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Human Gene Profiling in Response to the Active Protein Kinase, Interferon-induced Serine/threonine Protein Kinase (PKR), in Infected Cells

2006; Elsevier BV; Volume: 281; Issue: 27 Linguagem: Inglês

10.1074/jbc.m511983200

ISSN

1083-351X

Autores

Susana Guerra, Luis A. López‐Fernández, María Ángel García, Ángel Zaballos, Mariano Estéban,

Tópico(s)

Biochemical and Molecular Research

Resumo

The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were down-regulated. The ATF-3 transcription factor, involved in stress-induced β-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VVΔE3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR-/- cells. Using null cells for ATF-3 and for the p65 subunit of NF-κB, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-κB activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis. The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were down-regulated. The ATF-3 transcription factor, involved in stress-induced β-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VVΔE3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR-/- cells. Using null cells for ATF-3 and for the p65 subunit of NF-κB, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-κB activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis. The double-stranded RNA (dsRNA) 4The abbreviations used are: dsRNA, double-stranded RNA; VV, vaccinia virus; IFN, interferon; eIF-2α, eukaryotic initiation factor 2; PKR, interferon-induced serine/threonine protein kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; hpi, hours postinfection; EMSA, electrophoretic mobility shift assay; pfu, plaque-forming units; RT, reverse transcription; MEF, mouse embryo fibroblast; PARP, poly(ADP-ribose) polymerase. -dependent protein kinase (PKR) is a key mediator in the antiviral effects of interferon (IFN) and a dynamic participant in apoptosis induced by various stimuli (1Meurs E. Chong K. Galabru J. Thomas N.S. Kerr I.M. Williams B.R. Hovanessian A.G. Cell. 1990; 62: 379-390Abstract Full Text PDF PubMed Scopus (819) Google Scholar) (reviewed in Ref. 2Clemens M.J. Elia A. J. Interferon Cytokine Res. 1997; 17: 503-524Crossref PubMed Scopus (519) Google Scholar). PKR controls cell processes, such as growth (3Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (289) Google Scholar) differentiation (4Petryshyn R.A. Li J. Judware R. Prog. Mol. Subcell. Biol. 1994; 14: 1-14Crossref PubMed Scopus (16) Google Scholar), apoptosis (5Lee S.B. Esteban M. Virology. 1994; 199: 491-496Crossref PubMed Scopus (313) Google Scholar, 6Der S.D. Yang Y.L. Weissmann C. Williams B.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3279-3283Crossref PubMed Scopus (362) Google Scholar), stress response (7Williams B.R. Oncogene. 1999; 18: 6112-6120Crossref PubMed Scopus (700) Google Scholar), anti-tumor activity (8Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (496) Google Scholar, 9Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (417) Google Scholar), and antiviral functions (10Lee S.B. Esteban M. Virology. 1993; 193: 1037-1041Crossref PubMed Scopus (80) Google Scholar, 11Lee S.B. Bablanian R. Esteban M. J. Interferon Cytokine Res. 1996; 16: 1073-1078Crossref PubMed Scopus (39) Google Scholar). After PKR activation, the initiation of protein synthesis is inhibited by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2α) by PKR (12Hershey J.W. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (843) Google Scholar). This kinase regulates the activation of several transcription factors involved in virus-induced apoptosis (13Heylbroeck C. Balachandran S. Servant M.J. DeLuca C. Barber G.N. Lin R. Hiscott J. J. Virol. 