Portal de Periódicos da CAPES

Identification of 2′-Phosphodiesterase, Which Plays a Role in the 2-5A System Regulated by Interferon

2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês

10.1074/jbc.m400089200

1083-351X

Kazuishi Kubota, Kaori Nakahara, Toshiaki Ohtsuka, Shuku Yoshida, Junko Kawaguchi, Yoko Fujita, Yohei Ozeki, Ayako Hara, Chigusa Yoshimura, Hidehiko Furukawa, Hideyuki Haruyama, Kimihisa Ichikawa, Makoto Yamashita, Tatsuji Matsuoka, Yasuteru Iijima,

interferon and immune responses

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2′,5′-oligoadenylate synthetases (2′,5′-OASs), inactivated by 5′-phosphatase and completely degraded by 2′-phosphodiesterase (2′-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2′,5′-OASs and RNase L have been molecularly cloned and studied well, the identification of 2′-PDE has remained elusive. Here, we describe the first identification of 2′-PDE, the third key enzyme of the 2-5A system. We found a putative 2′-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2′-PDE by mass spectrometry. Based on the full-length sequence of the human 2′-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2′-PDE expressed in mammalian cells certainly cleaved the 2′,5′-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2′-PDE were found, the human 2′-PDE was considered to be a unique enzyme without isoform. Suppression of 2′-PDE by a small interfering RNA and a 2′-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2′-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2′-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments. The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2′,5′-oligoadenylate synthetases (2′,5′-OASs), inactivated by 5′-phosphatase and completely degraded by 2′-phosphodiesterase (2′-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2′,5′-OASs and RNase L have been molecularly cloned and studied well, the identification of 2′-PDE has remained elusive. Here, we describe the first identification of 2′-PDE, the third key enzyme of the 2-5A system. We found a putative 2′-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2′-PDE by mass spectrometry. Based on the full-length sequence of the human 2′-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2′-PDE expressed in mammalian cells certainly cleaved the 2′,5′-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2′-PDE were found, the human 2′-PDE was considered to be a unique enzyme without isoform. Suppression of 2′-PDE by a small interfering RNA and a 2′-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2′-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2′-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments. Virus infection to cells induces the production and secretion of interferons (IFNs), 1The abbreviations used are: IFN, interferon; A2′pA, adenylyl(2′,→5′)adenosine; A3′pA, adenylyl(3′→5′)adenosine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; dsRNA, double-stranded RNA; DTT, dithiothreitol; EST, expressed sequence tag; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; MEM, minimal essential medium; MOPS, 4-morpholinepropanesulfonic acid; 2′,5′-OAS, 2′,5′-oligoadenylate synthetase; pA2′pA2′pA, 5′-monophosphorylated adenylyl(2′→5′)adenylyl(2′→5′)adenosine; 2′-PDE, 2′-phosphodiesterase; 3′-PDE, 3′-phosphodiesterase; pppA2′pA, 5′-triphosphorylated adenylyl(2′→5′)adenosine; pppA2′pA2′pA, 5′-triphosphorylated adenylyl(2′→5′)adenylyl(2′→5′)adenosine; RT, reverse transcription; siRNA, small interfering RNA. which play a major role as the first line host defense against pathogens. IFNs are multifunctional cytokines with important roles in antiviral activity, cell growth, cell differentiation, and immunomodulation (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3398) Google Scholar, 2Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2511) Google Scholar). The 2-5A system is well known as one of the major pathways induced by IFNs, in which unusual oligoadenylates, referred to as 2-5A, modulate RNA degradation in cells (3Player M.R. Torrence P.F. Pharmacol. Ther. 1998; 78: 55-113Crossref PubMed Scopus (253) Google Scholar). 