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Journal of Virology, July 2005, p. 8388-8399, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8388-8399.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Flavivirus Activates Phosphatidylinositol 3-Kinase Signaling To Block Caspase-Dependent Apoptotic Cell Death at the Early Stage of Virus Infection

Chyan-Jang Lee,1,2 Ching-Len Liao,1,3 and Yi-Ling Lin1,2,4*

Graduate Institute of Life Sciences,1 Department of Microbiology and Immunology,3 National Defense Medical Center, Institute of Biomedical Sciences,2 Genomics Research Center, Academia Sinica, Taipei, Taiwan, Republic of China4

Received 29 October 2004/ Accepted 7 March 2005


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ABSTRACT
 
Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.


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INTRODUCTION
 
The genus Flavivirus comprises over 70 viruses, many of which have been associated with severe human diseases (8). Of particular importance for public health are the mosquito-borne flaviviruses, such as yellow fever virus, Japanese encephalitis virus (JEV) (65), West Nile virus (WNV) (29), and the dengue viruses (DENs) (40). Flaviviral virions are composed of a lipid bilayer with two or more envelope proteins surrounding a nucleocapsid, which consists of single-stranded positive-sense genomic RNA associated with multiple copies of capsid proteins (49).

Apoptotic cell death has been described in several flaviviral infections, such as DEN (22), JEV (46), and WNV (57). Furthermore, apoptosis has been implicated as a cytopathologic mechanism in response to DEN infection both in vitro and in vivo in several cell types (16). Growing evidence shows that flavivirus infections activate biochemically distinct apoptotic pathways. DEN serotype 2 (DEN-2) may trigger neuronal apoptosis through phospholipase A2 (PLA2) activation, superoxide anion generation, cytochrome c release, caspase-3 activation and NF-{kappa}B activation (34). In the apoptotic process triggered by JEV infection, it has been suggested that virus-induced endoplasmic reticulum (ER) stress may participate, via p38 mitogen-activated protein kinase (MAPK)-dependent and a death-related transcription factor CHOP (C/EBP homologous protein)-mediated pathways (66). Infection with WNV (13) and expression of WNV capsid protein lead to caspase-9 and -3 activation and induce apoptosis through the mitochondrial pathway.

Three major apoptosis pathways have been identified according to their initiator caspase (53). In the death receptor-mediated pathway, caspase-8 is recruited to a death-inducing signaling complex when death receptors such as Fas and the tumor necrosis factor are oligomerized after the binding of specific ligands (14, 52). The ER stress-mediated pathway attributes to activation of caspase-12 (56). In the mitochondrial pathway, caspase-9 is activated when cytochrome c is released into the cytoplasm from the intermembrane space of the mitochondria (44). Rather than being single linear mechanisms, these three apoptotic pathways may cross-interact. It has been suggested that cleavage of Bid into t-Bid by caspase-8 can activate mitochondrion-dependent apoptosis by releasing cytochrome c into the cytosol (43, 50). Caspase-9 could also be activated by ER stress-induced caspase-12 in a cytochrome c-independent manner (54). Once activated, these initiator caspases then undergo self-activation to start the downstream effector caspase signaling cascade (17).

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway regulates various cellular processes, such as metabolic regulation, cell growth, proliferation, and survival (6, 9, 25). The consequence of PI3K activation is the generation of phosphatidylinositol-3,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate in the membrane, which function as a second messenger to recruit pleckstrin homology domain-containing proteins, such as Akt and phosphoinositide-dependent kinase 1. Akt is then activated by phosphorylation at Thr308 by phosphoinositide-dependent kinase 1 and at Ser473 by an unidentified kinase (6). Activated Akt can then phosphorylate variety of substrates, including several proteins associated with cell death pathway, such as Bad (18, 20), NF-{kappa}B (37, 61), and caspase-9 (10). However, the precise molecular mechanisms whereby Akt inhibits apoptosis are not completely understood.

In recent years, accumulated evidence has shown that viruses modulate the PI3K/Akt signaling pathway (15). The gene products of viruses that are associated with oncogenesis such as polyoma virus (69), Epstein-Barr virus (19, 26), human papillomavirus (55), hepatitis B virus (64), and hepatitis C virus (31) have been shown to stimulate PI3K/Akt-mediated cell survival and thereby block apoptosis of infected cells, leading to viral survival and oncogenic transformation. In addition to its role in long-term survival, the PI3K/Akt pathway has also been implied in short-term cellular survival during the initial stages of acute infection when virus replication and protein synthesis take place. This short-term activation of PI3K/Akt signaling prolonged cell survival upon infections with encephalomyocarditis virus, herpes simplex virus (58), and respiratory syncytial virus (71). The PI3K/Akt pathway has also been involved in virus replication. For example, the optimal expression of some immediate-early genes of human cytomegalovirus depended on PI3K activity, and human cytomegalovirus DNA replication was strongly inhibited by treatment with the PI3K inhibitor, LY294002 (36). PI3K/Akt activation might also create a favorable environment for virus replication and virion assembly and appears to be favorable for infection with human immunodeficiency virus type 1 (24) and coxsackievirus B3 (23).

