribonucleotide reductase (RR), begins several hours before detectable expression of the immediate early (IE) genes (Nichol et al., 1996 ; Talsinger et al., 1997 ). In animal models, HSV can be reactivated by numerous stress conditions including NGF deprivation and hyperthermia, which are also known to induce neuronal cell apoptosis. Therefore, it might be advantageous for the virus to encode a protein(s) that is able to block the apoptotic pathways activated by these stimuli. Hence, it is not surprising that several HSV genes, including the E protein kinase US3, the glycoprotein gJ (US5) and the IE proteins ICP22 and ICP27, have been shown to be involved in the inhibition of apoptosis induced by diverse stimuli such as osmotic or thermal shock (Koyama & Miwa, 1997 ; Leopardi & Roizman, 1996 ), the anticancer drug cisplatin (Zachos et al., 2001 ) or by the virus itself (Asano et al., 1999 ; Aubert & Blaho, 1999 ; Jerome et al., 1999 ; Leopardi et al., 1997 ). In addition, such proteins could be important in counteracting the action of cytotoxic T lymphocytes and cytokines that limit virus dissemination in cells of the mucosal epithelia where the virus replicates after being released from neurons (Jones et al., 2000 ; Nash, 2000 ). Thus, it has been shown that HSV renders infected cells resistant to cytotoxic T lymphocyte-induced apoptosis (Jerome et al., 1998 ).

The HSV RR converts ribonucleoside diphosphates to the corresponding deoxyribonucleotides and plays a key role in the synthesis of viral DNA (reviewed in Conner et al., 1994 ). The association of two homodimeric subunits, denoted R1 and R2, the former containing the active site, forms the holoenzyme. The R1 subunits of HSV-1 and HSV-2 possess an N-terminal domain of about 400 amino acids that is not found in R1 of other species (Nikas et al., 1986 ). Because of its unique N-terminal domain and a pattern of expression different from that of its R2 partner, it has been suggested that HSV R1 could have a dual function. Interestingly, the cytomegalovirus (CMV)-encoded R1, which is thought not to be involved in ribonucleotide reduction, was recently shown to play a role in the control of apoptosis induced by the virus itself in endothelial cells (Brune et al., 2001 ). A study of the homologous gene of a related {beta}-herpesvirus, human herpesvirus 7 (HHV-7), revealed that the gene does not express a functional RR subunit, and it was concluded that R1 could have a different function, not only in HHV-7 but also in other {beta}-herpesviruses (Sun & Conner, 1999 ).

Studies with HSV-1 R1 mutants, first carried out with cultured cells, showed that the enzyme is required for efficient replication in non-dividing cells. Subsequently, experiments using animal models demonstrated that the enzyme is required for efficient pathogenicity and is essential for virus reactivation from the neurons, but is not essential for the establishment of latency (Brandt et al., 1991 ; Goldstein & Weller, 1988a , b ; Jacobson et al., 1989 ). The observations that a mutant virus bearing a deletion of the RR domain of the R1 gene (hrR3) exhibited the same phenotype in cell culture or in animal models as another virus with a deletion of both the N-terminal and RR domains (ICP6{Delta}) have suggested that the N-terminal domain may play only a minor role in virus pathogenesis (Goldstein & Weller, 1988a , b ; Jacobson et al., 1989 ). However, viral mutants that contain deletions only of the R1 N-terminal domain have not yet been characterized for their capacity to reactivate. Therefore, an important role of this domain in HSV reactivation could have been masked by the RR deficiency of the two mutants, which by itself completely prevents virus replication in latently infected neurons.

The view that a protein kinase activity could be intrinsic to the unique N-terminal domain of HSV R1 (Chung et al., 1989 ; Cooper et al., 1995 ; Paradis et al., 1991 ) has recently been ruled out by extensive biochemical work showing that R1 does not possess such an activity but rather is a good substrate for co-purifying protein kinases (Conner, 1999 ; Langelier et al., 1998 ). Following our unsuccessful attempts to select standard recombinant adenovirus (Ad) constitutively expressing an HSV-2 R1 bearing a deletion in its N-terminal domain, R1({Delta}2–357), we subsequently produced Ad recombinants using a tetracycline-regulated expression cassette, which led us to discover that this protein is cytotoxic (Massie et al., 1998a ). Here, we show that the full-length HSV-2 R1 has an anti-apoptotic function that is able to protect the cells against apoptosis induced by expression of R1({Delta}2–357) or by death receptor triggering. We also found that the R1 protein acts at, or upstream of, caspase-8 activation. Using HSV-1 R1 deletion mutants, we obtained evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis.