2-5A comprises 5′-triphosphorylated oligoadenylates containing adenosines linked with a 2′-5′-phosphodiester bond (pppA(2′pA)n, where n ≥ 2), which is resistant to most known RNases (4Kerr I.M. Brown R.E. Hovanessian A.G. Nature. 1977; 268: 540-542Crossref PubMed Scopus (127) Google Scholar, 5Kerr I.M. Brown R.E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 256-260Crossref PubMed Scopus (688) Google Scholar). The 2-5A system is composed of three kinds of enzymes: 2′,5′-oligoadenylate synthetases (2′,5′-OASs) activated by double-stranded RNA (dsRNA), which produce 2-5A from ATP (6Rebouillat D. Hovanessian A.G. J. Interferon Cytokine Res. 1999; 19: 295-308Crossref PubMed Scopus (188) Google Scholar); 2′-phosphodiesterase (2′-PDE), which degrades 2-5A to AMP and ATP (7Schmidt A. Zilberstein A. Shulman L. Federman P. Berissi H. Revel M. FEBS Lett. 1978; 95: 257-264Crossref PubMed Scopus (121) Google Scholar); and RNase L activated by 2-5A, which degrades RNA, resulting in inhibition of protein synthesis sometimes leading cells to apoptosis (8Zhou A. Hassel B.A. Silverman R.H. Cell. 1993; 72: 753-765Abstract Full Text PDF PubMed Scopus (463) Google Scholar, 9Dong B. Xu L. Zhou A. Hassel B.A. Lee X. Torrence P.F. Silverman R.H. J. Biol. Chem. 1994; 269: 14153-14158Abstract Full Text PDF PubMed Google Scholar, 10Zhou A. Paranjape J. Brown T.L. Nie H. Naik S. Dong B. Chang A. Trapp B. Fairchild R. Colmenares C. Silverman R.H. EMBO J. 1997; 16: 6355-6363Crossref PubMed Scopus (458) Google Scholar, 11Castelli J. Wood K.A. Youle R.J. Biomed. Pharmacother. 1998; 52: 386-390Crossref PubMed Scopus (85) Google Scholar). Because RNase L is expressed constitutively in a wide variety of cells, the amount of 2-5A is believed to be a primary factor that controls RNase L activity in the 2-5A system (3Player M.R. Torrence P.F. Pharmacol. Ther. 1998; 78: 55-113Crossref PubMed Scopus (253) Google Scholar, 12Minks M.A. Benvin S. Maroney P.A. Baglioni C. Nucleic Acids Res. 1979; 6: 767-780Crossref PubMed Scopus (71) Google Scholar, 13Williams B.R. Kerr I.M. Gilbert C.S. White C.N. Ball L.A. Eur. J. Biochem. 1978; 92: 455-462Crossref PubMed Scopus (91) Google Scholar). 5′-Phosphatase is indicated to have a role in the 2-5A system because the 5′-unphosphorylated 2-5A core has significantly reduced ability to activate RNase L (9Dong B. Xu L. Zhou A. Hassel B.A. Lee X. Torrence P.F. Silverman R.H. J. Biol. Chem. 1994; 269: 14153-14158Abstract Full Text PDF PubMed Google Scholar, 14Knight M. Cayley P.J. Silverman R.H. Wreschner D.H. Gilbert C.S. Brown R.E. Kerr I.M. Nature. 1980; 288: 189-192Crossref PubMed Scopus (154) Google Scholar). Since complete inactivation of 2-5A is only accomplished by cleavage of the 2′-5′ bond (9Dong B. Xu L. Zhou A. Hassel B.A. Lee X. Torrence P.F. Silverman R.H. J. Biol. Chem. 1994; 269: 14153-14158Abstract Full Text PDF PubMed Google Scholar), which is resistant to most known RNases (4Kerr I.M. Brown R.E. Hovanessian A.G. Nature. 1977; 268: 540-542Crossref PubMed Scopus (127) Google Scholar, 5Kerr I.M. Brown R.E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 256-260Crossref PubMed Scopus (688) Google Scholar), cleavage of the 2′-5′ bond by 2′-PDE appears to play an important role in inactivation of 2-5A. Viral infection of cells induces the secretion of IFNs which up-regulate 2′,5′-OASs severalfold to several hundredfold (3Player M.R. Torrence P.F. Pharmacol. Ther. 1998; 78: 55-113Crossref PubMed Scopus (253) Google Scholar, 6Rebouillat D. Hovanessian A.G. J. Interferon Cytokine Res. 1999; 19: 295-308Crossref PubMed Scopus (188) Google Scholar). Because viral infection frequently results in production of dsRNA, the amount of 2-5A in the infected cells is increased preferentially. As a result of RNA degradation by activated RNase L, the viral replication in the infected cells is suppressed by inhibition of protein synthesis and/or apoptosis (3Player M.R. Torrence P.F. Pharmacol. Ther. 1998; 78: 55-113Crossref PubMed Scopus (253) Google Scholar). dsRNA-dependent protein kinase (PKR). Mx protein, and other systems are also known as other mechanisms of antiviral action of IFNs (15Gale Jr., M. Katze M.G. Pharmacol. Ther. 1998; 78: 29-46Crossref PubMed Scopus (337) Google Scholar, 16Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar, 17Lee S.H. Vidal S.M. Genome Res. 2002; 12: 527-530Crossref PubMed Scopus (121) Google Scholar, 18Guo J. Hui D.J. Merrick W.C. Sen G.C. EMBO J. 2000; 19: 6891-6899Crossref PubMed Scopus (178) Google Scholar). Which pathway of the antiviral mechanism is predominant depends on the type of IFNs, cells, and viruses (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3398) Google Scholar, 19Chebath J. Benech P. Revel M. Vigneron M. Nature. 1987; 330: 587-588Crossref PubMed Scopus (219) Google Scholar). In addition to antiviral action, the 2-5A system is proposed to involve tumor suppression (20Lengyel P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5893-5895Crossref PubMed Scopus (195) Google Scholar), and indeed, recent studies showed an association between RNase L and prostate cancer (21Silverman R.H. Biochemistry. 2003; 42: 1805-1812Crossref PubMed Scopus (140) Google Scholar, 22Carpten J. Nupponen N. Isaacs S. Sood R. Robbins C. Xu J. Faruque M. Moses T. Ewing C. Gillanders E. Hu P. Bujnovszky P. Makalowska I. Baffoe-Bonnie A. Faith D. Smith J. Stephan D. Wiley K. Brownstein M. Gildea D. Kelly B. Jenkins R. Hostetter G. Matikainen M. Schleutker J. Klinger K. Connors T. Xiang Y. Wang Z. De Marzo A. Papadopoulos N. Kallioniemi O.P. Burk R. Meyers D. Gronberg H. Meltzer P. Silverman R. Bailey-Wilson J. Walsh P. Isaacs W. Trent J. Nat. Genet. 2002; 30: 181-184Crossref PubMed Scopus (434) Google Scholar, 23Rokman A. Ikonen T. Seppala E.H. Nupponen N. Autio V. Mononen N. Bailey-Wilson J. Trent J. Carpten J. Matikainen M.P. Koivisto P.A. Tammela T.L. Kallioniemi O.P. Schleutker J. Am. J. Hum. Genet. 2002; 70: 1299-1304Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 24Wang L. McDonnell S.K. Elkins D.A. Slager S.L. Christensen E. Marks A.F. Cunningham J.M. Peterson B.J. Jacobsen S.J. Cerhan J.R. Blute M.L. Schaid D.J. Thibodeau S.N. Am. J. Hum. Genet. 2002; 71: 116-123Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 25Rennert H. Bercovich D. Hubert A. Abeliovich D. Rozovsky U. Bar-Shira A. Soloviov S. Schreiber L. Matzkin H. Rennert G. Kadouri L. Peretz T. Yaron Y. Orr-Urtreger A. Am. J. Hum. Genet. 2002; 71: 981-984Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 26Casey G. Neville P.J. Plummer S.J. Xiang Y. Krumroy L.M. Klein E.A. Catalona W.J. Nupponen N. Carpten J.D. Trent J.M. Silverman R.H. Witte J.S. Nat. Genet. 2002; 32: 581-583Crossref PubMed Scopus (252) Google Scholar). It is of interest that RNase L and other proteins in the 2-5A system have a function in counteracting prostate cancer (21Silverman R.H. Biochemistry. 2003; 42: 1805-1812Crossref PubMed Scopus (140) Google Scholar). Among enzymes in the 2-5A system, 2′,5′-OASs and RNase L have been molecularly cloned and studied well (3Player M.R. Torrence P.F. Pharmacol. Ther. 1998; 78: 55-113Crossref PubMed Scopus (253) Google Scholar, 6Rebouillat D. Hovanessian A.G. J. Interferon Cytokine Res. 1999; 19: 295-308Crossref PubMed Scopus (188) Google Scholar, 8Zhou A. Hassel B.A. Silverman R.H. Cell. 1993; 72: 753-765Abstract Full Text PDF PubMed Scopus (463) Google Scholar). Although characterization of 2′-PDE such as substrate specificity using a purified enzyme has been investigated (27Schmidt A. Chernajovsky Y. Shulman L. Federman P. Berissi H. Revel M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4788-4792Crossref PubMed Scopus (150) Google Scholar, 28Johnston M.I. Hearl W.G. J. Biol. Chem. 1987; 262: 8377-8382Abstract Full Text PDF PubMed Google Scholar), molecular characterization of 2′-PDE has remained to be elucidated. To understand the molecular mechanism and biological role of the 