Because JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection (16, 34, 46), the possibility that the PI3K/Akt pathway participates in the preservation of host cell survival during viral infection has prompted us to investigate the interaction between JEV/DEN-2 and this pathway. In the present study, we show that Akt can be phosphorylated after JEV and DEN-2 infection in a PI3K- and lipid raft formation-dependent manner. When PI3K was blocked by specific inhibitors such as LY294002 and wortmannin, JEV and DEN-2 infections resulted in apoptosis at an early stage of infection; however, these inhibitors did not affect JEV and DEN-2 viral replication. Furthermore, this enhanced early apoptosis could be largely overcome by pan-caspase and caspase-9 inhibitors and partially by caspase-6 inhibition. We also found that Bcl-2 overexpression prevented the loss of Akt protein observed in JEV/DEN-2-infected cells and sustained a constant level of phosphorylated Akt, which might then protect cells from apoptosis even when PI3K was blocked. Our results provide evidence to suggest that flaviviruses such as JEV and DEN-2 not only induce cell apoptotic signaling but also activate a survival signaling involving the PI3K/Akt pathway.


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MATERIALS AND METHODS
 
Cell lines, viruses, and chemicals. N18 cells (a mouse neuroblastoma cell line) (1), baby hamster kidney BHK-21 cells, and B2-5 cells (a Bcl-2-overexpressing cell line derived from BHK-21 (45, 46) were cultured in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 2 mM L-glutamine. The human lung carcinoma cell line, A549 (71), which is susceptible to JEV and DEN-2 infection often with the consequence of severe cytopathic effects at around 3 to 4 days postinfection (p.i.), was cultured in F-12 medium (Gibco) supplemented with 10% FBS. JEV strain RP-9 (11) and DEN-2 strain PL046 (48) were used in the present study. Virus propagation was carried out in C6/36 cells in RPMI 1640 medium containing 5% FBS. The culture supernatants were clarified by centrifugation at 8,000 rpm for 30 min at 4°C. For Akt phosphorylation study, the virus was purified by ultracentrifugation through a 20% sucrose cushion at 27,000 rpm in a Beckman SW28 rotor for 3.5 h at 4°C. The virus pellets were resuspended in serum-free RPMI medium. For virus inactivation, we heated the sucrose cushion-purified virus at 56°C for 30 min, which resulted in a drop of virus titer by 5 log orders of magnitude. LY294002 and wortmannin were from Sigma and Calbiochem, respectively, and were dissolved in dimethyl sulfoxide. Toxin B, Clostridium difficile (catalog no. 616377), was obtained from Calbiochem. Pan-caspase (Z-VAD-FMK) and caspase-8 (Z-IETD-FMK) inhibitors purchased from Sigma were dissolved in dimethyl sulfoxide (DMSO). Inhibitors of caspase-9 (Z-LEHD-FMK), caspase-3 (Z-DQMD-FMK), caspase-6 (Z-VEID-FMK), and caspase-3/7 (5-[(S)-(–)-2-(methoxymethyl) pyrrolidino] sulfonylisatin) were purchased from Calbiochem and were dissolved in DMSO. The reactivity of these caspase inhibitors have been demonstrated previously (42, 68, 72). Cycloheximide (CHX) and methyl-ß-cyclodextrin (mßCD) were obtained from Sigma.

Virus infection and titration. To infect with JEV or DEN-2, monolayers of cells in 6- or 12-well plates were adsorbed with virus for 1 h at 37°C. After adsorption, unbound virus was removed by gentle washing with serum-free medium, followed by the addition of fresh medium and further incubation at 37°C. To determine virus titers, culture media were harvested for plaque-forming assays. Various virus dilutions were added onto 80% confluent BHK-21 cells and incubated at 37°C for 1 h. After adsorption, the cells were washed and overlaid with 1% agarose (SeaPlaque; FMC BioProducts) containing RPMI 1640 with 2% FBS. After incubation for 4 days for JEV and 7 days for DEN-2, cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet.

LDH assay. Cytotoxicity was assessed by the release of a cytoplasmic enzyme lactate dehydrogenase (LDH) by using a commercial kit (Cytotoxicity Detection Kit; Roche). The culture supernatants from cell samples were clarified by centrifugation, mixed with reaction mixture (diaphorase/NADH+, tetrazolium salt INT/sodium lactate), incubated at room temperature for about 30 min, and then read by an enzyme-linked immunosorbent assay reader at 490 nm (Molecular Devices). LDH release was calculated as a percentage versus the untreated mock-infected cells.

XTT assay. To determine the cell viability, a colorimetric sodium 3'-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) based assay was performed (Cell Proliferation Kit II; Roche) according to the manufacturer's instructions. The cells were incubated with the reaction mixture at 37°C for about 30 min and then read by an enzyme-linked immunosorbent assay reader at 490 nm (Molecular Devices). XTT activity was calculated as a percentage versus the untreated mock-infected cells.

Quantitative detection of DEN-2 RNA by using fluorogenic reverse transcription-PCR. The viral RNA used in the present study was extracted and quantified as previously described (76). Briefly, viral RNA was reverse transcribed by using a ThermoScript RT kit (Invitrogen) with a primer annealing to DEN-2 nucleotides 10723 to 10703 (5'-AGAACCTGTTGATTCAACAGC-3'). Real-time PCR to detect the DEN-2 genome was conducted as previously described (33). An ABI Prism 7700 sequence detection system (version 1.7; Perkin-Elmer Applied Biosystems), was used for PCR cycling reaction, real-time data collection, and analysis.

