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Journal of Virology, October 2005, p. 12332-12341, Vol. 79, No. 19
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.19.12332-12341.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation

Maitreyi S. Rajala,{dagger} Raju V. S. Rajala, Roger A. Astley, Amir L. Butt, and James Chodosh*

Molecular Pathogenesis of Eye Infection Research Center, Dean A. McGee Eye Institute, Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Received 26 March 2005/ Accepted 13 July 2005


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ABSTRACT
 
Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3ß and nuclear translocation of the p65 subunit of NF-{kappa}B, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.


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INTRODUCTION
 
Epidemic keratoconjunctivitis is an explosive and highly contagious ocular infection caused by the subgroup D adenovirus serotypes 8, 19, and 37 (15) and is associated with recurrent bouts of corneal stromal inflammation long after the acute infection has resolved. Critical to adenoviral entry into target cells, and following a primary interaction with the adenovirus receptor, arginine-glycine-aspartic acid amino acid sequences in the adenoviral capsid penton base interact with and cluster target cell integrins {alpha}vß3 and {alpha}vß5 (38, 51) to mediate activation of an intracellular signaling cascade involving focal adhesion kinases, Src family kinases, phosphoinositide 3-kinase (PI3K), and Rho family GTPases; actin polymerization; and clathrin-mediated endocytosis of the virus (30, 31). Therefore, internalization of the virus is mediated by an intracellular signaling cascade in the target cell. However, the potential roles of cell signaling molecules in adenovirus pathogenesis aside from viral internalization remain largely uninvestigated.

PI3K is a heterodimeric lipid kinase consisting of 85-kDa and 110-kDa regulatory and catalytic subunits, respectively (8, 19). PI3K regulates diverse cellular functions, including cell survival, cellular metabolism, and apoptosis (41, 49, 52). PI3K is activated upon recruitment to the plasma membrane via interactions between Src homology 2 (SH2) domains in its p85 regulatory subunit and phosphotyrosine residues located on membrane-bound receptors (3, 7, 43). Activated PI3K phosphorylates cellular inositol phospholipids at the D3 position of their inositol ring, and the resultant phospholipid products, phosphatidylinositol 3,4,5 triphosphate (PIP3) and 4,5 biphosphate, serve as second messengers to bind and regulate proteins containing pleckstrin homology domains (29). Akt, a serine/threonine protein kinase and downstream target of PI3K, is activated by binding of its pleckstrin homology domain to PIP3 and subsequent recruitment to the plasma membrane, where Akt becomes phosphorylated by phosphoinositide-dependent kinase 1 (2, 16, 20). Akt is an important regulator of apoptosis in neurons (13), fibroblasts (26), epithelial cells (27), and endothelial cells (17). Activated Akt promotes cell survival by phosphorylation and down-regulation of the activity of proapoptotic proteins BAD, glycogen synthase kinase 3 (GSK-3), and Forkhead transcription factor, along with specific caspases. Notably, the transcription factor NF-{kappa}B, identified in viral infections as an important regulator of apoptosis (34) and inflammation (6, 48), is also regulated by the PI3K/Akt pathway (47).

We have previously shown that infection of primary human corneal fibroblasts (HCF) with adenovirus type 19 results in activation of focal adhesion kinase, cSrc, and ERK1/2 and that these signaling proteins mediate the expression of inflammatory mediators, such as interleukin-8, from virus-infected cells (36, 37). Because PI3K has been reported to act in conjunction with cSrc to mediate adenoviral entry (30, 31), we turned to the study of PI3K and Akt in adenovirus-infected human corneal cells. Herein, we show for the first time that adenovirus type 19 infection of HCF results in activation of PI3K, Akt, and GSK-3ß and increases nuclear translocation of the p65 subunit of NF-{kappa}B. Internalization of adenovirus type 19 is not PI3K dependent in corneal fibroblasts, but inhibition of Akt by the PI3K inhibitor LY294002 or by a dominant negative Akt expression vector during adenovirus infection results in early apoptotic cell death and reduced virus titers. In the absence of virus infection, no such effect on cell viability of inhibiting PI3K or Akt is seen. Furthermore, overexpression of active Akt aborts the apoptotic effect of jointly administered LY294002 and virus. Adenoviral activation of the PI3K/Akt pathway maintains host cell viability during viral replication and therefore benefits the virus rather than the host.


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MATERIALS AND METHODS
 
Reagents. Antibodies to phospho-Akt (Ser 473 and Thr 308), total Akt, phospho-BAD (Ser 112 and 136), total BAD, phospho-GSK-3ß, and total GSK-3ß; the Akt kinase assay kit; PI3K inhibitor LY294002; anti-rabbit horseradish peroxidase (HRPO); and anti-mouse HRPO conjugates were obtained from Cell Signaling (Beverly, MA). The antiphosphotyrosine (PY99) and anti-p85 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphoserine and -phosphothreonine antibodies were purchased from Calbiochem (San Diego, CA). Phospho- and total Akt enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioSource International (Camarillo, CA). SYBR green master mix for real-time PCR was purchased from Applied Biosystems (Foster City, CA). The NF-{kappa}B subunit p65 ActivELISA kit was purchased from Imagenex (San Diego, CA). Lipofectamine 2000 and OptiMEM cell culture medium were obtained from Invitrogen Life Technologies (Carlsbad, CA). The dominant negative and constitutively active Akt1 expression vectors, BAD phosphorylation kit, and anti-Src antibodies were obtained from Upstate Biotechnology (Charlottesville, VA). The in situ apoptosis detection kit was purchased from Apotech (San Diego, CA). The Apo-ONE Homogeneous caspase-3/7 assay kit was purchased from Promega (Madison, WI). [{gamma}-32P]ATP was obtained from Perkin-Elmer Life Sciences, and D-myo-phosphatidylinositol-4-5-biphosphate (PI-4,5-P2) was obtained from Echelon Research Laboratories Inc. (Salt Lake City, UT). All other reagents were analytical grade and were from Sigma (St. Louis, MO).