   Methods
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Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells, plasmids, Ad recombinants and HSV.
The conditions for the culture of human 293A, 293S, HeLa S3 and A549 cells and for the selection of A549-tTA, HeLa-rtTA and HeLa-tTA cell lines to complement the tetracycline (Tet)-regulated expression cassette were as described previously (Massie et al., 1998a ). A549-tTA cells were maintained in medium containing 30 µg/ml phleomycin until one passage before the experiments, whereas medium for HeLa-tTA and HeLa-rtTA cells contained 350 µg/ml G-418.

Recombinants Ad5TR5-R1 and Ad5CMV5-R1 for expression of R1 of the HSV-2 RR, Ad5TR5-R1({Delta}2–357), an Ad recombinant expressing a truncated R1 with amino acids 2–357 deleted, and Ad5TR5-GFPQ, an Ad recombinant expressing a mutated green fluorescent protein (GFPQ), have been described previously (Massie et al., 1998a , b ). AdTR5-R1(1–398), AdTR5-R1(1–446)-GFPQ, and AdTR5-R1(1–496) are Ad recombinants expressing mutants of R1 deleted at various positions in the N-terminal domain and were constructed as detailed previously (Massie et al., 1998b ). Ad5CMV-Fas-L is a recombinant expressing the rat Fas ligand (Fas-L) under the control of the CMV IE promoter. Ad5TR5-Bax expresses the mouse Bax protein under the control of the TR5 promoter. Plasmids pAdTR5-R1-K7-GFPQ, pAdTR5-p35-K7-GFPQ and pAdTR5-19K-K7-GFPQ were obtained by inserting the coding sequences of the HSV-2 R1, the baculovirus p35 and the adenovirus E1B-19K genes in the BamHI site of the pAdTR5-K7-GFPQ transfer vector (Massie et al., 1998b ).

Adenovirus stocks were prepared by infecting 293S cells in suspension culture and titrated by plaque assay on 293A cells. To ensure optimal entry of the virus, the adsorption conditions for infection and titration were based on the protocol described by Mittereder et al. (1996) . High-titre stocks of HSV-1 (strain F) and HSV-2 (strain HG-52) were prepared by infecting confluent BHK-21/C13 cells with a low m.o.i., as described previously (Langelier & Buttin, 1981 ). The RR null HSV-1 mutants ICP6{Delta} and hrR3 (Goldstein & Weller, 1988a , b ) and its parental strain KOS were propagated and titrated on subconfluent Vero cells. The HSV-1 deletion mutant of the viral host shut-off gene (pvhs-) is derived from the KOS strain (Jones et al., 1995 ).

{blacksquare} Apoptosis assays.
Cells were seeded 1 day before infection either in 6-well plates at 3x105 cells per well, in 60 mm dishes at 1x106 cells per dish or in 96-well plates at 5x103 cells per well. The Ad recombinants Ad5TR5-R1({Delta}2–357) or Ad5CMV-Fas-L suspended in 1 ml of medium were adsorbed on to the cells for 18 h. For the co-infection experiments with Ad5TR5-R1 or Ad5CMV5-R1, these recombinants were first adsorbed on to the cells for 7 h. HSV was adsorbed on to the cells for 1 h. Doxycycline was added at 30 ng/ml when used to inhibit the recombinant protein synthesis in tTA-expressing cells, and at 3 µg/ml to induce protein expression in rtTA-expressing cells. To trigger apoptosis, cycloheximide (CHX; 30 µg/ml) plus either human recombinant tumour necrosis factor (TNF) (2·5 ng/ml; Sigma) or anti-human Fas mAb CH-11 (50 ng/ml; Upstate Biotechnology) were used.

To quantify the percentage of apoptotic cells, the detached cells, which had a strong tendency to aggregate, were dispersed by gently pipetting the medium. The cells were then counted in at least five randomly selected fields in each duplicate dish using a Nikon Diaphot inverted photomicroscope (magnification x200). The percentage of apoptotic cells was evaluated by dividing the number of cells with apoptotic morphology by the total number of cells. Cell viability was also assessed using the Cell Proliferation Reagent WST-1 (Roche Molecular Biochemicals) with cells seeded in 96-well plates (four wells per assay). For protein analysis, cells were scraped, washed with PBS, resuspended in 80 mM Tris–HCl (pH 6·8), 2% SDS, 6 M urea, and frozen at -80 °C until extraction.