2-5A system more precisely, identification of 2′-PDE is indispensable. In this study, we partially purified 2′-PDE from bovine liver and molecularly cloned the human homologous protein. Overexpression of human 2′-PDE suppressed antiproliferative activity of IFNs and dsRNA. Moreover, suppression of 2′-PDE by a small interfering RNA (siRNA) and a 2′-PDE inhibitor exhibited antiviral activity. Our findings suggest 2′-PDE as a potential novel target for therapeutics against replication of viruses and development of tumors. Purification and Identification of 2′-PDE from Bovine Liver—All purification steps were conducted at 4 °C. A portion of bovine liver (200 g, Tokyo Shibaura Zouki) was homogenized in 700 ml of 10 mm MOPS, pH 7.4, containing 10 mm MgCl2, 40 mm NaCl, 0.2 mm dithiothreitol (DTT), and 0.1 mm phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 15,000 × g for 20 min, and the supernatant was centrifuged further at 100,000 × g for 60 min. The 30-50% saturated ammonium sulfate precipitates of the supernatant were collected, dissolved in 200 ml of HEPES buffer (20 mm HEPES, pH 7.0, containing 5 mm MgCl2, 1 mm DTT, and protease inhibitor mixture (complete, Roche Applied Science)), and dialyzed against 10 liters of HEPES buffer for 3 h followed by a change of buffer to 10 liters of HEPES buffer containing 2 m NaCl. The dialyzed sample was filtered and loaded onto a hydrophobic interaction column (HiPrep 16/10 Phenyl FF (High sub), Amersham Biosciences) and eluted with a linear gradient of 2-0 m NaCl in HEPES buffer. The active fractions were then serially purified on an affinity column (HiTrap Blue HP 5 ml, Amersham Biosciences) with a linear gradient of 0-1 m NaCl, an anion exchange column (Resource Q 1 ml, Amersham Biosciences) with a linear gradient of 0-1 m NaCl and an anion exchange column (Mono Q PC 1.6/5, Amersham Biosciences) in HEPES/CHAPS buffer (HEPES buffer containing 0.05% CHAPS). Between each step, the active fractions were dialyzed against the HEPES/CHAPS buffer. A portion of the active fraction from the Mono Q column was loaded onto a gel filtration column (Superdex 75 PC 3.2/30, Amersham Biosciences) equilibrated with HEPES/CHAPS buffer containing 150 mm NaCl. A portion of the active fractions from the Mono Q column and the gel filtration column was loaded onto SDS-polyacrylamide gel under reduced conditions, and the gel was stained with SYPRO Ruby (Molecular Probes) and scanned by Molecular Imager FX (Bio-Rad). The 2′-PDE activity was measured by NH3 assay as described below, and the protein concentration was determined by the modified Bradford method (Coomassie Plus Protein Assay, Pierce) with bovine serum albumin as a standard. The band on the SDS-polyacrylamide gel was excised, and the protein in the gel was reduced, alkylated, and digested as described previously (29Kubota K. Sakikawa C. Katsumata M. Nakamura T. Wakabayashi K. J. Bone Miner. Res. 2002; 17: 257-265Crossref PubMed Scopus (107) Google Scholar). The resultant peptides were subjected to reverse-phase liquid chromatography with tandem mass spectrometry as described previously (30Kubota K. Wakabayashi K. Matsuoka T. Proteomics. 2003; 3: 616-626Crossref PubMed Scopus (78) Google Scholar) with slight modification. The tandem mass spectra were searched against the GenBank non-redundant protein data base using the Mascot program (Matrix Sciences). Molecular Cloning of Human 2′-PDE—To obtain the full-length sequence of human 2′-PDE, we performed a BLAST search against the human EST data base using the corresponding nucleotide sequence of BAB85079.1 (AK074423.1, GenBank) as a query. We found a series of EST fragments with obvious identity. By assembling these EST sequences (AK074423.1, BM894197.1, H06019.1, and H42112.1, all in GenBank), we obtained a virtual longest nucleotide sequence (1,939 bp, AB115695, DDBJ/EMBL/GenBank), which revealed an open reading frame predicted to encode 609 amino acids with the C-terminal extension of BAB85079.1. The cDNA was synthesized using reverse