Cell culture, transfection, and virus infection. Primary HCF were derived from donor corneas (North Carolina Eye Bank, Winston-Salem, North Carolina) as previously described (11) and were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. Our use of human corneas of deceased donors was approved by the Institutional Review Board of the University of Oklahoma Health Sciences Center and is consistent with the principles expressed in the Declaration of Helsinki. For inhibitor analysis, HCF were pretreated overnight with the PI3K inhibitor LY294002 (20 µM) prior to virus infection. The inhibitor was included in all subsequent steps. Cell toxicity due to 20 µM LY294002 was ruled out by trypan blue exclusion. For expression of mutant and constitutively active Akt in HCF prior to adenoviral infection, pUSE constructs (Upstate Biotechnology) containing Myc-His-tagged mouse Akt1 cDNA (dominant negative), Akt1 cDNA (activated), or no expressed gene were transiently transfected into HCF with Lipofectamine 2000 (Invitrogen) 24 h prior to viral infection. Transfection efficiency was determined by transfection of a green fluorescent protein-expressing vector, and successful expression was confirmed by Western blotting.

Adenovirus type 19 was cultured and grown as previously described (9). The virus was CsCl gradient purified and dialyzed against a 10 mM Tris (pH 8.0) buffer with 80 mM NaCl, 2 mM MgCl2, and 10% glycerol. For viral infection, monolayer HCF cultures were grown to 90 to 95% confluence, washed thrice, incubated in serum-free DMEM for 4 h prior to infection, and then infected with purified virus or mock infected with virus-free dialysis buffer. A multiplicity of infection of 50 was chosen to ensure complete and synchronous activation of host cell receptors. To evaluate viral replication in the presence of PI3K inhibitor, we used a previously described method (21) in which HCF were pretreated overnight with LY294002 and then infected with purified virus in LY294002 at the same concentration. After 1 h of adsorption at 37°C, the cultures were washed and fed, and the supernatants were collected every 48 h thrice for titer determination by the tissue culture infectious dose (TCID) method. LY294002 was included in all steps.

Preparation of cell extracts. Cells were harvested in 200 µl of chilled lysis buffer consisting of phosphate-buffered saline, 1% Triton X-100, 2 mM EDTA, and 0.2 mM sodium orthovanadate, along with protease inhibitors, including phenylmethylsulfonyl fluoride (1 mM), pepstatin A (5 µg/ml), leupeptin (10 µg/ml), and aprotinin (10 µg/ml), and incubated on ice for 10 min. Cells were dislodged from the plates with a cell scraper, and the cell lysates were centrifuged at 13,000 x g for 30 min at 4°C.

SDS-PAGE and Western blot analysis. Cell lysates were boiled in 2x sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad, Hercules, CA) and loaded on separating gels. Spectrophotometric bicinchoninic acid analysis was performed to normalize protein loading. After SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer, nitrocellulose membranes were blocked overnight at 4°C in 5% bovine serum albumin. Incubations with primary antibody diluted in blocking buffer were performed for 2 h at room temperature. Immunoblots were washed thrice with Tris-buffered saline after both antibody incubation, and antibody reactivity was determined with enhanced chemiluminescent reagents (Amersham Biosciences, Piscataway, NJ) using horseradish peroxidase-coupled secondary antibodies. Prior to reprobing of developed blots, blots were incubated in ImmunoPure immunoglobulin G elution buffer (Pierce, Rockford, IL) for 60 min.

Immunoprecipitation. Cell lysates were precleared by incubation with 50 µl of protein A agarose beads (5 mg/ml) with gentle mixing for 2 h at 4°C, followed by centrifugation at 10,000 x g. Cleared supernatants were mixed with 2.5 µg of PY99 antibody, 2.5 µg of anti-p85 antibody, or 2.5 µg of anti-cSrc antibody and incubated overnight at 4°C. Then, 50 µl of protein A agarose beads was added for an additional 2 h at 4°C. Immunoprecipitates were washed twice with solubilization buffer (0.1% Triton X-100, 137 mM NaCl, 20 mM Tris-HCl [pH 8.0], 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 1 mM Na3VO4, 10 µg/ml leupeptin, and 1 µg/ml aprotinin) and once with phosphorylation buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 2 mM MgCl2, and 0.2 mM Na3VO4). Immune complexes were solubilized with equal volumes of 2x SDS sample buffer and boiled for 5 min. Insoluble material was removed by centrifugation, and the supernatants were subjected to SDS-PAGE. After transfer, separated proteins were probed with anti-p85, anti-PY99, or anti-cSrc antibodies. All Western blotting and immunoprecipitation experiments were performed at least three times with similar results.

PI3K assay. The PI3K assay was performed in a final volume of 50 µl containing immune complexes, 0.2 mg/ml PI-4,5-P2, 50 µM ATP, 50 µCi [{gamma}-32P]ATP, 5 mM MgCl2, and 10 mM HEPES buffer (pH 7.5) and incubated at room temperature for 15 min, and the reaction was terminated by adding 100 µl of 1 N HCl followed by 200 µl of chloroform-methanol (1:1 [vol/vol]). Radiolabeled phospholipids were extracted, spotted onto a potassium oxalate-coated silica gel 60 thin-layer chromatography plate that had been coated with 1% potassium oxalate in 50% methanol, and then baked at 100°C for 1 h prior to use. Spotted lipids on the silica gel plate were resolved by thin-layer chromatography in a solvent system of 2-propanol-2 M acetic acid (65:35 [vol/vol]) and then air dried, and the incorporation of [{gamma}-32P]ATP was detected by autoradiography. Densitometric scans of kinase assay autoradiographs were analyzed by ONE-DSCAN software (Scanalytics, Billerica, MA) in the linear range of detection and absolute values then normalized. Densitometry parameters included threshold and bandwidth and were set identically for tested bands.