{blacksquare} Protein extraction for SDS–PAGE and Western blot analysis.
SDS–PAGE and Western blotting of total protein extracts were performed as described previously (Lamarche et al., 1996 ). For HSV R1 detection, 168R1, a rabbit polyclonal anti-R1 antiserum (Langelier et al., 1998 ) or 932, a mouse mAb (Ingemarson & Lankinen, 1987 ), were used; P9, a polyclonal antiserum directed against the HSV R2 C terminus (Cohen et al., 1986 ), was used for HSV R2; the mouse mAb C15 (Scaffidi et al., 1997 ) was used for caspase-8; the mouse mAb PARP(Ab-2) (Calbiochem) was used for poly(ADP–ribose) polymerase (PARP). Quantification of the percentage of recombinant protein was carried out as detailed by Lamarche et al. (1996) and Poon et al. (1996) .

{blacksquare} In vitro caspase assays.
The ApoAlert (Clontech) caspase-3 and -8 fluorescent assay kits, which detect the shift in fluorescence emission of 7-amino-4-trifluoromethyl coumarin (AFC), were used to evaluate caspase-3 and -8 activity by measuring the initial rate of release of free AFC with a 96-well plate fluorometer.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
HSV-2 R1 protects cells against cytokine-induced apoptosis
In previous studies, we had observed that the inducible expression of R1({Delta}2–357), an HSV-2 R1 subunit with its first 357 amino acids deleted, resulted in cell death by apoptosis (Massie et al., 1998a ). Subsequently, we found that the full-length HSV-2 R1 has an anti-apoptotic function able to protect the cells against death triggered by the expression of the R1({Delta}2–357) (data not shown). As R1({Delta}2–357) is not a natural degradation product of R1, the physiological relevance of the above-described observation was at first sight not evident. However, since it has been reported that HSV could block apoptosis induced by diverse stimuli, we first tested whether R1 could block apoptosis induced by activation of receptors of the TNF family. To this end, A549-tTA and HeLa cells, which are sensitive to Fas- and TNF-mediated apoptosis, were chosen, as their normally flat morphology facilitates the scoring of apoptotic cells, characterized by membrane blebbing and cell body condensation. Hoechst staining indicated that cells scored apoptotic by morphology also exhibited nuclear condensation and fragmentation (data not shown).

A549-tTA cells massively undergo apoptosis when exposed to TNF in the presence of CHX, more than 95% of the cells being seen floating in the medium 18 h after the treatment. In contrast, no more than 5% of the cells that had been infected with Ad5TR5-R1 for 7 h prior to the application of the pro-apoptotic stimulus exhibited apoptotic morphological appearance (Fig. 1A). Similar results were obtained with HeLa-rtTA cells when they were infected in the presence of doxycycline to induce R1 expression (data not shown). The protection conferred to A549-tTA cells by Ad5TR5-R1 infection was confirmed by the observation that the TNF-induced PARP cleavage was prevented in infected cells (Fig. 1B). Moreover, as shown in Fig. 1(C), the curve showing the decrease in the percentage of apoptotic cells as a function of the m.o.i. of Ad5TR5-R1 was similar to the curve of the percentage of cells expected to be infected by the Ad recombinant according to the Poisson distribution. This result indicates that infection by only one infectious virion was sufficient to confer protection against TNF. Evidence that R1 expression is necessary for the protective effect came from the observation that infection with Ad5TR5-R1 in the presence of 30 ng/ml doxycycline (not shown) or with Ad5TR5-GFPQ (Fig. 1C) did not protect cells. By repressing R1 expression with doxycycline, the minimal amount necessary for protection was evaluated to be ~0·06 % [available as supplementary data (Fig, s1) at JGV Online (http://vir.sgmjournals.org)]. During an HSV-2 infection, a similar R1 level can be attained at 3 h post-infection and at its maximal value the R1 accumulation can reach 1% of total cell protein.



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Fig. 1. Full-length R1 prevents apoptosis induced by TNF. (A) Morphological appearance of cells photographed using a phase-contrast microscope. A549-tTA cells were mock-infected (Mock) or infected for 7 h with Ad5TR5-R1 (R1) at an m.o.i. of 5 p.f.u. per cell. After 7 h, the cells received new medium either without any additive (Control), with 30 µg/ml CHX (CHX) or with 30 µg/ml CHX plus 2·5 ng/ml TNF (CHX+TNF). Photographs were taken 18 h later. (B) Cell lysates were prepared from the cell populations described in (A) and analysed for PARP (116K) cleavage by immunoblotting. (C) A549-tTA cells were infected with increasing m.o.i.s of Ad5TR5-R1 ({blacksquare}, {square}) or Ad5TR5-GFPQ ({blacktriangleup}) and 7 h later received CHX ({square}) or CHX+TNF ({blacksquare}, {blacktriangleup}) as in (A); mock-infected untreated control ({circ}). The percentage of apoptosis was evaluated from 24 to 26 h after the first infection by counting apoptotic and non-apoptotic cells under microscopic observation. The dotted line (right axis) represents the percentage of cells expected to be infected by the Ad recombinant according to the Poisson distribution.