transcriptase (SuperScript II reverse transcription (RT) Kit, Invitrogen) from human liver poly(A)+ RNA (Clontech), and the assembled nucleotide sequence of human 2′-PDE was amplified by PCR using a 5′-primer (5′-CTCCTCAGCTCCACCTGACAGTAGG-3′), a 3′-primer (5′-TACTTCCTTTTCAGACTTCAATTCC-3′) and KOD-plus (Toyobo). The resulting PCR product was then cloned into a pCR-Blunt II-TOPO vector (Invitrogen), and the sequence was confirmed using an ABI PRISM 3700 DNA analyzer (Applied Biosystems). Construction of Classification Tree—The human proteins containing the PF03372 domain (31Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2024) Google Scholar) in the Swiss Protein data base and the human hypothetical proteins with 20-30% identity to human 2′-PDE in GenBank data bases were retrieved. Partial sequences corresponding to the PF03372 domain were extracted from these proteins and subjected to multiple alignment using ClustalW (32Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56228) Google Scholar). A dendrogram was subsequently drawn using the Treeview program. Expression Analysis of 2′-PDE Transcript—The expression profile of 2′-PDE in human tissues was determined by RT-PCR. RT-PCR was performed with RNA from 24 human tissues using a 5′-primer (5′-GTCATCAATGGCAGCATTCCAGAG-3′) and a 3′-primer (5′-TAAATCACATACAAGTGC-3′). Poly(A)+ RNA of the human adult adrenal gland, bone marrow, brain, heart, kidney, liver, lung, pancreas, placenta, prostate, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, fetal brain, fetal kidney, and fetal liver were purchased from Clontech, and human adult colon was from BioChain Institute. The amplified DNA (309 bp) was subjected to 1.5% agarose gel electrophoresis and stained with ethidium bromide. Northern blot analysis was performed for various cell lines. Total RNA from the cells was prepared using TRIzol reagent (Invitrogen), and 15 μg of the RNA was loaded and separated on 1% agarose gel containing 6% formaldehyde and transferred onto nylon membranes (Hybond-N+, Amersham Biosciences). About a 1-kbp fragment of the human 2′-PDE cDNA or human β-actin cDNA was 32P labeled using Rediprime II Random Prime Labeling Kit (Amersham Biosciences), and then the membrane was hybridized with these probes in ExpressHyb buffer (Clontech). The expression levels of each mRNA were determined using a bioimage analyzer (Fujix BAS2000, Fujifilm). Expression of Human 2′-PDE in Mammalian Cells—The open reading frame of the full-length human 2′-PDE was subcloned into the expression vector pEF6/V5-His TOPO (Invitrogen), which encodes human 2′-PDE with the C-terminal V5 epitope and His6 (V5-His) tags. 20 μg of the DNA was transfected into semiconfluent COS-1 cells (1 × 106 cells) using 30 μl of FuGENE 6 (Roche Applied Science) in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS). As a negative control, an equal amount of pEF6/V5-His-TOPO/LacZ (Invitrogen) and FuGENE 6, or only FuGENE 6, was transfected into the COS-1 cells. The transfected cells were cultured for 72 h, washed twice with phosphate-buffered saline, and lysed with CelLytic-M (Sigma) containing 1 mm DTT and protease inhibitor mixture at 25 °C for 5 min. The lysate was centrifuged, and the supernatant was stored at -80 °C until use. For experiments to determine 2′-PDE activity in the high performance liquid chromatography (HPLC) assay or analysis, recombinant human 2′-PDE with V5-His tags was partially purified using a HiTrap Chelating HP column (Amersham Biosciences) and used as an enzyme source. 2′-PDE Activity Assay—We measured 2′-PDE activity by monitoring the NH3 amount (NH3 assay) or using HPLC (HPLC assay). In the NH3 assay, 10 μl of sample was added to 40 μl of HEPES buffer containing 1 mg/ml bovine serum albumin and 1.5 mm A2′pA (Seikagaku), and the mixture was incubated at 37 °C for 1 h. The produced adenosine was converted to inosine and NH3 at 37 °C for 20 min by adding 10 μl of 0.5 m succinic acid, pH 6.0, containing 3.5 μg/ml adenosine deaminase (Sigma) and 2.5 m KCl. The NH3 produced was assayed with an ammonia test kit (Wako Pure Chemicals). One unit/ml 2′-PDE activity was defined as the concentration required for 1 mm adenosine production. 