TUNEL assay. Apoptosis-induced DNA strand breaks were end-labeled with dUTP by using terminal deoxynucleotidyl transferase from a commercial kit (In Situ Cell Death Detection kit, TMR red; Roche) according to the manufacturer's instructions. Briefly, the cells were fixed with paraformaldehyde solution (2% in phosphate-buffered saline [pH 7.4]) for 60 min at room temperature and permeabilized in 0.1% Triton X-100-0.1% sodium citrate for 2 min on ice. The TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) reaction was performed by using TMR red dUTP at 37°C for 60 min, and the labeling was analyzed under a Leica fluorescence microscope.

Antibodies and Western immunoblot analysis. Cell monolayers were rinsed and lysed with lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1 mM EDTA) containing a cocktail of protease inhibitors (Roche) and phosphatase inhibitor cocktail I (Sigma). The concentration of total protein in the cell lysates was quantified by using the DC protein assay kit (Bio-Rad). Equal amounts of protein in samples from each experimental group were mixed with sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue), boiled, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hybond-C Super; Amersham Biosciences). To improve resolution, a 4 to 12% gradient gel (NuPAGE Novex Bis-Tris Gel; Invitrogen) was used for the study of poly(ADP-ribose) polymerase (PARP) cleavage. The nonspecific antibody-binding sites were blocked with 5% skimmed milk in phosphate-buffered saline and reacted with various primary antibodies as indicated for each experiment. Monoclonal anti-JEV E, NS3 antibodies (11) and anti-DEN-2 E, NS3 antibodies (48) have been described previously. Akt antibody (antibody 9272), which detects total levels of endogenous Akt1, Akt2, and Akt3 proteins, and phospho-Akt (Ser473) antibody (antibody 9271), which recognizes Akt1, Akt2, and Akt3 only when they are phosphorylated at Ser473, were purchased from Cell Signaling Technology, Inc. An antibody (antibody 9332) that detects glycogen synthase kinase-3ß was also purchased from Cell Signaling Technology, Inc. Caspase-3 antibody (antibody 9662; Cell Signaling Technology, Inc.) detects endogenous levels of full-length caspase-3 (35 kDa) and the large fragment of caspase-3 resulting from cleavage (17 kDa). Caspase-6 antibody (antibody 9762; Cell Signaling Technology, Inc.) recognizes endogenous levels of both full-length (35 kDa) and the small subunit of caspase-6 (15 kDa). PARP antibody (antibody 9542; Cell Signaling Technology, Inc.) reacts with full-length PARP (116 kDa), as well as the large (89 kDa) and small fragments (24 kDa) of PARP resulting from caspase cleavage. The anti-actin antibody (MAB1501) was from Chemicon International, Inc., and the anti-human Bcl-2 antibody (sc-509) was from Santa Cruz. The blots were then reacted with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin or Biotin-SP-conjugated AffiniPure goat anti-rabbit immunoglobulin G combined with peroxidase-conjugated streptavidin (Jackson Immunoresearch). Finally, the blots were developed by using an ECL system (Amersham Biosciences). The intensities of protein bands were quantified by using MetaMorph software from Universal Imaging Corp.


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RESULTS
 
PI3K-dependent Akt phosphorylation was induced by JEV and DEN-2 infections. Because JEV and DEN-2 infections induce apoptosis in the infected cells late after infection (46, 67), we speculated that a survival signal might be triggered at an early stage of viral infection. The activation of PI3K/Akt signaling was studied in JEV- and DEN-2-infected mouse neuroblastoma N18 cells by Western blotting with antibody specific to phospho-Akt (Ser473). To minimize the effects of exogenous growth factors in our system, we purified the virus through a sucrose cushion and resuspended it in serum-free medium. As shown in Fig. 1A, phosphorylated Akt was evident in JEV- and DEN-2-infected cells starting from around 15 to 30 min p.i., and this elevated phosphorylation was still detectable at 60 min p.i., but not in the mock-infected cells. The activation of Akt was dependent on PI3K activity, since addition of the PI3K inhibitor, LY294002 (75) (Fig. 1B, lanes 4 and 5) or wortmannin (2) (data not shown), but not a Rho GTPase inhibitor toxin B (Fig. 1B, lanes 6 and 7), blocked the virus-induced Akt phosphorylation. Since Akt phosphorylation occurred soon after virus cell encounter, virus binding, but not virus replication per se, likely triggers this event. This notion was supported by the findings in Fig. 1B, in which neither CHX (lane 12), a protein synthesis inhibitor, nor heat inactivation (lane 15) could diminish the level of Akt phosphorylation induced by DEN-2 infection. Since membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to serve as platforms for the initiation of signaling cascades (7, 28, 63), we further examined the requirement of lipid raft formation for the flavivirus-induced Akt phosphorylation. Indeed, disruption of cholesterol-rich lipid raft formation by mßCD (77) reduced the DEN-2-induced Akt phosphorylation (Fig. 1B, lane 13), suggesting an essential role of lipid raft in flavivirus-triggered Akt activation. During the 48-h kinetic study, JEV/DEN-2-induced Akt phosphorylation was found to peak in the first few hours postinfection and then subside (Fig. 1C). In addition, compared to the amounts of glycogen synthase kinase-3ß that is also a downstream component of the PI3K pathway, the protein levels of Akt were found to be reduced at the later time points after virus infection (Fig. 1C). These results suggest that JEV and DEN-2 induced Akt phosphorylation soon after the virus-cell encounter and that Akt phosphorylation was dependent on PI3K activation, through the interaction of virion and cell membrane in a lipid raft-dependent manner.