Akt kinase assay. Akt kinase activity was measured using a kit (Cell Signaling) following the manufacturer's instructions. In brief, 100 µg of total protein was immunoprecipitated with 20 µl of immobilized anti-Akt antibody and incubated overnight at 4°C. The immune complexes were washed twice with lysis buffer and once with kinase buffer, then suspended in 40 µl of kinase buffer supplemented with 200 µM ATP and 1 µg of paramyosin fused to the GSK-3 fusion peptide as a target substrate, and incubated for 30 min at 30°C. The reaction was terminated with 20 µl of 3x SDS sample buffer, and the samples were pulse centrifuged for 1 min. Twenty-five microliters from each supernatant was subjected to SDS-PAGE followed by immunoblot analysis using anti-phospho-GSK-3{alpha}/ß (Ser 21/9) antibody. The kinase assays were each performed three times with similar results.

Phospho-Akt ELISA. Cell lysates collected from mock- or virus-infected cells in the presence or absence of LY294002 were subjected to total and phospho-Akt ELISAs 20 and 30 min after infection. Protein concentrations were adjusted to 0.1 µg/µl with sample diluent. Phosphorylated Akt (Ser 473) protein was used as the standard. One hundred microliters of each sample was incubated overnight at 4°C in a microtiter plate coated with anti-Akt monoclonal antibody. After three washes, each plate was incubated for 1 h at room temperature with 100 µl of anti-phospho-Akt (Ser 473). The plate was washed again, HRPO-labeled anti-rabbit immunoglobulin G was added, and the plate was incubated for 30 additional minutes. The plate was washed and treated with 100 µl of chromogen substrate (stabilized chromogen-tetramethylbenzidine). The reaction was stopped, and the plate was read at 405 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). The means of triplicate ELISA values, normalized by adjustment for total Akt levels as measured by ELISA on the same cell lysates used for the phospho-Akt ELISA, were compared by analysis of variance (ANOVA). The Akt ELISA was performed twice with similar results.

Real-time PCR. Real-time PCR was performed as previously described (37). Briefly, HCF cultures were infected with adenovirus type 19 or mock infected with virus-free buffer in MEM-2% fetal bovine serum in the presence or absence of LY294002. Virus was adsorbed at 37°C for 1 h and incubated for one additional hour. Total RNA was isolated by a single-step RNA isolation method with TRIzol reagent (GIBCO-BRL Life Technologies) using instructions provided by the manufacturer. Proteins were removed by chloroform extraction, RNA was precipitated with ethanol, and the RNA pellet was resuspended in diethyl pyrocarbonate H2O. RNAseIn (Promega) and DNase I (Promega) were used to prevent RNase action and remove contaminating DNA, respectively, followed by phenol-chloroform extraction, ethanol precipitation, and resuspension of the RNA. Spectrophotometric readings at 260 nm were used to determine RNA concentrations. The quality of each RNA sample was determined by calculating the ratio of the optical density at 260 nm to that at 280 nm (a ratio of approximately 1.8:1 or greater indicated only nondegraded RNA). Two micrograms of total RNA was reverse transcribed to yield single-stranded cDNA using 200 U Moloney murine leukemia virus reverse transcriptase (Promega), 2 mM concentrations of deoxynucleoside triphosphates, 20 U of rRnasin (Promega), and 250 nM reverse primer specific for the adenoviral early region 1A (E1A) 10s transcript (see below). The mixture containing RNA template and primer was first heated at 70°C for 10 min, chilled on ice, and then, after the addition of the remaining components of the reaction mix, incubated at 37°C for 1 h followed by enzyme inactivation at 70°C for 15 min.

Quantitative real-time PCR amplification was performed using an ABI Prism 7000 sequence detection system (PE Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. PCR primers for the E1A 10s product were designed using Primer Express software (PE Applied Biosystems) using the known adenovirus type 9 (subgroup D) adenovirus sequence (46) (GenBank accession no. AF099665): Forward (nucleotides 715 to 735), 5' GGA GGT AGA TGC CCA TGA TGA 3'; and Reverse (nucleotides 805 to 784), 5' GTT GGC TAT GTC AGC CTG AAG A 3'. Three microliters of cDNA was subjected to real-time PCR in a final volume of 50 µl containing 25 µl of 2x SYBR green master mixes and 250 nM concentrations of specific forward and reverse primers. Samples were then analyzed by comparison of the number of PCR cycles required to reach the midpoint of each amplification curve or threshold cycle (CT). Comparison of gene expressions between two samples was performed by calculating the difference (n-fold) in mRNA abundance using the formula 2–{Delta}{Delta}CT, where {Delta}{Delta}CT = (CTtarget – CTGAPDH)virus – (CTtarget – CTGAPDH)buffer. Reactions lacking template were used to control for primer-dimer formation. To control for contamination by residual genomic DNA, reverse transcriptase was omitted from parallel cDNA synthesis reactions in all experiments. The real-time PCR experiment was performed three times.

BAD phosphorylation. One hundred micrograms of total protein harvested from virus- or mock-infected HCF with or without LY294002 was incubated for 30 min at 30°C with His-tagged murine Bad fusion protein bound to agarose beads, as per the manufacturer's instructions. The mixture was then centrifuged at 10,000 x g, and the beads were washed thrice with cold Tris-buffered saline and suspended and boiled for 5 min in 2x SDS sample buffer. After repeat centrifugation, the supernatants were subjected to Western blot analysis using anti-phospho-BAD antibodies (Ser 112 and 136).