2000; 74: 3781-3792Crossref PubMed Scopus (150) Google Scholar), including p53 (14Cuddihy A.R. Li S. Tam N.W. Wong A.H. Taya Y. Abraham N. Bell J.C. Koromilas A.E. Mol. Cell. Biol. 1999; 19: 2475-2484Crossref PubMed Scopus (125) Google Scholar), IRF-1 (15Kumar A. Yang Y.L. Flati V. Der S. Kadereit S. Deb A. Haque J. Reis L. Weissmann C. Williams B.R. EMBO J. 1997; 16: 406-416Crossref PubMed Scopus (315) Google Scholar), and IRF-3, and activation of c-Jun (16Chu W.M. Ostertag D. Li Z.W. Chang L. Chen Y. Hu Y. Williams B. Perrault J. Karin M. Immunity. 1999; 11: 721-731Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). By regulating the expression of genes involved in cell proliferation, NF-κB induction is important in mediating PKR function (16Chu W.M. Ostertag D. Li Z.W. Chang L. Chen Y. Hu Y. Williams B. Perrault J. Karin M. Immunity. 1999; 11: 721-731Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). NF-κB activation by PKR also triggers production of IFN-β (16Chu W.M. Ostertag D. Li Z.W. Chang L. Chen Y. Hu Y. Williams B. Perrault J. Karin M. Immunity. 1999; 11: 721-731Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 17Visvanathan K.V. Goodbourn S. EMBO J. 1989; 8: 1129-1138Crossref PubMed Scopus (214) Google Scholar), an essential component in PKR functionality. The genes involved in PKR-induced apoptosis in infected human cells have not been defined. We previously described an isopropyl-β-d-thiogalactopyranoside-inducible VV system in which PKR expression triggers apoptosis (5Lee S.B. Esteban M. Virology. 1994; 199: 491-496Crossref PubMed Scopus (313) Google Scholar) (for a review, see Ref. 18Gil J. Esteban M. J. Interferon Cytokine Res. 2004; 24: 637-646Crossref PubMed Scopus (8) Google Scholar). When PKR was expressed, the antiviral action against VV and vesicular stomatitis virus provoked a translational block by phosphorylation of eIF-2α and triggered NF-κB activation through the IκB kinase complex (5Lee S.B. Esteban M. Virology. 1994; 199: 491-496Crossref PubMed Scopus (313) Google Scholar, 10Lee S.B. Esteban M. Virology. 1993; 193: 1037-1041Crossref PubMed Scopus (80) Google Scholar, 11Lee S.B. Bablanian R. Esteban M. J. Interferon Cytokine Res. 1996; 16: 1073-1078Crossref PubMed Scopus (39) Google Scholar, 19Lee S.B. Rodriguez D. Rodriguez J.R. Esteban M. Virology. 1997; 231: 81-88Crossref PubMed Scopus (117) Google Scholar, 20Gil J. Alcami J. Esteban M. Oncogene. 2000; 19: 1369-1378Crossref PubMed Scopus (121) Google Scholar). Results using this inducible virus-cell system have been validated in transfected cells or cells derived from PKR gene knock-out mice (6Der S.D. Yang Y.L. Weissmann C. Williams B.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3279-3283Crossref PubMed Scopus (362) Google Scholar, 18Gil J. Esteban M. J. Interferon Cytokine Res. 2004; 24: 637-646Crossref PubMed Scopus (8) Google Scholar). The VV inducible system was also used to identify apoptotic viral genes (21Suarez P. Diaz-Guerra M. Prieto C. Esteban M. Castro J.M. Nieto A. Ortin J. J. Virol. 1996; 70: 2876-2882Crossref PubMed Google Scholar), as well as cellular and viral inhibitors of apoptosis (18Gil J. Esteban M. J. Interferon Cytokine Res. 2004; 24: 637-646Crossref PubMed Scopus (8) Google Scholar, 19Lee S.B. Rodriguez D. Rodriguez J.R. Esteban M. Virology. 1997; 231: 81-88Crossref PubMed Scopus (117) Google Scholar, 22Brun A. Rivas C. Esteban M. Escribano J.M. Alonso C. Virology. 1996; 225: 227-230Crossref PubMed Scopus (95) Google Scholar). To identify host genes involved in PKR-induced apoptosis of cells infected with VV recombinants, we used cDNA microarray high-throughput screening of over 15,000 human genes. With RNAs obtained from VV-PKR-infected HeLa cells at late but not at early times postinfection, we found transcriptional alteration in defined gene subsets. Comparison of VV-PKR with the catalytically inactive VV-PKR-K296R mutant showed that PKR catalytic activity was required for regulation of these genes. When grouped into functional categories, a significant proportion of altered transcripts consisted of genes involved in cell cycle, apoptosis, stress, defense, and immune response. The genes up-regulated by PKR activity included ATF-3, a stress-inducible gene that encodes a member of the ATF/cAMP-response element-binding protein family of transcription factors (23Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar, 24Hai T. Hartman M.G. Gene (Amst.). 