 
The R1 anti-apoptotic potential against death receptor activation was further demonstrated by showing that it could block cell death induced either by Fas triggering with the anti-Fas antibody CH11 in the presence of CHX in A549-tTA and HeLa-rtTA cells or when apoptosis was induced by more physiological conditions (without CHX) with an Ad recombinant that expresses Fas-L. Infecting HeLa cells or MDA-MB 231 breast cancer cells with the recombinant Ad5CMV5-R1, a constitutive Ad with an improved CMV-based expression cassette (Massie et al., 1998a ), ruled out the possible involvement of the tTA protein in the protective effect of the R1 [supplementary data (Fig. s2) at JGV Online (http://vir.sgmjournals.org)]. Taken together, these results demonstrate that HSV-2 R1 expressed at physiological levels is able to protect several types of cells against apoptosis induced by the activation of death receptors.

Adenovirus proteins are not involved in the anti-apoptotic action of HSV-2 R1
The involvement of Ad protein(s) in the protective effect was ruled out by transfecting HeLa-tTA cells with the shuttle plasmids used to construct the Ad recombinants. These experiments, described in detail in supplementary data [Fig. s3; JGV Online (http://vir.sgmjournals.org)], showed that the R1 protective activity did not require the co-expression of any Ad protein and that it was as potent as the two well-known anti-apoptotic proteins Ad E1B-19K and baculovirus p35.

HSV-2 R1 blocks caspase-8 activation induced by TNF and Fas-L
Two principal pathways for apoptosis have been described: the mitochondrial (intrinsic) pathway requires the participation of mitochondria, which activate caspases by releasing cytC, and the death receptor (extrinsic) pathway, in which mitochondria can be bypassed and caspases are activated directly by the triggering of death receptors. Both pathways converge on effector pro-caspases such as pro-caspase-3 (reviewed in Ashkenazi & Dixit, 1998 ). In several types of cell, the death receptor pathway is amplified by the mitochondrial pathway through the cleavage of the protein Bid by caspase-8 (Korsmeyer et al., 2000 ). Signalling through death receptors results in the assembly of the death-inducing signalling complex (DISC), which leads to the recruitment of the pro-caspase form of caspase-8 through adapter death-domain-containing proteins FADD and TRADD. Oligomerization of the pro-caspase leads to its auto-proteolytic activation (Ashkenazi & Dixit, 1998 ). Indication that HSV-2 R1 could act specifically in the extrinsic pathway came from testing the effect of the protein against stimuli known to induce cell death via the intrinsic pathway: Bax overexpression (Gross et al., 1999 ), etoposide (Engels et al., 2000 ), menadione (Samali et al., 1999 ) or staurosporine (Li et al., 2000 ). For these experiments, as some of these agents such as menadione are known to induce cell death both by apoptosis and necrosis (Samali et al., 1999 ), cell killing was scored not only by microscopic observation of cells but also using the colorimetric WST-1 viability assay. As shown in Fig. 2A, both scorings indicated that R1 expression could not prevent cell death induced by any of these stimuli. For staurosporine, cell death was even increased in the presence of R1.



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Fig. 2. HSV-2 R1 does not impair cell death induced by agents acting via the mitochondrial pathway. (A) A549-tTA cells infected for 24 h with an m.o.i. of 25 p.f.u. of Ad5TR5-R1 (R1+) or mock-infected (R1-) were either reinfected with Ad5TR5-Bax with an m.o.i. of 200 p.f.u. (Bax) or treated with 100 µM menadione, 400 µM etoposide, 250 nM staurosporine, 30 µg/ml CHX (CHX control) or 30 µg/ml CHX+2·5 ng/ml TNF (CHX+TNF). The percentage of viability was determined 24 h after treatment by scoring cells exhibiting normal morphology under microscopic observation in ten randomly selected fields (grey bars) or by the cell viability WST-1 assay (black bars). Results are expressed as percentage of the control value without R1 infection; for CHX+TNF, the value of CHX treated cells was used as control. (B) HSV-2 R1 does not block staurosporine-induced caspase activation. A549-tTA cells infected with Ad5TR5-R1 or uninfected were treated with staurosporine or CHX+TNF as described in (A). At the indicated times, cells were harvested for caspase-8 (grey bars) and -3 (black bars) determination with the ApoAlert kits.