NF-{kappa}B activation (p65 subunit translocation). Nuclear extracts were tested for the presence of the NF-{kappa}B subunit p65, using the NF-{kappa}B ActivELISA kit from Imagenex, according to the manufacturer's instructions. Proteins were harvested in cold phosphate-buffered saline using a cell scraper at 60 and 90 min postinfection and centrifuged at 3,000 x g. The cell pellet was suspended in 400 µl of hypotonic lysis buffer, incubated on ice for 15 min, mixed with 30 µl of 10% NP-40, and centrifuged at 10,000 x g for 1 min. Following centrifugation, 220 µl of cold nuclear extraction buffer was added, and the mixture was incubated on ice for 30 min followed by centrifugation at 10,000 x g. ELISA plates were precoated with anti-p65 antibody, and the presence of p65 was detected by the addition of a second anti-p65 antibody followed by alkaline phosphatase-conjugated secondary antibody and colorimetric analysis at 405 nm. Each experimental condition was duplicated in three wells, and the means for each experimental condition were compared by ANOVA. The NF-{kappa}B p65 translocation assay was performed three times with similar results.

Detection of apoptosis by TUNEL staining. HCF were cultured overnight in OptiMEM and 20 µM LY294002 or dimethyl sulfoxide (DMSO). Treated cells were infected with virus or mock infected with virus-free buffer. At 24, 48, and 72 h postinfection, cells were fixed with 1% paraformaldehyde. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis for DNA fragmentation was carried out using the Apoptag peroxidase-in situ apoptosis detection kit (Apotech) following the manufacturer's instructions. Each experimental condition was duplicated in three wells, and the number of apoptotic cells in 10 high-power fields in each well was counted in masked fashion and averaged. The means for each experimental condition were compared by ANOVA. The TUNEL assay was performed four times with similar results.

Detection of caspase-3/7 activation. Caspase-3/7 activation with and without LY294002 was determined using the Apo-ONE Homogeneous caspase-3/7 assay kit (Promega) according to manufacturer's instructions. For each experimental condition, 100 µl of each cell lysate collected from ~4 x 105 cells/well was incubated with Apo-ONE caspase-3/7 substrate (Z-DEVD-R110) in a black multiwell plate in the dark for 10 h and the fluorescence of each well measured with an excitation wavelength of 485 nm and an emission wavelength of 531 nm on a microplate reader. Infections were done in triplicate, and the mean values from triplicate wells were compared by ANOVA.

GST pull-down experiments. Pull-down experiments were carried out using glutathione S-transferase (GST)-full-length p85 (GST p85-FL) and N-terminal SH2 domain (GST p85-N-SH2) fusion proteins as described previously (40). Cell lysate proteins from virus- or mock-infected corneal cells were precleared by incubation for 1 h with 50 µl of glutathione Sepharose beads with gentle mixing followed by centrifugation at 10,000 x g for 1 min at 4°C. The cleared supernatants were incubated overnight with GST, GST p85-FL, or GST p85-N-SH2 fusion proteins under constant mixing. The Sepharose beads were washed thrice with 500 µl of HNTG buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) and centrifuged at 13,000 x g for 1 min at 4°C. The proteins were boiled in 2x SDS sample buffer for 5 min, and the eluted proteins were subjected to SDS-PAGE followed by immunoblotting with anti-PY99 or anti-cSrc antibody.


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RESULTS
 
PI3K activation in human corneal fibroblasts upon adenovirus type 19 infection. To evaluate the possible role of PI3K in adenovirus-infected corneal cells, lysates from virus- or mock-infected cells were immunoprecipitated with PY99 antibody followed by Western blot analysis with antibody against the p85 subunit of PI3K. We found relatively more p85 in virus-infected cells (Fig. 1A) and increased PI3K activity within 10 min after virus infection (Fig. 1B). Pretreatment of cells with LY294002, a specific inhibitor of PI3K, blocked the kinase activity. Addition of purified virus directly to p85 immunoprecipitates from uninfected cells did not induce PI3K activity, and immunoprecipitation with anti-p85 antibody of lysates from virus- and mock-infected cells and probing with anti-PY99 antibody revealed no phosphorylation of p85 in either virus- or mock-infected cells (data not shown).



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  		FIG. 1. PI3K is activated in adenovirus-infected human corneal fibroblasts. HCF were serum starved overnight and infected with purified adenovirus type 19 or mock infected with virus-free dialysis buffer. Cell lysates were prepared 10 min after infection. (A) Total protein (200 µg) was immunoprecipitated with antiphosphotyrosine antibody (PY99) followed by Western blot analysis with anti-p85 antibody (upper panel). Five micrograms of the same protein was separated by SDS-PAGE and probed with anti-p85 antibody to assess the amount of p85 in virus- and mock-infected protein lysates (lower panel). (B) Cells were pretreated overnight with LY294002 (20 µM) or DMSO as a control prior to infection. Infected cell lysates in the presence or absence of LY294002 were immunoprecipitated with antiphosphotyrosine antibody and assayed for PI3K activity using PI-4,5-P2 and [{gamma}-32P]ATP as substrate. Densitometric values normalized to allow direct comparison of band intensity are shown above each lane.

Cell lysates were subjected to Western blot analysis using phospho-specific antiserine and antithreonine antibodies. In virus-treated cells, a 60-kDa protein was found to be serine and threonine phosphorylated within 20 to 30 min after infection (Fig. 2A). Western blot analyses with an anti-phospho-Akt (Ser 473) antibody revealed increased Akt phosphorylation in virus-infected cells within 20 to 30 min after infection, while total cellular Akt levels remained unchanged (Fig. 2B). Pretreatment of corneal cells with LY294002 reduced virus-induced phosphorylation of Akt at Ser 473 and Thr 308 but did not alter the quantity of total Akt (Fig. 3A). By ELISA, Akt phosphorylation (Ser 473) was also shown to be increased in infected over mock-infected cells (P < 0.05); this effect was blocked by pretreatment of cells with LY294002 (Fig. 3B). By kinase assay, increased Akt activity was observed at 20 and 30 min after viral infection (Fig. 3C) and was abrogated by pretreatment with LY294002 (Fig. 3D).