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  		FIG. 1. Flaviviral infection induces PI3K-dependent Akt phosphorylation. (A) Akt phosphorylation in JEV- and DEN-2-infected cells. N18, a mouse neuroblastoma cell line, cultured in medium with 0.5% FBS for 24 h was mock infected or infected with the sucrose cushion-purified JEV or DEN-2 at a multiplicity of infection (MOI) of 5 under the same culture condition (medium with 0.5% FBS). After 5, 15, 30, and 60 min of viral infection, the cell lysates were analyzed by Western blotting with the antibodies specific for Ser473-phospho-Akt (top panel) and Akt (bottom panel), respectively. (B) LY294002 and mßCD blocked the flavivirus-induced Akt phosphorylation. N18 cells pretreated with 50 µM LY294002 (lanes 4 and 5) or 100 ng of toxin B/ml (lanes 6 and 7) for 30 min were infected with the sucrose cushion-purified JEV or DEN-2 (MOI = 5) for 60 min before the cell lysates were harvested for Western immunoblotting as described in panel A. N18 cells treated with CHX (10 µg/ml; lanes 9 and 12) or MßCD (5 mM; lanes 10 and 13) were mock infected (lanes 8~10) or infected with the cushion-purified DEN-2 (MOI = 5; lanes 11 to 13) for 30 min. The purified DEN-2 in RPMI medium (lane 15) or just the medium alone (lane 14) were heated at 56°C for 30 min and then adsorbed to N18 cells for 30 min before the cell lysates were harvested for Western immunoblotting. (C) Kinetics of Akt phosphorylation and Akt and glycogen synthase kinase-3 protein levels in JEV- and DEN-2-infected cells. N18 cells were infected with JEV or DEN-2 (MOI = 5), and then the cells were cultured in medium with 0.5% FBS for various periods (in hours) as indicated at the top. The cell lysates were harvested for Western blotting with the antibodies indicated at the right of each panel. The intensities of Akt protein bands were quantified by using MetaMorph (Universal Imaging Corp.). The relative intensities of the Akt protein bands were calculated by using the band intensities at 1 h p.i. for each group as the denominators and are indicated for each lane (1.00, 1.09, etc.).

Blockage of PI3K activation enhanced cell death in JEV- and DEN-2-infected cells but had no effect on viral replication. To evaluate the effects of PI3K/Akt signaling in flavivirus-infected cells, we checked for virus-induced cytopathic effects and virus replication in N18 cells treated with the PI3K inhibitor LY294002 (75). As shown in Fig. 2A, inhibition of PI3K activity greatly increased the cell death induced by JEV and DEN-2 infections, whereas virus production was not affected by LY294002 treatment (Fig. 2B). Lowering the serum concentration in culture medium from 2 to 0.5% further sensitized the cells to undergo cell death triggered by flavivirus infection plus PI3K blockage, as measured by LDH releases (Fig. 2C) and XTT assay (Fig. 2D). Treatment of the cells by wortmannin, another specific PI3K inhibitor (2), gave results similar to those observed for LY294002 (data not shown). These observations were not limited to a single cell type. In A549 cells, a human lung carcinoma cell line that is susceptible for JEV and DEN-2 infection, both wortmannin (data not shown) and LY294002 accelerated JEV- and DEN-2-induced cell death (Fig. 3) without affecting viral protein expression (Fig. 4A and B), viral RNA replication (Fig. 4C), or infectious virus production, since the virus replicated to ca. 106 PFU/ml with or without PI3K inhibitor (data not shown). We also noticed that blocking of the PI3K activity has shorten the time period of active viral production (data not shown), since infected cells continued to produce infectious virus up to 3 days p.i., whereas, in the presence of PI3K inhibitor, the level of newly synthesized virus progeny was greatly reduced at late stage of infection, probably due to the obvious loss of cells, as shown in Fig. 2 and 3. These results suggest that, although not involved in virus replication, flavivirus-induced PI3K/Akt signaling protects the host cells from early death induced by JEV and DEN-2.



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  		FIG. 2. Effects of PI3K activation in flavivirus-infected N18 cells. (A) Morphology of flavivirus-infected N18 cells when PI3K is blocked. N18 cells were cultured in medium containing 2% FBS for overnight. The cells were then pretreated with various concentrations of LY294002 or just solvent control for 30 min and then infected with JEV or DEN-2 (MOI = 5). After 1 h of viral adsorption, the cells were washed and replenished with fresh media with 2% FBS and various concentrations of LY294002. (A and B) At 36 h p.i., the cell morphology was photographed (A), and the culture supernatants were collected for viral titration by plaque formation assays (B). The virus titers shown as PFU per milliliter were the averages and standard deviations from three independent experiments. N18 cells, cultured in medium supplemented with 0.5 or 2% FBS, were JEV, DEN-2, or mock infected in the presence of various concentrations of LY294002 for 30 h. (C and D) The cytotoxicity was quantified by LDH release (C), and the cell survival was measured by XTT assay (D). Data are shown as the percentage versus the mock-infected cells without drug treatment. Representative results from two independent experiments are shown here.