 
To elucidate further the molecular mechanism underlying the R1 anti-apoptotic activity, we evaluated the activation of caspase-8 induced either by CHX+TNF or by Fas-L expression in A549-tTA cells with or without prior infection with Ad5TR5-R1. Caspase-8 activation was monitored by immunoblot analysis with a mAb visualizing the inactive 56 kDa pro-form and the active 18 kDa species (Fig. 3A) and by an in vitro assay using IETD–AFC as a caspase-8-specific fluorescent substrate (Fig. 3B, C). In Fig. 3(A), caspase-8 activation induced by an 8 h CHX+TNF treatment is clearly evidenced by the complete disappearance of the inactive 56 kDa pro-form and the presence of the 18 kDa active species. Time-course studies of the IETD–AFC substrate cleavage (Fig. 3B, C) showed that the activation reached a maximum between 4 and 6 h post-treatment and was no longer detectable at 16 h, a time when most of the cells had been destroyed. Both caspase assays revealed that R1 protein expression impaired the activation of caspase-8 induced either by CHX+TNF or Fas-L expression. Additional experiments using increasing m.o.i. of Ad5TR5-R1, as for the experiment described in Fig. 1(C), showed that the percentage of protection correlated with the extent of caspase-8 impairment (data not shown).



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Fig. 3. HSV-2 R1 impairs caspase-8 activation. A549-tTA cells were either mock-infected (MOCK) or infected with the recombinant Ad5TR5-R1 (R1) at an m.o.i. of 5 for 7 h before addition of CHX (CHX), CHX+TNF (CHX+TNF), control medium (Control) or reinfection with Ad5CMV-Fas-L (Fas L) at an m.o.i. of 25. After 8 h, 16 h or at the indicated time points in the time-course experiment presented in panel (B), the percentage of apoptosis ({lozenge}, grey bars) was scored as described in Fig. 1(C), and cells were harvested and cytoplasmic extracts were prepared for caspase-8 determination. Caspase-8 activation was monitored either by immunoblotting 20 µg of protein extract with the monoclonal antibody mAb C15 (A) or by measuring caspase-8 activity with the ApoAlert kit ({square}, black bars) (B, C).

 
The R1 segment 190–240 contains sequences exhibiting weak similarities with preferred recognition motifs of some caspases. Also, we observed previously that both HSV-1 and -2 R1 were cleaved after one of these motifs at HSV-2 position 240. These facts led us to assess a possible inhibitory effect of the purified RR–active R1 on mature caspases 8 and 3. Our experiments demonstrated that purified HSV-2 R1 added up to 2 µM was unable on its own to inhibit these enzymatic activities in tube assays (data not shown). These results suggest that R1, which does not act by preventing caspase-8 enzymatic activity, specifically functions to interrupt death receptor-mediated signalling at, or upstream of, caspase-8 activation. Additional evidence in favour of that conclusion was provided by an experiment aimed at determining whether R1 could influence the activation of caspases induced by staurosporine. It has been recently demonstrated that, in staurosporine-induced apoptosis, caspase-8 is activated by a cytochrome C-dependent activation of caspase-3 (Engels et al., 2000 ; Tang et al., 2000 ). As expected from the observation that staurosporine-induced cell death was increased by R1 (Fig. 2A), caspase-3 activity measured using DEVD–AFC as specific substrate was higher in cells expressing R1 than in the mock-infected cell control (Fig. 2B). Surprisingly, the activation of caspase-8 was also increased in the presence of R1. In control cells treated in parallel with CHX+TNF, the activation of these two caspases was blocked. Thus, it can be concluded that R1, which efficiently interferes with death receptor-dependent activation of caspase-8, cannot block the caspase-3-dependent activation of caspase-8 that is triggered by staurosporine.

HSV inhibits TNF-induced apoptosis
We next investigated whether, during HSV infection, the HSV R1 protein could prevent apoptosis induced by TNF. In a first series of experiments, A549-tTA or HeLa cells were infected with either HSV-1 strains KOS and F or HSV-2 strain HG-52 at an m.o.i. of 10 for 8 h before addition of CHX+TNF. Apoptosis was scored 20 h later by microscopic examination of the cells. The three HSV strains diminished the level of apoptosis from 95% in the mock-infected control to 15–25 %, levels that were similar to those obtained in HSV-infected cells treated only with CHX (see Fig. 4 for A549-tTA cells). The protective effect was confirmed by the observation that these viruses prevented the TNF-induced PARP cleavage (see Fig. 5B for KOS strain, and data not shown). In order to determine the time-course of the appearance of the protective effect in HSV-infected cells, A549-tTA cells were infected for increasing periods of time before addition of the lethal cocktail. As can be seen in Fig. 4, protection became detectable between 2·5 and 4 h, and appeared about 1–2 h earlier with the HSV-2 strain. However, for all three of the strains tested, maximal protection was reached within 6 h when the levels of apoptosis became comparable with those obtained in HSV-infected cells treated only with CHX. These results suggest that the protective effect is mediated by the synthesis of IE or E viral protein(s).