2001; 273: 1-11Crossref PubMed Scopus (656) Google Scholar). ATF-3 is induced by PERK and GCN4 (25Jiang H.Y. Wek S.A. McGrath B.C. Lu D. Hai T. Harding H.P. Wang X. Ron D. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (390) Google Scholar), but this is the first report of ATF-3 up-regulation by PKR. Here, we provided evidence that ATF-3 is specifically induced by PKR and is involved in PKR-induced apoptosis and that induction of apoptosis by PKR is regulated by NF-κB. These findings add the transcription factor ATF-3 to the specific mechanisms used by PKR to induce apoptosis. Cells and Viruses—HeLa cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% newborn bovine serum and antibiotics. Mouse 3T3-like fibroblasts derived from homozygous PKR-deficient mice (PKR-/-) (26Yang Y.L. Reis L.F. Pavlovic J. Aguzzi A. Schafer R. Kumar A. Williams B.R. Aguet M. Weissmann C. EMBO J. 1995; 14: 6095-6106Crossref PubMed Scopus (566) Google Scholar) were obtained from C. Weissmann (University of Zurich, Switzerland) and cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. ATF-3-/- cells and their wild type counterparts were kindly provided by T. Hai (Ohio State University, Columbus, OH). Briefly, MEF cells were isolated by trypsinization of embryos from ATF-3-/- mice and immortalized by infection with a recombinant retrovirus expressing simian virus 40 large T antigen (25Jiang H.Y. Wek S.A. McGrath B.C. Lu D. Hai T. Harding H.P. Wang X. Ron D. Cavener D.R. Wek R.C. Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (390) Google Scholar). p65-/- cells and wild type counterparts were a gift from A. Martín (Centro de Investigación Príncipe Felipe, Valencia, Spain). p65-/- MEF cells were also isolated from embryos of p65-/- mice and immortalized by several passages (27Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1638) Google Scholar). MEF cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. VV recombinants expressing PKR (wild-type enzyme, VV-PKR), VV-PKRK296R (the catalytically inactive point mutant K296R), and VV-Hsp70 (VV expressing the human heat shock protein Hsp-70) were grown on monkey BSC-40 cells, purified by sucrose gradient banding, and titrated on BSC-40 cells by plaque assay. Unless otherwise indicated, VV infections were performed at a multiplicity of infection of 5 pfu/cell. Microarray Production—To generate cDNA arrays, we used the Research Genetics 40K sequence-verified clone human cDNA library (available on the World Wide Web at www.resgen.com/products/SVHcDNA.php3), as described (28Guerra S. Lopez-Fernandez L.A. Pascual-Montano A. Munoz M. Harshman K. Esteban M. J. Virol. 2003; 77: 6493-6506Crossref PubMed Scopus (101) Google Scholar). Slides contained 15,360 cDNAs, of which 13,295 corresponded to known genes and 2,257 corresponded to control genes. DNA was printed on CMT-GAPS II slides (Corning) with a Microgrid II (BioRobotics) at 22 °C and 40-45% relative humidity. Microarray Hybridization—Total RNA was isolated from VV-PKR-, VV-, or VV-PKR-K296R-infected HeLa cells cultured in 10-cm plates with Ultraspect-II RNA (Biotecx) following the manufacturer's instructions. At 6 and 16 h postinfection (hpi), two RNA samples (1 μg each) were processed for analysis from each infected HeLa cell culture; each sample was used for two distinct hybridizations (dye swapping). Two different microarray approaches were used. In the first approach, we hybridized cDNA from VV-PKR-infected cells against VV-infected HeLa cells; in the second, we hybridized cDNA from VV-PKR-infected cells against VV-PKR-K296R-infected cells. In both cases, we performed four hybridizations for each postinfection time. Labeling and hybridization conditions were as described (28Guerra S. Lopez-Fernandez L.A. Pascual-Montano A. Munoz M. Harshman K. Esteban M. J. Virol. 