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  		FIG. 3. Blocking of PI3K activation enhances cell death in flavivirus-infected A549 cells. A549, a human lung carcinoma cell line, cultured in medium containing 2% FBS was pretreated with LY294002 (12.5 to 50 µM) or solvent alone for 30 min and then infected with JEV or DEN-2 (MOI = 5). After 1 h of viral adsorption, the cells were washed and replenished with fresh media with 2% FBS and LY294002 (12.5, 25, and 50 µM). (A) Cell morphology at 48 h p.i. (B) LDH release at 24, 36, and 48 h p.i. was determined as described in Materials and Methods. Representative results from two independent experiments are shown.



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  		FIG. 4. Flaviviral protein expression and RNA replication are not affected by PI3K inhibitor. A549 cells pretreated with LY294002 (0, 12.5, 25, and 50 µM) for 30 min were infected with JEV or DEN-2 (MOI = 5). At 12 and 24 h after infection with JEV (A) and DEN-2 (B), cell lysates were prepared for Western blotting with anti-E (top panels)- and anti-NS3 (bottom panels)-specific antibodies. (C) Viral RNA prepared from the DEN-2-infected cells treated with various doses of LY294002 was quantified by a fluorogenic real-time reverse transcription-PCR as described in Materials and Methods. The values indicate the threshold cycle (Ct), the calculated fractional cycle number at which the PCR product crosses a threshold of detection for each reaction.

Inhibition of PI3K resulted in apoptotic cell death in the early stages of JEV and DEN-2 infection through a caspase-dependent pathway. To elucidate the role of the PI3K/Akt pathway in regulating apoptosis in JEV- and DEN-2-infected neuronal N18 cells, the levels of apoptosis were assessed by measuring caspase-3 cleavage by using Western blotting. Caspase-3 is one of the key executioners of apoptosis, and its activation requires proteolytic processing of its inactive form into activated p17 and p12 fragments. Using an antibody recognizing the endogenous levels of full-length caspase-3 (35 kDa) and the large fragment of caspase-3 resulting from cleavage (17 kDa), JEV and DEN-2 infections caused caspase-3 cleavage at 36 h p.i. (Fig. 5A, top panel, lanes 8 and 9); however, when PI3K was blocked by LY294002, DEN-2 induced caspase-3 cleavage at the earlier time of 24 h p.i. (Fig. 5B, top panel, lane 6). Furthermore, the extent of caspase-3 cleavage was much more evident at 36 and 48 h p.i. with JEV and DEN-2 when LY294002 was present (Fig. 5A and B, top panels, lanes 8 and 9 and lanes 11 and 12). A low level of caspase-3 cleavage was also noticed in the mock-infected cells after 48 h of blockage of PI3K with LY294002 (Fig. 5B, top panel, lane 10), a finding similar to that noted in a recent report (23) in which a low level of PARP and caspase-3 cleavage was observed in HeLa cells treated with LY294002. In fact, not only caspase-3 but also caspase-6 was activated earlier in flavivirus-infected cells when treated with LY294002. Caspase-6 activation resulted in cleavage of its full-length precursor (35 kDa) in DEN-2- (at 24 h p.i., Fig. 5B, bottom panel, lane 6) and in JEV-infected cells (at 36 h p.i., Fig. 5B, bottom panel, lane 8) treated with LY294002.



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  		FIG. 5. LY294002 treatment promotes the cleavage of caspase-3 and caspase-6 in flavivirus-infected cells. N18 cells pretreated with or without LY294002 (10 µM) for 30 min were mock infected or infected with JEV or DEN-2 (MOI = 5) in the presence of solvent (A) or 10 µM LY294002 (B). At various time points after virus infection, the cell lysates were harvested for Western blotting with anti-caspase-3 (top panels) or anti-caspase-6 (bottom panels) antibody to detect the cleavage of the caspase precursor forms.

To further define the apoptotic pathway caused by flavivirus infection, we used TUNEL assays to reveal which caspase was involved. As shown in Fig. 6, in the treatment with LY294002, JEV and DEN-2 induced severe apoptotic cell death (66 and 56% of infected cells, respectively, at 26 h p.i.), which could be greatly reduced to <5% by the pan-caspase inhibitor, Z-VAD-FMK. Furthermore, inhibition of caspase-9 (Z-LEHD-FMK), but neither caspase-8 (Z-IETD-FMK) nor caspase-3 (Z-DQMD-FMK), substantially inhibited the apoptotic rate caused by JEV and DEN-2 when PI3K was blocked (Fig. 6). A lower dose of caspase inhibitor at 75 µM also gave complete protection for Z-VAD-FMK and some degrees of protection for Z-LEHD-FMK (~20% TUNEL-positive signals for LY294002 treated JEV- and DEN-2-infected cells). In order to verify that the caspase-8 inhibitor Z-IETD-FMK was functional at the concentration used, we demonstrated that Z-IETD-FMK was able to reduce cell death in MCF-7 cells treated with tumor necrosis factor alpha and CHX (data not shown). These results suggest that JEV and DEN-2 likely activate PI3K/Akt signaling to protect cells from an apoptotic pathway mainly initiated by caspase-9.