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Fig. 4. HSV infection protects A549-tTA cells against TNF-induced apoptosis. A549-tTA cells were mock-infected (x, {triangleup}, {blacktriangleup}) or infected with either HSV-1 strains KOS ({square}, {blacksquare}) or F ({circ}, {bullet}) or HSV-2 strain HG-52 ({lozenge}, {diamondsuit}) at m.o.i.s of 10 for increasing periods before addition of either CHX ({triangleup}, {square}, {circ}, {lozenge}), CHX+TNF ({blacktriangleup}, {blacksquare}, {bullet}, {diamondsuit}) or control medium (x). Apoptosis was scored 20 h after addition of the lethal cocktail.

 


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Fig. 5. The R1 mutant ICP6{Delta} has a reduced anti-apoptotic potential against TNF. A549-tTA cells were either mock-infected ({circ}, {bullet}, {blacksquare}) or infected with increasing m.o.i.s of either the parental HSV-1 KOS ({lozenge}, {diamondsuit}) or the R1 null mutant ICP6{Delta} ({triangleup}, {blacktriangleup}) for 7 h before addition of either control medium ({circ}, {lozenge}, {triangleup}), CHX ({bullet}, {diamondsuit} dotted line, {blacktriangleup} dotted line), or CHX+TNF ({diamondsuit} continuous line, {blacktriangleup} continuous line). (A) Apoptosis was scored 20 h after addition of the lethal cocktail. (B) PARP cleavage and R2 protein were detected by immunoblotting of protein extracts harvested at 20 h post-infection from cells infected at 7·5 or 20 p.f.u. per cell. Extracts of cells infected with Ad5TR5-R1 (AdR1) at an m.o.i. of 25 for 7 h before addition of CHX or CHX+TNF were added as control.

 
Protection against TNF is reduced in cells infected by ICP6{Delta} and hrR3, two R1 deletion mutant viruses
Next, the effect of deleting the HSV-1 R1 gene was studied by infecting A549-tTA cells with increasing m.o.i.s of either ICP6{Delta} or its WT parent, KOS. At the three m.o.i.s tested, ICP6{Delta} showed a roughly twofold decreased protection against CHX+TNF (Fig. 5A). In similar experiments, the extent of apoptosis was also evaluated by examining PARP cleavage. These experiments, as in the one presented in Fig. 5(B), showed that the TNF-induced PARP degradation was completely impaired in KOS-infected cells but only partially impaired in ICP6{Delta}-infected cells. In addition, as it was reported previously that the R1 deletion does not affect the synthesis of other viral polypeptides (Goldstein & Weller, 1988a , b ), we could compare the infectivity of both viruses by measuring the accumulation of R2 (the other subunit of RR). As similar levels were detected in both series of infected cells at 7 h post-infection, we concluded that the anti-apoptotic defect of the ICP6{Delta} viral stock was not due to a lower infectivity. Finally, the effect of virus infection on caspase-8 activation induced by the CHX+TNF treatment was monitored by measuring the cleavage of the caspase-8-specific substrate at 4 and 24 h post-treatment (Fig. 6). The caspase-8 activation induced by the cytokine was completely blocked in A549-tTA cells infected with the WT virus, KOS. In sharp contrast, it occurred significantly at the two different time points in cells infected with the mutant ICP6{Delta}, the activity measured being at least threefold higher than in the corresponding KOS-infected cells. Therefore, it can be concluded that the R1 null mutant is partially defective in blocking TNF-induced caspase-8 activation. Taken together, these results, which demonstrate that HSV-1 R1 plays an important role in the protection of HSV-infected cells against TNF, also indicate that other viral protein(s) are involved.



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Fig. 6. The R1 mutant ICP6{Delta} is defective in blocking TNF-induced caspase-8 activation. A549-tTA cells were either mock-infected or infected at an m.o.i. of 10 with either the parental HSV-1 KOS or the R1 null mutant ICP6{Delta} for 8 h, followed by addition or not (Control) of CHX+TNF. The percentages of apoptosis (grey bars) and caspase-8 activity (black bars) were measured at 4 and 24 h after addition of the lethal cocktail.

 
The HSV-2 R1 N-terminal domain is not anti-apoptotic by itself
When the protection assays were performed with hrR3, the mutant expressing the first 428 amino acids of R1 fused to {beta}-galactosidase, results similar to those observed with ICP6{Delta} were obtained (data not shown). This suggested that the N-terminal domain by itself could not be sufficient for the R1 anti-apoptotic activity. Using Ad recombinants that express variable lengths of the R1 N-terminal domain (1–398, 1–446-GFPQ and 1–496), we confirmed this hypothesis. The three proteins, which were found to be as soluble as the WT R1 as determined by centrifugation (Fig. 7B) or immunofluorescence (not shown), were not protective at all against apoptosis induced by cytokines (Fig. 7A for CHX+TNF, not shown for Fas-L). Hence, both the N-terminal domain and the RR domain, or parts of them, contribute to the HSV-2 R1 anti-apoptotic activity.