2003; 77: 6493-6506Crossref PubMed Scopus (101) Google Scholar, 29Guerra S. Lopez-Fernandez L.A. Conde R. Pascual-Montano A. Harshman K. Esteban M. J. Virol. 2004; 78: 5820-5834Crossref PubMed Scopus (73) Google Scholar). Slides were dried by centrifugation and scanned on a ScanArray 4000 (Packard Biosciences) using ScanArray 3.1 software. Raw data were obtained from Cy5 and Cy3 images using QuantArray 3.0 software (Packard Biosciences) and processed using SOLAR software (BioALMA, Spain). Briefly, background was subtracted from the signal, and the log 10 (signal) was plotted versus log 2 (ratio) and Lowess-normalized to adjust most spots to log ratio 0. This was calculated for all four replicates, and a table was obtained with mean signal, x-fold change, log ratio, S.D. of the log ratio, and z-score (30Quackenbush J. Nat. Genet. 2002; 32 (suppl.): 496-501Crossref PubMed Scopus (1500) Google Scholar). Gene Expression Analysis—The original data set contained 13,295 clones/slide. In each analysis, genes with an interreplicate S.D. value of >1 were removed. The resulting data set was reduced to the transcripts that showed a consistent expression value among the four replicates. The z-score value (a measure of the proximity of one value (log ratio) to other values with a similar signal) was used to eliminate genes that did not show significant expression in at least one experimental condition (30Quackenbush J. Nat. Genet. 2002; 32 (suppl.): 496-501Crossref PubMed Scopus (1500) Google Scholar). Quantitative Real Time Reverse Transcription-PCR (RT-PCR)—RNA (1 μg) was reverse-transcribed using the Superscript first-strand synthesis system for RT-PCR (Invitrogen). A 1:40 dilution of the RT reaction mixture was used for quantitative PCR. Primers and probe sets used to amplify H2BFB, caspase-9, ATF-3, NFκBIA, NFκB-2, interleukin-6, and IFN-γ were purchased from Applied Biosystems. RT-PCRs were performed according to Assay-on-Demand, optimized to work with TaqMan Universal PCR MasterMix, No AmpErase UNG, as described (29Guerra S. Lopez-Fernandez L.A. Conde R. Pascual-Montano A. Harshman K. Esteban M. J. Virol. 2004; 78: 5820-5834Crossref PubMed Scopus (73) Google Scholar). All samples were assayed in duplicate. Threshold cycle (Ct) values were used to plot a standard curve in which Ct decreased in linear proportion to the log of the template copy number. The correlation values of standard curves were always >99%. Immunoblotting—HeLa cells were infected in 6-well plates with VVPKR, VV, or VV-PKR-K296R (5 pfu/cell) and collected at 6 and 16 hpi in lysis buffer (50 mm Tris-HCl, pH 8.0, 0.5 m NaCl, 10% Nonidet P-40, 1% SDS). Protein lysates were resuspended in 2× Laemmli buffer, and equal amounts of protein (100 μg) were separated by 14 or 8% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary anti-PKR (31Gil J. Garcia M.A. Esteban M. FEBS Lett. 2002; 529: 249-255Crossref PubMed Scopus (48) Google Scholar), anti-PKR-P (Calbiochem), anti-Hsp70 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-caspase-9 (Oncogene), anti-ATF-3 (Santa Cruz Biotechnology), anti-ATF-4 (Santa Cruz Biotechnology), anti-GADD34 (Santa Cruz Biotechnology), anti-PARP-1 (Cell Signaling), eIF-2α (Santa Cruz Biotechnology), eIF-2α-P (BIOSOURCE), actin (Sigma), or NF-κB (Santa Cruz Biotechnology), followed by peroxidase-conjugated mouse or rabbit secondary antibodies. Blots were developed using ECL (Amersham Biosciences). Metabolic Labeling of Proteins—HeLa cell monolayers in 12-well plates were infected with VV-PKR, VV, or VV-PKR-K296R (5 