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  		FIG. 2. Adenovirus infection induces increased phosphorylation of Akt in human corneal fibroblasts. Serum-starved HCF were infected with purified adenovirus type 19 or mock infected with virus-free dialysis buffer. Lysates were prepared from infected cells at the indicated times after infection. Ten micrograms of total protein was separated by SDS-PAGE. (A) Western blot analysis was carried out with specific antibodies to phosphoserine (top) and phosphothreonine (bottom). (B) Ten micrograms of total protein isolated from infected or mock-infected cells was separated by SDS-PAGE followed by Western blot analysis with phospho-specific anti-Akt antibody (top). The blot was stripped and reprobed with an antibody against total Akt (bottom).



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  		FIG. 3. The PI3K inhibitor LY294002 inhibits Akt activation in adenovirus-infected human corneal fibroblasts. Serum-starved HCF were treated overnight with LY294002 (20 µM) or DMSO as a control prior to infection with purified adenovirus type 19 or mock infection with virus-free dialysis buffer. Cell lysates were prepared at 20 and 30 min after infection. (A) Ten micrograms of total protein was isolated and subjected to Western blot analysis with anti-Akt antibodies specific to phosphorylation at Ser 473 (top panel) and Thr 308 (middle panel). The latter membrane was stripped and reprobed with antibody to total Akt (lower panel). (B) Ten micrograms (0.1 µg/µl) of total protein lysates collected from infected cells at the indicated times in the presence or absence of LY294002 was subjected to phospho-Akt ELISA to quantify phosphorylation on the Ser 473 site. Error bars represent the standard deviation (SD) of each mean (virus-DMSO versus mock-DMSO, P < 0.05; virus-LY294002 versus virus-DMSO, P < 0.05 at both time points). (C) One hundred micrograms of total protein from infected and mock-infected cells was subjected to an Akt kinase assay using immobilized Akt antibody and GSK-3 fusion peptide as the substrate. Immune complexes were solubilized in sample buffer and separated by SDS-PAGE. Phosphorylation of the GSK-3 fusion peptide was determined by Western blot analysis using anti-phospho-GSK-3{alpha}/ß (Ser 21/9) antibody. (D) An Akt kinase assay was also performed on corneal fibroblasts pretreated with LY294002 (20 µM) or DMSO. Densitometric values normalized to allow direct comparison of band intensity are shown above each lane.

Because prior studies suggested that inhibition of PI3K blocked internalization of adenovirus type 2 (subgroup C) into human lung carcinoma cells (31), we sought to determine if inhibition of PI3K would similarly block adenovirus type 19 internalization into corneal fibroblasts. Expression of adenoviral E1A and other early genes can be demonstrated by real-time PCR within 1 h of adenovirus type 19 infection of corneal fibroblasts and continues to rise over the next several hours (data not shown). Preinfection treatment of HCF with PP2, a specific inhibitor of Src, reduced E1A expression at 1, 2, and 3 h postinfection to levels comparable with uninfected cells and markedly less than infected DMSO-pretreated controls (1, 2, and 3 h postinfection, 2.8-fold less, 17.2-fold less, and 49.6-fold less, respectively) (Table 1), while LY294002 pretreatment at a dose shown to block Akt phosphorylation in adenovirus-infected HCF (20 µM) was associated with a modest increase or no difference in E1A mRNA expression compared to infection in the presence of DMSO solvent (1, 2, and 3 h postinfection, 2-fold greater, 2.3-fold greater, and no change, respectively) (Table 1). Although the absence of adenovirus type 19 early gene expression in cells treated with PP2 could be due to a mechanism other than failure of viral internalization, the normal levels of E1A expression we observed in the presence of LY294002 suggest that PI3K activity is not essential to adenovirus type 19 internalization in HCF.


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  		TABLE 1. Change in early adenoviral gene expression as a surrogate marker for viral internalization in the presence of inhibitors of cSrc and P13K

Activation of downstream targets of the PI3K/Akt pathway. Immunoblots of protein lysates from mock- and virus-infected cells probed with a phospho-specific antibody against GSK-3ß revealed an increased level of GSK-3ß phosphorylation within 30 min of infection; this effect was blocked by LY294002 (Fig. 4A). In in vitro experiments, PI3K-dependent phosphorylation of exogenous BAD at Ser 112 was observed within 15 min after infection (data not shown). In separate experiments, nuclear protein harvested from virus- or mock-infected cells and subjected to ELISA for the presence of the p65 subunit of NF-{kappa}B revealed increased nuclear translocation of the p65 subunit of NF-{kappa}B in virus-infected cell nuclear extracts at 60 and 90 min after infection (P < 0.05 for both time points), whereas infection-induced nuclear NF-{kappa}B translocation was blocked by LY294002 treatment (Fig. 4B) (P < 0.05 for both time points).



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  		FIG. 4. Adenovirus infection results in GSK-3ß phosphorylation and induces NF-{kappa}B p65 subunit nuclear translocation in a PI3K-dependent manner. Serum-starved HCF were pretreated with LY294002 (20 µM) or DMSO prior to infection and infected with adenovirus type 19 or mock infected with virus-free dialysis buffer. (A) At 40 min after infection, the cells were lysed, and 10 µg of total protein was subjected to SDS-PAGE followed by Western blot analysis using a phospho-specific anti-GSK-3ß antibody (upper panel). The blot was stripped and reprobed with total GSK-3ß antibody (lower panel). (B) Virus- or mock-infected cells, pretreated with LY294002 or DMSO, were collected 60 and 90 min after infection. The cell pellet was prepared and subjected to ELISA for p65 nuclear translocation as described in Materials and Methods. Error bars represent the SD of each mean (virus-DMSO versus mock-DMSO, P < 0.05; virus-DMSO versus virus-LY294002, P < 0.05 at both time points). OD, optical density.