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  		FIG. 6. Inhibitors against pan-caspase and caspase-9 reduces the apoptotic cell death caused by JEV and DEN-2 infections under LY294002 treatment. N18 cells cultured in medium containing 0.5% FBS overnight were pretreated with LY294002 (10 µM) and various caspase inhibitors (150 µM) for 30 min. The cells were then infected with JEV or DEN-2 (MOI = 5) in the presence of LY294002 (10 µM) and various caspase inhibitors (150 µM). At 26 h p.i., the cells were fixed with paraformaldehyde solution (2% in phosphate-buffered saline [pH 7.4]) and stained by TUNEL assay. Cell labeling was then analyzed under a Leica fluorescence microscope. The percentage of cells that stained positive for TUNEL was determined from about 100 cells derived from two independent experiments, and the values are shown at the right side of each image.

To further reveal the role of caspase in JEV- and DEN-2-induced apoptosis, we used Western blotting to monitor the cleavage of PARP (12). PARP antibody detects endogenous levels of full-length PARP (116 kDa), as well as the C-terminal catalytic domain (89 kDa) and the N-terminal DNA-binding domain (24 kDa) of PARP resulting from caspase cleavage. Similar to the observations of caspase-3 and -6 cleavage shown in Fig. 5, PARP cleavage was also much more evident in the virus-infected N18 cells simultaneously treated with LY294002 (Fig. 7A and B, lanes 3 and 4). The pan-caspase (Z-VAD-FMK) and caspase-9 (Z-LEHD-FMK) inhibitors significantly blocked the cleavage of PARP (Fig. 7A and B, lanes 5 and 7). Neither caspase-8 (Z-IETD-FMK) nor caspase-3 (Z-DQMD-FMK) inhibitor markedly reduced PARP cleavage (Fig. 7A and B, lanes 6 and 8). Besides caspase-3, we also tested the roles in JEV and DEN-2 infections of two other effector caspases, caspase-6 and caspase-7. We found that the caspase-6 inhibitor (Z-VEID-FMK) blocked the cleavage of PARP (Fig. 7C and D, compare lane 4 to lanes 6 and 7), especially at higher doses, whereas the caspase-3/7 inhibitor (5-[(S)-(–)-2-(methoxymethyl) pyrrolidino] sulfonylisatin) had no significant effect on blocking PARP cleavage (Fig. 7C and D, lanes 8 to 10). The PARP cleavage induced by viral infection alone was also greatly reduced by either pan-caspase (Z-VAD-FMK), caspase-9 (Z-LEHD-FMK), or caspase-6 (Z-VEID-FMK) inhibitor at 150 µM in the JEV- and DEN-2-infected N18 cells (data not shown). These results suggest that an apoptotic pathway mainly involving caspase-9 and caspase-6 may play an important role in JEV- and DEN-2-induced cell death.



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  		FIG. 7. Caspase inhibitors block the cleavage of PARP in flavivirus-infected cells even when PI3K activation is blocked. N18 cells pretreated with LY294002 (10 µM) and various caspase inhibitors for 30 min were infected with JEV or DEN-2 (MOI = 5) for 1 h. After viral adsorption, the cells were washed and changed to fresh medium containing 0.5% FBS, LY294002 (10 µM), and caspase inhibitors as indicated. At 30 h p.i., the cell lysates were harvested for Western blotting with anti-PARP antibody. For better resolution, 4 to 12% gradient gels (NuPAGE Novex Bis-Tris Gel; Invitrogen) were used. The concentrations of inhibitors against various caspases used in panels A and B were 150 µM; in panels C and D the concentrations of caspase-6 inhibitor were 75 and 150 µM, and caspase-3/7 inhibitor concentrations were 1, 10, and 25 µM.

Bcl-2 overexpression sustained Akt protein level and reduced virus-induced PARP cleavage. Caspase-9 is primarily involved in the mitochondrion-dependent apoptosis pathway, and Bcl-2, a proto-oncogene, also plays an important role in mitochondrion-induced apoptosis. Previously, we found that overexpression of bcl-2 in BHK-21 cells appeared to delay the process of JEV (46)- and DEN-2-induced apoptosis (67). Furthermore, the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 plays a role in the establishment of JEV persistence (45). To study further the mechanisms of how Bcl-2 protects cells from flavivirus-induced apoptosis, we examined the role of PI3K/Akt signaling in cells overexpressing Bcl-2. As shown in Fig. 8A, in BHK-21 cells at 24 h p.i. JEV (lane 5) and especially DEN-2 (lane 6) infections lowered the Akt protein levels, similar to what was observed in N18 cells at 36 to 48 h p.i. (Fig. 1C). In great contrast, in B2-5, a human Bcl-2-overexpressing BHK-21 cell line (46), the Akt protein levels remained constant even after viral infection (Fig. 8A, lanes 7 to 12). Furthermore, the levels of Akt phosphorylation were also maintained at a higher level in Bcl-2-overexpressing cells after JEV and DEN-2 infections. Apparently, the overexpressed Bcl-2 could compensate for PI3K/Akt signaling in protecting cells from flavivirus-induced apoptosis, since the virus-induced PARP cleavage was greatly repressed in B2-5 cells even in the presence of LY294002, especially in JEV-infected cells (Fig. 8B and C). Further testing of other Bcl-2-overexpressing cell clones, besides B2-5 might rule out the potential cell clone effect and support a direct role of Bcl-2 in this protection pathway. Our results suggest that Bcl-2 is one of the major downstream mediators of the PI3K/Akt pathway in protecting cells from apoptotic cell death induced by JEV and DEN-2 infections.