pfu/cell). At the postinfection times indicated, cells were washed with methionine-free medium and incubated in methionine-free medium containing [35S]methionine (50 μCi/well, 30 min, 37 °C). Proteins from cell extracts prepared in lysis buffer were fractionated by 12% SDS-PAGE and developed by autoradiography. Immunofluorescence—HeLa or PKR-/- cells cultured on coverslips were infected with the recombinant viruses indicated. At 16 hpi, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized (10 min, room temperature) with 0.1% Triton X-100 in PBS, washed, and blocked with 20% bovine serum albumin in PBS. Cells were incubated (1 h, 37 °C) with anti-PKR, anti-Hsp-70, or anti-ATF-3 antibody; coverslips were washed extensively with PBS and were further incubated (1 h, 37 °C) with ToPro (Molecular Probes, Inc.) and appropriate fluorescein- or Texas Red-conjugated isotype-specific secondary antibodies. After washing with PBS, coverslips were mounted on microscope slides using Mowiol (Calbiochem). Images were obtained using a Bio-Rad Radiance 2100 confocal laser microscope. Apoptosis Measurement by ELISA—HeLa, ATF-3-/-, and p65-/- cells and their wild type counterparts were grown in 12-well plates, infected (5 pfu/cell) with the viruses indicated, and harvested at 24 hpi. We used the cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Applied Science) to determine the absorbance at 405 nm. This assay is based on the quantitative sandwich enzyme immunoassay principle and uses mouse monoclonal antibodies against DNA and histones to estimate the amount of cytoplasmic histone-associated DNA. Duplicate samples were measured in two independent experiments. Measurement of Apoptotic Cell Death by Cell Cycle Analysis—Cell cycle stages and the percentage of cells with sub-G0 DNA content were analyzed by propidium iodide staining (32Martin A.G. Fearnhead H.O. J. Biol. Chem. 2002; 277: 50834-50841Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). ATF-3-/- and p65-/- cells and their wild type counterparts were uninfected or infected at 5 pfu/cell with VV or VV-PKR strains in the presence or absence of benzyloxycarbonyl-VAD-fluoromethyl ketone, a general caspase inhibitor (40 μm; Calbiochem), or staurosporine, an apoptosis activator (0.5 μm; Sigma). Cells were permeabilized at 24 hpi with 70% ethanol in PBS (4 °C, 30 min). After three washes with PBS, cells were incubated with RNase A (0.1 mg/ml, 37 °C, 45 min) and stained with propidium iodide (10 μg/ml). The percentage of cells with hypodiploid DNA content was determined by flow cytometry. Data were acquired on 6000 cells/sample and analyzed as described (32Martin A.G. Fearnhead H.O. J. Biol. Chem. 2002; 277: 50834-50841Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Gel Retardation Assay (EMSA)—Nuclear extracts (3 μg) from the indicated cells grown in 6-cm well plates, either uninfected or infected with VV or VV-PKR at 5 pfu/cell for the indicated times, were analyzed using the synthetic [α-32P]dCTP-labeled double-stranded wild-type HIV enhancer oligonucleotide 5′-AGCTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA-3′, containing two κB consensus motifs, following protocols described previously (33Arenzana-Seisdedos F. Thompson J. Rodriguez M.S. Bachelerie F. Thomas D. Hay R.T. Mol. Cell. Biol. 1995; 15: 2689-2696Crossref PubMed Google Scholar). RT-PCR Analysis—Total RNA (1 μg) was used to identify ATF-3 and ATF-3ΔZip2a mRNAs using the Superscript first-strand synthesis system for reverse transcription (Invitrogen). The PCR was performed at 95 °C for 1 min and 72 °C for 2 min for 26 cycles. To amplify the full-length ATF-3 and ATF-3ΔZip2a mRNAs, the pair of primers used were 5′-ATGATGCTTCAACACCCAGGC-3′ and 5′-TTAGCTCTGCAATGTTCCTTC-3′ (described in Ref. 34Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). Bands of 546 and 858 bp represented products of full-length ATF3 and the spliced form ATF-3ΔZip2a. Gene Profiling of HeLa Cells Infected with VV Recombinants Expressing Wild Type PKR or the Catalytically Inactive PKR Mutant K296R— Isopropyl-β-d-thiogalactopyranoside-inducible VV recombinants that express wild type (VV-PKR) or the K296R mutant PKR (VV-PKRK296R) were used to study the cell transcriptional response after PKR expression in HeLa cells. In this virus-cell system, we previously reported that PKR is produced between 2 and 4 hpi, it is autophosphorylated by 6-9 hpi (10Lee S.B. Esteban M. Virology. 