Premature cell death due to PI3K/Akt inhibition in adenovirus-infected corneal fibroblasts. Because PI3K-mediated Akt activation is important for cell survival (33), we wished to determine whether activation of the PI3K/Akt signaling pathway by adenovirus infection would affect cell viability. Pretreatment of cells with LY294002 followed by adenovirus infection caused cytopathic effect as early as 24 h after infection—a time when cells infected with adenovirus but not pretreated with LY294002 showed minimal morphological changes (data not shown). TUNEL staining at 48 h postinfection in cells that had been pretreated with LY294002 revealed a significantly greater number of TUNEL-positive cells than either adenovirus-infected cells pretreated with DMSO or mock-infected cells pretreated with LY294002 (Fig. 5A) (P < 0.05). The proportion of adherent cells found to be TUNEL positive among adenovirus-infected, LY294002-pretreated corneal cells increased from 8% at 48 h to 34% at 72 h postinfection (data not shown). A significant increase in caspase-3/7 activation in cells pretreated with LY294002 was also observed at 24 h after infection (Fig. 5B) (P < 0.05).



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  		FIG. 5. Inhibition of PI3K or Akt in adenovirus-infected human corneal fibroblasts results in premature cell death due to apoptosis and is associated with activation of cellular caspases. (A) HCF were cultured overnight in LY294002 (20 µM) or DMSO prior to infection with purified adenovirus type 19 or virus-free dialysis buffer. At 48 h after infection, photomicrographs were taken and apoptosis was evaluated by TUNEL staining. At this time postinfection, cell death was marked in LY294002-pretreated adenovirus-infected HCF (far right photomicrograph), corresponding to an increase in TUNEL staining (virus-DMSO versus virus-LY294002, P < 0.05). Error bars represent the SD of the mean number of apoptotic cells per high-power field (hpf) for each treatment group. (B) Serum-starved HCF were pretreated with LY294002 or DMSO and infected with purified adenovirus or virus-free buffer. Cell lysates collected from ~4 x 105 cells per experimental condition were analyzed for caspase-3/7 activation as described in Materials and Methods. Error bars represent the SD of mean optical density values obtained from the triplicate wells of mock- or virus-infected cells in the presence of DMSO or LY294002 (virus-DMSO versus virus-LY294002, P < 0.05). (C) HCF were transiently transfected with Myc-His-tagged mouse Akt1 cDNA (dominant negative) or empty expression vector. At 24 h after transfection, cells were infected with purified adenovirus type 19 or mock infected with virus-free dialysis buffer. Apoptotic cells were assessed by TUNEL staining at 48 h postinfection (virus-infected dominant negative Akt versus mock-infected dominant negative Akt, P < 0.05; virus-infected dominant negative Akt versus virus-empty vector, P < 0.05). (D and E) HCF were transiently transfected with Myc-His-tagged mouse Akt1 cDNA (active Akt) or empty expression vector or mock transfected without DNA, followed by overnight treatment of all cells with LY294002; 24 h after transfection, HCF were infected with purified adenovirus type 19 or mock infected with virus-free dialysis buffer. (D) Western blotting with an antibody against phospho-Akt (Ser 473) was performed to confirm the overexpression of active Akt. (E) Under the same experimental conditions, a TUNEL assay was performed 48 h after infection, as described above. Error bars represent the SD of each mean number of apoptotic cells per high-power field (hpf). The means of the number of apoptotic cells detected per high-power field were compared by t test (mock-active Akt versus virus-active Akt, P = 0.46; mock-empty vector versus virus-empty vector, P < 0.05; mock infected and not transfected versus virus infected and not transfected, P < 0.05).

In subsequent experiments, dominant negative Akt-transfected cells coinfected with adenovirus died well before those transfected with an empty expression vector, similar to that seen with LY294002 treatment. TUNEL staining at 48 h postinfection revealed that virus-infected HCF pretransfected with dominant negative Akt contained significantly greater numbers of TUNEL-positive cells than virus-infected cells transfected with an empty vector or mock-infected cells transfected with dominant negative Akt (Fig. 5C) (P < 0.05).

To determine whether overexpression of Akt would circumvent the effect of PI3K inhibition in adenovirus-infected HCF, cells were transiently transfected with either constitutively active Akt or an empty expression vector, treated overnight with LY294002, and then mock or virus infected. Overexpression of active Akt in transfected cells was confirmed by Western blot analysis using a phospho-specific anti-Akt antibody (Fig. 5D). TUNEL assays demonstrated no significant change in the number of apoptotic cells between mock- and virus-infected cells transfected with active Akt and exposed to LY294002 (Fig. 5E) (P = 0.46). A significantly increased degree of TUNEL staining between mock- and virus-infected cells was evident in LY294002-treated cells transfected with the empty expression vector and in LY294002-treated cells treated with Lipofectamine alone without DNA transfection (P < 0.05 for both comparisons).

Because inhibition of PI3K and Akt appeared to reduce cell viability in adenovirus-infected cells, we wished to determine if LY294002 would also render the cells less susceptible to viral replication. Virus titers in the DMSO-treated cells increased between 4 and 6 days postinfection, at a time when the virus titer in the LY294002-treated cells fell to the lower limit of detection of the assay (Fig. 6) (9 x 103 versus 10 TCID/ml, respectively; P < 0.05).



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  		FIG. 6. Inhibition of PI3K in adenovirus-infected human corneal fibroblasts results in reduced viral replication. To evaluate viral replication in the presence of PI3K inhibition, HCF were pretreated overnight with LY294002 (20 µM) and then infected with purified virus in LY294002 at the same concentration. After 1 h of adsorption at 37°C, the cultures were washed and fed with media also containing LY294002, and the supernatants were collected every 48 h for titer determination by the TCID method. Error bars represent the SD of each mean. The means of the titers at each time point were compared by t test (mock- versus virus-infected HCF, P < 0.05 at 6 days postinfection).