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Fig. 7. The HSV-2 R1 N-terminal domain is not anti-apoptotic by itself. A549-tTA cells were infected at an m.o.i. of 25 with the Ad recombinants Ad5TR5-R1 [R1], Ad5TR5-R1(1–398) [R1(1–398)], Ad5TR5-R1(1–446)-GFPQ [R1(1–446)-GFPQ], Ad5TR5-R1(1–496) [R1(1–496)] or mock-infected (Mock). In (A), the cells received medium containing CHX (grey bars) or CHX+TNF (black bars) after 7 h. Apoptosis was scored 14 h later. In (B), extracts from the CHX-treated cells were centrifuged at 100000 g for 1 h at 4 °C; supernatants (S) and pellets (P) were analysed for recombinant protein expression by immunoblotting. Extract containing the R1({Delta}2–357) was included as reference for a low-solubility protein.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The major finding of the present work is that HSV-2 R1 very efficiently prevents apoptosis induced by activation of death receptors. It came from the serendipitous observation that a large deletion in the N-terminal domain of HSV-2 R1 produces a pro-apoptotic protein. Our data supporting an anti-apoptotic function for HSV-2 R1 were mostly accumulated using Ad recombinants. Thus, effects of gene expression could be observed with high reproducibility in a larger proportion of cells than could be attained by transfection. Moreover, the inducible Ad5TR5 system is very powerful as it permits the regulatable expression of a gene of interest in nearly 100% of the cells. Hence, using doxycycline to repress R1 expression we were able to determine that maximal protection against TNF could be seen at R1 levels ~20-fold lower than what is maximally accumulated during an HSV infection. However, as Ad does express some of its genes, even in non-permissive cells, one could suspect the involvement of Ad genes in the observed phenomenon. Here, it was clearly shown that the R1 protective effect did not necessitate the co-expression of any Ad protein because R1 protein expression from transfected plasmids conferred protection against CHX+TNF to most of the transfected cells.

The majority of cell lines, including A549, HeLa and MDA-MB-231 cells, which naturally express low levels of TNF receptor and Fas at their surface, are resistant to TNF and Fas cytotoxicity unless co-treated with protein or RNA synthesis inhibitors, such as CHX and actinomycin D. The sensitizing effect of these inhibitors has recently been ascribed to a direct strong down-regulation of accumulation of apoptosis regulatory proteins such as FLIP or RIP (Fulda et al., 2000 ; Kreuz et al., 2001 ; Wajant et al., 2000 ). Most of our data demonstrating the R1 anti-apoptotic potential were obtained with CHX to increase the cytotoxicity of TNF or Fas agonist mAb. However, even when apoptosis was induced by infecting cells with the Ad5CMV-Fas-L recombinant without the use of CHX, pre-infection by Ad5TR5-R1 was fully protective. Therefore, it can be concluded that the R1 anti-apoptotic action is not dependent on the inhibition of the synthesis of short-lived regulatory proteins.

The second important observation of the present work is that HSV-2 R1 prevents caspase-8 activation induced via death receptor triggering. HSV-2 R1 did not inhibit: (i) cell death mediated by a variety of agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione); (ii) active caspase-8 and -3 in tube assays; and (iii) the caspase-3-dependent activation of caspase-8 induced by staurosporine in A549-tTA cells; thus, it is most likely that HSV-2 R1 specifically interrupts death receptor-mediated signalling at, or upstream of, caspase-8 activation. The mechanism by which HSV-2 R1 impairs the process of caspase-8 activation is currently unknown. One possible mode of action of the R1 protein could be that, through an interaction with either caspase-8 itself or the adapter molecules TRADD, FADD or FLASH, it prevents pro-caspase-8 oligomerization. This mechanism could be similar to viral or cellular FLIP proteins that act by inhibition of caspase-8 recruitment to the DISC (Krueger et al., 2001 ). However, our co-immunoprecipitation studies could not detect HSV-2 R1 interacting with pro-caspase-8 using conditions similar to those recently used to detect interaction between this caspase and the CMV anti-apoptotic protein vICA (Skaletskaya et al., 2001 ).