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  		FIG. 8. Bcl-2 overexpression sustains Akt protein levels and reduces the PARP cleavage in flavivirus-infected cells. (A) BHK-21 and B2-5, a human Bcl-2-overexpressing BHK-21 cell line, were infected with JEV or DEN-2 (MOI = 5). At 12 and 24 h p.i., the protein lysates were analyzed by Western blotting with the antibodies indicated at the left side of each panel. The intensities of the Akt protein bands were quantified by MetaMorph (Universal Imaging Corp.). The relative intensities of Akt protein bands were calculated by using the band intensities of the 12-h-mock-infected BHK-21 and B2-5 cells as the denominators, respectively, and are indicated for each lane (1.00, 0.91, etc.). (B and C) BHK-21 and B2-5 cells were pretreated with solvent or LY294002 (7.5 µM) for 30 min, and then the cells were infected with JEV (B) or DEN-2 (C) (MOI = 5). After viral adsorption, the cells were washed and replenished with fresh media with 0.5% FBS and LY294002 (7.5 µM) as indicated. At 24 h p.i., the cell lysates were harvested for Western blotting with anti-PARP antibody.


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DISCUSSION
 
In this study, we found that flaviviruses such as JEV and DEN-2 not only trigger apoptotic signaling to kill the infected cells but also initiate survival signaling to hold the cells in a favorable condition for longer virus progeny production. Soon after the flavivirus-cell encounter, Akt is phosphorylated via lipid-raft formation and PI3K activation (Fig. 1), protecting the cells from caspase-mediated apoptotic cell death during the early stage of viral infection (Fig. 2, 3, and 5 to 7). However, PI3K/Akt signaling is not essential for JEV and DEN-2 replication, as the PI3K inhibitors LY294002 and wortmannin did not reduce viral RNA replication, viral protein expression, or infectious virus production (Fig. 2 and 4). Furthermore, overexpression of Bcl-2 overcomes the cell death induced by flavivirus, even when PI3K is blocked (Fig. 8), indicating that Bcl-2 serves as an important mediator of flavivirus-induced PI3K/Akt signaling.

Akt has been shown to inhibit mitochondrial cytochrome c release (38), suggesting that Akt inhibits apoptosis by maintaining the integrity of mitochondria. Bcl-2 family members either maintain or disrupt the integrity of mitochondrial membranes and thereby promote or inhibit the release of proapoptotic molecules from the mitochondrial intramembrane space. The Bcl-2 family of proteins consists of proapoptotic Bad, Bak, Bid, etc., and the antiapoptotic Bcl-2 and Bcl-xL. Akt has been shown to negatively regulate the activity of several proapoptotic members of the Bcl-2 family. Akt can phosphorylate and inhibit the activity of the BH3-only protein Bad (18, 20). Akt also antagonizes tBid-mediated Bax activation and mitochondrial Bak oligomerization, two downstream events thought to be critical for tBid-mediated apoptosis, via a glucose-dependent mechanism involving mitochondrial hexokinases (51). On the other hand, the enhanced cyclic AMP response element-binding protein activity induced by Akt signaling leads to upregulation of antiapoptotic Bcl-2 expression and cell survival (59). Moreover, Bcl-2 overexpression appears to inhibit the Akt degradation induced by daunorubicin, a broad-spectrum antitumor chemotherapeutic drug (39). In our study, JEV and DEN-2 infections likely activate PI3K/Akt signaling to protect mitochondrial integrity through a Bcl-2-related mechanism, preventing the cells from dying through a mainly caspase-9-mediated pathway during the early stage of infection. After large quantities of viral products such as viral RNA, viral proteins, and viral particles are produced, these viral molecules might trigger other stress responses such as ER stress (66) and cause another wave of cell death signaling, which finally and inevitably kills the infected cells.

Recently, a special role of caspase-6 in neuronal cell death was described. Caspase-6 is activated and responsible for neuronal apoptosis induced by serum deprivation (41). By direct microinjection of active recombinant enzymes, caspase-6, but not caspase-3, -7, or -8, induces human neuronal cell death (78). Downregulation of caspase-6 completely prevented neurotrophin-induced death, and depletion of caspase-3 gave only partial protection (73). Furthermore, cyclic AMP response element-binding protein, a transcription coactivator, has been identified as a new caspase-6 substrate and is specifically targeted during the onset of neuronal apoptosis (62). Our findings that caspase-6 but not caspase-3 and -7 inhibitors more efficiently blocks the cleavage of PARP induced by JEV and DEN-2 infections (Fig. 7C and D) in a mouse neuronal cell line, N18, is consistent with this line of findings that caspase-6 is strongly implicated in neuronal apoptosis. It has also been shown that astrocytes undergo apoptosis when microinjected with caspase-3 but not with caspase-6, -7, or -8 (78), suggesting a cell type-specific vulnerability to caspases in the central nerve system. Whether caspase-6 also plays a dominant role in flavivirus-induced cell death in different types of cells remains elusive.