Interaction between cSrc and p85 in adenovirus type 19-infected cells. Our previous studies demonstrated phosphorylation and activation of cSrc in adenovirus type 19-infected HCF (37). Therefore, we wished to determine if cSrc plays a role in activation of PI3K. We performed GST pull-down assays with full-length p85 (GST p85-FL) and subjected the recovered proteins to Western blot analysis with anti-PY99 antibody. As shown in Fig. 7A, top panel, a 60-kDa protein was present to a greater degree in pull-downs from the virus-infected cell lysates. When stripped and reprobed with anti-cSrc antibody, the resultant blot indicated increased cSrc in the virus-infected cell lysates (Fig. 7A, middle). An identical GST p85-FL pull-down experiment carried out and probed directly with anti-cSrc antibody also demonstrated increased cSrc in the GST p85-FL pull-down from virus-infected cells (data not shown), and Western blotting performed directly on cell lysates demonstrated no difference in total cSrc levels between virus- and mock-infected cells (Fig. 7A, bottom). The p85 subunit of PI3K can interact with Src phosphotyrosine through its SH2 domains (8) or Src proline-rich regions through its SH3 domain (23, 39). To determine whether the observed interaction between cSrc and p85 was phosphorylation dependent, we performed pull-down experiments with the N-SH2 domain of p85, which specifically mediates phosphorylation-dependent binding. Virus-infected cell lysate proteins recovered from p85-N-SH2 pull-downs probed with PY99 demonstrated increased immunoreactivity at 60 kDa (Fig. 7B, top). The identity of the 60-kDa protein was confirmed after membrane stripping and reprobing as cSrc (Fig. 7B, middle). Western blotting, performed directly on cell lysates from virus- and mock-infected cells in this second experiment, again demonstrated no difference in total cSrc levels (Fig. 7B, bottom). Finally, to determine whether the observed relationship between p85 and phosphorylated cSrc after adenovirus infection is associated with PI3K activation, virus- and mock-infected cell lysates immunoprecipitated with anti-cSrc antibody were subjected to PI3K assays. A demonstrable increase in PI3K activity was associated with anti-cSrc immunoprecipitates (Fig. 7C).



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  		FIG. 7. cSrc and p85 interact in adenovirus-infected corneal cells. Serum-starved HCF were infected with adenovirus type 19 or mock infected with virus-free dialysis buffer, and lysates were prepared at 10 min postinfection. (A) Two hundred micrograms of total protein harvested from mock- or virus-infected cell lysates was incubated with GST p85-FL fusion protein followed by Western blot analysis of bound protein with antiphosphotyrosine (PY99) antibody (upper panel). The same blot was stripped and reprobed with anti-cSrc antibody (middle panel). At the same time, 5 µg of total protein from mock- and virus-infected cells was subjected directly to SDS-PAGE and Western blot analysis with anti-cSrc antibody to compare total cSrc in the samples in the absence of GST pull-down (lower panel). (B) The same experiment as in panel A was performed, except that the GST p85-N-SH2 fusion protein was used for the pull-down. (C) One hundred micrograms of total protein was immunoprecipitated with anti-cSrc antibody and assayed for PI3K activity using PI-4,5-P2 and [{gamma}-32P]ATP as substrate. Densitometric values normalized to allow direct comparison of band intensity are shown above each lane.


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DISCUSSION
 
We have suggested that leukocyte infiltration into the human cornea after adenovirus type 19 infection depends upon an intracellular signaling cascade initiated by binding of the virus to cells within the superficial corneal stroma and culminating in the expression of leukocyte chemoattractants and adhesion molecules (9, 36, 37). The data presented in the present paper suggest that productive infection of corneal stromal fibroblasts by adenovirus type 19 further depends upon activation of the antiapoptotic PI3K/Akt signaling cascade. We have not studied whether other adenoviruses utilize PI3K activation to ensure cell viability. In this context, Flaherty and coworkers recently demonstrated that infection of airway epithelial cells with a replication-deficient adenoviral vector with no transgene was protective against the proapoptotic effects of actinomycin D plus tumor necrosis factor alpha and suggested that activation of Akt by infection was a potential mechanism (14). Zhang and coworkers found in endothelial cells that the adenoviral early 4 (E4) region in adenoviral gene vectors activates the PI3K/Akt pathway and prolongs cell survival; no such effect of E4 expression was seen in epithelial cells or fibroblasts (53). These latter lines of evidence using subgroup C-based adenoviral expression vectors are consistent with the idea that it may be advantageous to adenoviruses to prolong cell viability early in acute infection. Emerging evidence also indicates that other viruses activate PI3K/Akt signaling with variable effects during acute infection, chronic illness, and/or viral latency (reviewed in reference 10). Infection with human respiratory syncytial virus (47), human immunodeficiency virus (32), or coxsackievirus B3 (54) activates the PI3K/Akt pathway in acute infection but induces premature apoptosis in cells in which PI3K has been inhibited with LY294002, whereas human cytomegalovirus activates the PI3K/Akt pathway during acute infection but does not induce early apoptosis under blockade of PI3K with LY294002 (22). These reports suggest a role for the PI3K/Akt signaling pathway in the pathogenesis of several acute viral infections and further suggest that the underlying basic mechanisms, although perhaps common, are not universal.