Our data with A549-tTA cells showing that activation of caspase-3 and -8 induced by staurosporine was not blocked by R1 contradict a recent report indicating that 293 cells constitutively expressing HSV-2 R1 exhibited resistance to staurosporine-induced apoptosis, as shown by an absence of caspase-3 activation and PARP cleavage (Perkins et al., 2002 ). The reason for this discrepancy is unknown. As we were also unable to observe protection either in HeLa or 293A cells transiently expressing HSV-2 R1 or in 293S stable cell lines inducibly expressing the protein (S. Chabaud, C. Ablasou, L. Bourget, B. Massie & Y. Langelier, unpublished results), it is unlikely that it could be attributable to the use of different cell lines.

The third important finding of the present work is that both HSV-1 and HSV-2 infection impair apoptosis induced by TNF-R or Fas activation in the presence of CHX. The role of the R1 protein in this protection was substantiated by our data showing that the protective effect appears in both HSV-1- and HSV-2-infected cells with a time-course compatible with the synthesis of IE or E viral protein(s), and that deleting R1 in HSV-1 strain KOS (ICP6{Delta} and hrR3 mutants) decreased the anti-apoptotic potential by 50%. Our observation that half of the cells infected with the R1 null mutants were resistant to TNF treatment suggests that other viral gene(s) also contribute to the phenomenon. Among them, the vhs gene appears to play a significant role as we have observed that deleting it reduced the anti-apoptotic potential by about 30% (C. Guilbault & Y. Langelier, unpublished observations). The vhs protein, by inhibiting cellular protein synthesis, could act, for example, by decreasing the amount of cytokine receptors at the cell surface. Direct evidence has also been provided that HSV glycoprotein gJ can also antagonize Fas-induced apoptosis (Jerome et al., 2001 ). Other viral genes, which have been shown to be involved in the control of apoptosis induced by diverse stimuli, could also contribute to the partial resistance of the HSV R1 mutants. Among them are the protein kinase Us3, which acts through the phosphorylation of the apoptosis regulatory protein Bad (Munger & Roizman, 2001 ), the glycoprotein gD and the IE gene ICP27 (Aubert et al., 1999 ).

Recent observations showing the predominance of TNF in the trigeminal ganglion during primary infection and following reactivation suggest that the protection of HSV-infected cells against TNF could be important during the establishment of latency and/or reactivation (Kodukula et al., 1999 ; Shimeld et al., 1997 , 1999 ). As HSV R1 begins to be synthesized at an early stage during both the lytic cycle and reactivation, and accumulates throughout the productive cycle in amounts larger than needed for DNA replication (N. Lamarche & Y. Langelier, unpublished observations), it could contribute to slowing down the antiviral activity of TNF-family cytokines. It could also counteract the action of cytotoxic lymphocytes that are maintained for long periods at mucosal sites of virus shedding (Liu et al., 2000 ; Nash, 2000 ; Posavad et al., 2000 ). Further work to assess the role of the R1 anti-apoptotic domain in latency will require an HSV mutant defective in R1 anti-apoptotic activity, without being pro-apoptotic as is R1({Delta}2–357), but still functional in RR activity, since the R1 RR domain is required to allow deoxyribonucleotide synthesis, which is essential to viral DNA replication during reactivation in animal ganglion neurons.

Finally, our initial thought that the anti-apoptotic function could be ascribed to its unique N-terminal domain has proved to be wrong and studies are under way to delineate which parts of the two domains are important for the control of apoptosis. As the R1 protein does not exhibit clear homology to any of the known anti-apoptotic proteins, it could act through a new mechanism of action. Our results indicate that HSV R1 possesses an anti-apoptotic function, which we propose is important in HSV pathogenesis. Further understanding of this mechanism may suggest new approaches to control HSV pathogenicity and contribute to a better understanding of the control of apoptosis.


   Acknowledgments
 
We gratefully acknowledge Sandra Weller for providing us with HSV-1 mutants ICP6{Delta} and hrR3, James Smiley for the viral host shut-off mutant pvhs-, Marcus Peter for the anti-caspase-8 antibody, Lars Thelander for 932 mAb, Nadine Pavloff for the Ad5CMV-Fas-L recombinant and Gordon Shore for the mouse Bax cDNA. We also thank Nadine Jabbour for the construction of Ad5TR5-Bax. This work was supported by grant MT-14686 from the Canadian Institutes of Health Research to Y.L. and by an operation grant to B.M. from the National Research Council of Canada (NRC). This is an NRC publication no. 37697.


   Footnotes
 
a Present address: Shire-Biochem inc., 275 Armand-Frappier Blvd, Québec, Canada H7V 4A7.

b Present address: Molecular Biology and Genetics, The University of Guelph, Guelph, Ontario, Canada N1G 2W1.


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Received 7 March 2002; accepted 3 July 2002.