1993; 193: 1037-1041Crossref PubMed Scopus (80) Google Scholar, 35Gil J. Alcami J. Esteban M. Mol. Cell. Biol. 1999; 19: 4653-4663Crossref PubMed Google Scholar), and its substrate eIF-2α is phosphorylated from 6 to 10 hpi, leading to severe protein synthesis inhibition by 16 hpi (10Lee S.B. Esteban M. Virology. 1993; 193: 1037-1041Crossref PubMed Scopus (80) Google Scholar) (reviewed in Ref. 19Lee S.B. Rodriguez D. Rodriguez J.R. Esteban M. Virology. 1997; 231: 81-88Crossref PubMed Scopus (117) Google Scholar). As shown in Fig. 1, in HeLa cells infected with VV-PKR, protein synthesis is strongly inhibited at late times postinfection, and this translational block correlated with phosphorylation of PKR and of eIF-2α. The reduced protein synthesis was not due to a decrease in protein load in the gels, since the levels of actin and of total eIF-2α were similar in all lanes of Fig. 1. A clear phosphorylation of wild type PKR was shown from 6 hpi. However, the absence of catalytic activity of the mutant PKR correlates with the lack of autophosphorylation (Fig. 1). We used cDNA microarray technology to compare the relative abundance of specific PKR-induced mRNAs in VV-PKR-versus VV-infected HeLa cells at 6 and 16 hpi. At 6 hpi, we observed, with the exception of PKR mRNA, very few altered genes after PKR expression, validating the virus-cell system (not shown). At 16 hpi, analysis of the list of cellular genes up-regulated by PKR expression indicated several gene families with distinct biological functions (supplemental Table 1); these included genes involved in cell cycle, apoptosis, stress, defense, and immune modulation. The levels of PKR were enhanced 15-fold in VVPKR-versus VV-infected HeLa cell, indicating the correct induction of PKR (supplemental Table 1). In parallel, we analyzed the specific mRNAs induced by PKR catalytic activity, for which we performed a microarray analysis comparing VV-PKR-with VV-PKRK296R-infected HeLa cells at 6 and 16 hpi. Again, very few altered genes were found at 6 hpi (not shown). However, clear differences were observed at 16 hpi. Genes regulated by PKR catalytic activity were classified according to biological function, which included cell cycle regulation and immune modulation (supplemental Table 2). We chose selected regulated genes identified in microarray analysis for target verification by quantitative RT-PCR. The RNA preparation used was the same as that used in the microarray. We analyzed five up-regulated genes (NFκBIA, NFκB-2, interleukin-6, ATF-3, caspase-9, and H2BFB) and one unaltered gene (IFN-γ); HPRT was used as internal control. The RT-PCR data confirmed microarray results, showing the same relative transcription regulation of the selected genes (Table 1) and validating microarray analysis. Absolute values are not identical when comparing microarray and RT-PCR data, probably due to differences intrinsic to the techniques.TABLE 1Validation of microarray data by real time RT-PCR RT-PCR conditions are described under "Experimental Procedures." The name of each gene product, values of microarray data at different experimental conditions, and values obtained after RT-PCR are indicated.Gene product-Fold changeVV-PKR versus VVVV-PKR versus VV-PKR-K296RMicroarrayRT-PCRMicroarrayRT-PCR-foldH2BFB3.482.244.214.78NFκBIA2.923.22.313.10Interleukin9.38.712.610.5IFN-γ1.21.121.131.03NFκB-24.525.273.694.03Caspase-92.593.211.251.35ATF-37.016.53.694.82 