PI3K is a versatile signaling molecule with a complex means of regulation, including tyrosine phosphorylation of its p85 subunit (24) in response to stimuli including platelet-derived growth factor (25) and insulin (18). However, PI3K may also be recruited to signaling complexes in the absence of p85 phosphorylation (39). We demonstrated an increase in total p85 in PY99 immunoprecipitates from adenovirus-infected corneal cells, where we also showed increased PI3K activity, but did not find an increase in p85 phosphorylation with infection. It has been shown previously that activation of PI3K can occur due to a direct interaction between the p85 subunit and specific viral gene products (28, 45). However, the addition of adenovirus directly to p85 immunoprecipitates did not alter PI3K activity. Our experiments demonstrate that adenoviral infection does increase PI3K activity but not p85 phosphorylation in corneal fibroblasts, and the mechanism of PI3K activation does not appear to involve a direct interaction between the virus and the kinase.

In our previous work, we reported that infection of primary corneal fibroblasts with adenovirus type 19 results in the expression of inflammatory mediators through the activation of cell signaling molecules, including the 60-kDa tyrosine kinase cSrc (37). To identify and characterize the 60-kDa tyrosine-phosphorylated protein in adenovirus-infected cells that we identified by PY99 immunoprecipitation and p85 immunoblotting, we utilized GST fusion proteins containing either full-length or isolated N-terminal p85 SH2 domains. SH2 domains mediate signals from cell surface receptors to specific intracellular targets (35) and recognize and bind to proteins and linear peptide sequences containing phosphorylated tyrosine residues (50). GST pull-down assays on mock- and virus-treated HCF analyzed by Western blot analysis with anti-PY99 and Src antibodies indicated the binding of cSrc to both fusion proteins (more so in virus-infected cells). The binding of cSrc to the N-SH2 domain of p85 indicates that the binding event is cSrc phosphorylation dependent. Src kinase-dependent interactions between the p85 subunit of PI3K and other tyrosine-phosphorylated proteins, including FAK and Cas, have been shown to activate PI3K (1, 42). We also demonstrated increased PI3K activity in cSrc immunoprecipitates. Taken together, these results suggest that adenovirus infection promotes the phosphorylation of cSrc and that this leads to binding to the p85 subunit of PI3K and its subsequent activation. Our data also demonstrate PI3K-dependent activation of Akt in adenovirus-infected human corneal cells. Activation of Akt likely mediates the subsequent phosphorylation of GSK-3ß and BAD, nuclear translocation of NF-{kappa}B, and suppression of caspase-3/7 activity. It has been reported previously that Akt-dependent BAD phosphorylation occurs at Ser 136 (12). Although we could induce BAD phosphorylation at Ser 112 in vitro by interaction of exogenous BAD with infected cell lysates, we were unable to demonstrate Ser 136 phosphorylation either in vitro or in vivo, suggesting that PI3K/Akt-dependent phosphorylation of BAD at Ser 136 may be cell specific.

Blockade of PI3K/Akt activity by chemical inhibition of PI3K or transfection of dominant negative Akt in adenovirus-infected corneal cells led to early cell death by apoptosis. In our prior work, inhibition of cSrc by the chemical inhibitor PP2 blocked subsequent ERK1/2 activation in adenovirus-infected HCF (37), consistent with prior reports of a relationship between cSrc and ERK (44). Interestingly, both Akt and ERK1/2 have been shown to promote cell survival by inhibition of apoptosis (4). Our results suggest, more specifically, that Akt activation in adenovirus-infected corneal fibroblasts is central to the maintenance of cell viability during the early stages of viral infection. This hypothesis was supported by the finding that constitutively active Akt protected against the proapoptotic effects of combined adenovirus infection and PI3K inhibition by LY294002. It has also been shown previously that respiratory syncytial virus infection of airway epithelial cells instigates the degradation of inhibitor peptides I{kappa}B{alpha} and I{kappa}Bß (5), and this in turn initiates NF-{kappa}B activation. Furthermore, inhibition of the PI3K pathway in respiratory syncytial virus-infected airway epithelial cells results in premature cell death (47). Activation of an antiapoptotic signaling pathway including Akt and NF-{kappa}B may be a fundamental mechanism by which specific viruses ensure their ability to replicate before the onset of cell death, suggesting that artificial blockade of the PI3K/Akt pathway might reduce viral replication in susceptible cells (10). We found a 3-log reduction in viral growth titers when PI3K was inhibited by LY294002, consistent with the idea that adenoviral activation of the PI3K/Akt signaling pathway represents subversion of a cellular pathway by the virus.

Based on our previous studies showing increased activation of cSrc in response to virus infection as early as 5 min after infection (37), we propose that viral interaction with host cell receptors induces cSrc activation and promotes an interaction between cSrc, the p85 subunit of PI3K, and possibly other as-yet-unidentified proteins. This interaction then leads directly or indirectly to activation of the PI3K/Akt signaling pathway and to multiple downstream effects, not least the prolonged survival of the infected cell (Fig. 8). Further studies will be necessary to better delineate the relationships among activated signaling molecules in ocular adenovirus infection and determine their downstream effects on adenoviral pathogenesis.



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  		FIG. 8. Adenovirus type 19 infection of human corneal fibroblasts induces activation of signaling molecules that mediate cell survival (PI3K/Akt) and inflammation (cSrc/Erk). Cell signaling initiated by viral binding is critical to adenoviral ocular pathogenesis.


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ACKNOWLEDGMENTS
 
We thank Madeleine Cunningham for her kind review of the manuscript.

This work was supported by P30-EY12190, R01-EY13124 (to J.C.), and a Lew Wassermann Award from Research to Prevent Blindness (to J.C.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Dean McGee Eye Institute, 608 Stanton L. Young Blvd., Oklahoma City, OK 73104. Phone: (405) 271-1095. Fax: (405) 271-3680. E-mail: james-chodosh{at}ouhsc.edu. Back

{dagger} Present address: Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110 067, India. Back


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Journal of Virology, October 2005, p. 12332-12341, Vol. 79, No. 19
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.19.12332-12341.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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