Cross talk between PI3K/Akt and p38 MAPK has been described and may contribute to the balance of apoptosis and cell survival. Expression of active mutants of PI3K and Akt inhibit p38 activation and the apoptosis induced by the inhibition of extracellular signal-related kinase pathway (3). PI3K/Akt signaling has been shown to promote endothelial cell survival by inhibiting p38-dependent apoptosis (30). Activation of p38 and inactivation of Akt play a role in adenoviral early region 1A-mediated sensitization to apoptosis (47). Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 and inhibition of PI3K/Akt pathways (70). Previously, we have shown that JEV infection triggered p38 MAPK activation and a p38-specific inhibitor, SB203580, partially blocked JEV-induced apoptosis (66). Our finding in the present study that blocking of PI3K activation in JEV- and DEN-2-infected cells resulted in more severe apoptotic cell death might be due to the prominent apoptotic effect of p38 activation when PI3K/Akt pathway is blocked as observed in other systems.

The activation of the PI3K/Akt pathway in JEV- and DEN-2-infected cells appeared to be transient (Fig. 1), probably due to the following mechanism. The decrease of Akt protein levels in JEV- and DEN-2-infected cells during the late stage of viral infection (Fig. 1 and 8) is likely mediated by the virus-activated caspases, since downregulation of Akt levels during apoptosis could be blocked by a caspase inhibitor (60). However, before Akt degradation, it is very likely that viruses might trigger certain negative regulators to inactivate Akt by dephosphorylation, since Akt phosphorylation peaked at the rather early stage of JEV and DEN-2 infection of during the first few hours postinfection and then subsided afterward (Fig. 1C). The termination of PI3K/Akt activation can be achieved by phosphatidylinositol-3,4,5-trisphosphate phosphatases such as PTEN, SHIP1, and SHIP2, as well as other protein phosphatases such as PP1 and PP2A (6, 9, 25, 74). The mechanism involved in the negative regulation of the PI3K/Akt pathway in flavivirus-infected cells remains to be further studied.

PI3K/Akt signaling has also been implicated in translation control. Activation of TOR kinase by PI3K/Akt signaling regulates translation through two independent pathways, involving its downstream targets p70 S6 kinase (S6K) and the initiation factor 4E (eIF-4E)-binding protein 1 (4E-BP1) (4). S6K phosphorylation enhances the translation of 5'TOP mRNA, a class of mRNAs containing an oligopyrimidine tract at their transcriptional start, encoding ribosomal proteins and elongation factors (35). Phosphorylation of 4E-BP1, which is sensitive to wortmannin and LY294002, inhibits its interaction with eIF-4E, which binds the m7GpppN cap of mRNA and directs the correct positioning of ribosomal subunits to relieve the translation block (27). Our results that the life cycle of JEV and DEN-2 infections was not hindered by wortmannin (data not shown) and LY294002 (Fig. 2 and 4) strongly suggest that these viruses are resistant to the translational blockage triggered by PI3K inhibitors on cap-dependent translation, even though there is a cap structure on the 5' end of flaviviral RNA (49). The 3'-untranslated region of flaviviral RNA, which has been shown to bind various host factors such as translation elongation factor 1{alpha} and La antigen (5, 21) and is important in controlling DEN-2 translational efficiency (32), is likely to play a role in viral translation when PI3K activation is blocked.

In summary, the results presented here add JEV and DEN-2 to the growing list of viruses modulating the PI3K/Akt signaling pathway. JEV and DEN-2 could inhibit apoptosis by activating the PI3K/Akt in various cell types, such as neuronal (N18), epithelial (A549), and fibroblast (BHK-21) cells. Once the PI3K activity is blocked by LY294002 or wortmannin, the JEV- and DEN-2-infected cells would die at earlier time points of infection through a caspase-mediated pathway. However, the replication of JEV and DEN-2 was not affected by PI3K inhibition, indicating that a cap-independent translation mechanism might be adopted by these viruses when PI3K is blocked. Bcl-2, which might lead to persistent flaviviral infection (45), appears to be an important mediator in the PI3K/Akt survival signal triggered by JEV and DEN-2 infections since both the protein and the phosphorylation levels of Akt were higher in Bcl-2-overexpressing cells during the late stage of viral infection. A balance between the apoptotic and antiapoptotic signaling triggered by the interplay between host and virus likely regulates the outcomes of flaviviral infections.


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ACKNOWLEDGMENTS
 
Y.-L.L. was supported by grants from the National Science Council (NSC-92-2320-B-001-051 and NSC-93-2320-B-001-019) and the National Health Research Institute (NHRI-CN-CL9302P) and by Academia Sinica, Taiwan, Republic of China.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biomedical Sciences, Academia Sinica, No. 128, Sec. 2, Academy Rd., Nankang, Taipei 11529, Taiwan, Republic of China. Phone: (886)-2-2652-3902. Fax: (886)-2-2785-8847. E-mail: yll{at}ibms.sinica.edu.tw. Back


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Journal of Virology, July 2005, p. 8388-8399, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8388-8399.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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