Open table in a new tab Although changes in mRNA levels do not necessarily represent changes in protein expression, we analyzed whether changes in gene expression detected in microarray analysis correlated with expression and activation of selected gene products. To confirm the microarray data, we analyzed two proteins, one that is up-regulated (caspase-9) and another that is down-regulated (Hsp70). Transcriptional activation of caspase-9 detected by microarray does not correlate with an apparent increase in the caspase-9 protein level, as measured by Western blot. However, in VV-PKR-infected HeLa cells, there is enhanced caspase-9 activation, as indicated by cleavage of the procaspase-9 (Fig. 2A). Activation of caspase-9 was not observed in cells infected with VV-PKR-K296R or VV or in uninfected HeLa cells (Fig. 2A). Apoptosis is mediated by activation of caspase-8 or -9, leading to induction of effector caspase-3 and -7, which cleave specific substrates, including PARP-1. This enzyme catalyzes formation of poly(ADP-ribose) polymers on acceptor proteins involved in the maintenance of chromatin structure, indicating activation of the apoptotic cascade (36Soldani C. Scovassi A.I. Apoptosis. 2002; 7: 321-328Crossref PubMed Scopus (597) Google Scholar). Since PKR expression induced changes in caspase-9 activation, we analyzed whether PKR activation correlated with PARP-1 cleavage using an antibody that recognizes only the cleaved protein. After VV-PKR infection, we found the 89-kDa PARP-1 cleavage product, indicating that effector-caspases were activated (Fig. 2A). To confirm catalytic activation of PKR, we measured phosphorylation of eIF-2α as a PKR substrate. Phosphorylation of eIF-2α was observed only in VV-PKR-infected cells (Fig. 2A). In contrast to caspase-9 activation, Hsp70 protein levels decreased after PKR expression, concurring with the Hsp70 transcript down-regulation observed in microarray analysis (Fig. 2A). Since Hsp70 suppresses PKR activity in hematopoietic cells (37Pang Q. Christianson T.A. Keeble W. Koretsky T. Bagby G.C. J. Biol. Chem. 2002; 277: 49638-49643Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we analyzed the subcellular localization of Hsp70 and PKR in HeLa cells by triple confocal microscopy, using anti-PKR and anti-Hsp70 antibodies and ToPro to detect DNA (Fig. 2B). Hsp70 expression by VV resulted in characteristic punctate cytosolic localization of endogenous protein (not shown). PKR expression was mainly cytosolic, with strong perinuclear localization. When PKR and Hsp70 were coexpressed, we detected PKR and Hsp70 colocalization in the merged images (Fig. 2B). To evaluate the functional effect of Hsp70 on PKR activity, we coexpressed these proteins and measured PKR-induced cell death. VV-PKR infection induced apoptosis in HeLa cells, whereas in the presence of Hsp70, the percentage of PKR-triggered apoptotic cells was reduced by more than 70%. Apoptosis was not detected following expression of Hsp70 alone (Fig. 2C). These data indicate that Hsp70 inhibits the proapoptotic effect of PKR. Since PKR down-regulates Hsp70, reduced Hsp70 synthesis would promote more active PKR in the cells. PKR Induces ATF-3 Protein Expression—ATF-3, a 181-amino acid protein, is a member of the ATF/cAMP-response element-binding protein family of transcription factors and is maintained at low levels in quiescent cells (23Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar). To confirm the increase in the ATF-3 messenger after PKR expression, we used Western blot to analyze the effect of PKR on ATF-3 protein levels in PKR-/- cells. Whereas overall protein levels were reduced, ATF-3 was increased after VV-PKR infection in comparison to mock-, VV-, or VV-P

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