3′-PDE activity was determined by using 1.5 mm A3′pA (Seikagaku) in place of A2′pA. In the HPLC assay, 80 μl of 0.75 mm A2′pA in the assay buffer (20 mm HEPES, pH 7.0, containing 1 mg/ml bovine serum albumin, 1 mm DTT, and 5 mm MgCl2), 10 μl of partially purified human 2′-PDE, and 10 μl of inhibitor dissolved in dimethyl sulfoxide were mixed and incubated at 37 °C for 1 h. The samples were mixed with 200 μl of methanol to inactivate the enzyme, and 10 μl of the filtrate (0.45 μm) was subjected to a C18 column (Inertsil ODS-2, 5 μm, 4.6 × 150 mm, GL Science) and analyzed with isocratic elution in 50 mm ammonium acetate, pH 5.0, containing 5% acetonitrile. Absorbance at 260 nm was monitored, and 2′-PDE activity was determined by measuring the decreased peak area of A2′pA. HPLC Analysis of Degradation of 2-5A Trimer by Recombinant 2′-PDE—2-5A trimer, pppA2′pA2′pA, was enzymatically synthesized by chicken 2′,5′-OAS (33Yamamoto A. Iwata A. Koh Y. Kawai S. Murayama S. Hamada K. Maekawa S. Ueda S. Sokawa Y. Biochim. Biophys. Acta. 1998; 1395: 181-191Crossref PubMed Scopus (38) Google Scholar, 34Tatsumi R. Hamada K. Sekiya S. Wakamatsu M. Namikawa T. Mizutani M. Sokawa Y. Biochim. Biophys. Acta. 2000; 1494: 263-268Crossref PubMed Scopus (27) Google Scholar, 35Tatsumi R. Sekiya S. Nakanishi R. Mizutani M. Kojima S. Sokawa Y. J. Interferon Cytokine Res. 2003; 23: 667-676Crossref PubMed Scopus (19) Google Scholar), purified using an anion exchange column (TSKgel DEAE-2SW, 4.6 × 250 mm, Tosoh), and confirmed by mass spectrometry. 90 μl of 50 μm pppA2′pA2′pA in the assay buffer (20 mm HEPES, pH 7.0, containing 1 mg/ml bovine serum albumin, 1 mm DTT, and 5 mm MgCl2) and 10 μl of partially purified human 2′-PDE were mixed and incubated at 37 °C for specified times. As a negative control, pppA2′pA2′pA was incubated for 7 h with 10 μl of H2O instead of the enzyme. The samples were incubated at 95 °C for 5 min to inactivate the enzyme and centrifuged briefly, and 50 μl of the supernatant was subjected to a C18 column (XTerra MS C18,5 μm, 4.6 × 150 mm, Waters) with a guard column (3.9 × 20 mm) of the same material equilibrated with 50 mm triethylamine acetate, pH 7.0. After injection, the column was washed with the same buffer for 5 min, and the nucleotides were eluted with a linear gradient of 0-10% acetonitrile over 20 min. Absorbance at 260 nm was monitored, and each eluted peak was fractionated and analyzed by mass spectrometry. Peaks corresponding to AMP and ATP were further confirmed using standards. Protection from IFN- or dsRNA-induced Cell Death in Human Prostate Cancer Cells Expressing 2′-PDE—Human prostate cancer PC-3 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin (all from Invitrogen) in humidified 5% CO2 at 37 °C. To establish stable clones expressing 2′-PDE, a human 2′-PDE cDNA fragment was inserted into the mammalian expression vector pEF-DEST51 containing the blasticidin-resistant determinant to express 2′-PDE with C-terminal V5-His tags. The resulting construct was introduced into PC-3 cells using LipofectAMINE Plus reagent (Invitrogen), and then the blasticidin-resistant transformants were selected in RPMI 1640 medium containing 10% FBS and 10 μg/ml blasticidin S. Stable transformant colonies were isolated and confirmed by Western blot analyses. Whole cell extracts were subjected to 10% SDS-PAGE and then electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad). After having been blocked with Block Ace (Dainippon Pharmaceutical), the membrane was incubated first with indicated antibodies to a V5 epitope tag (Invitrogen) or actin (Oncogene), and then with peroxidase-conjugated secondary antibody followed by an ECL Plus Western blotting detection system (Amersham Biosciences). For the cell viability assay, cells (3.0 × 103 cells/well) were seeded onto 96-well plates in a volume of 100 μl of RPMI 1640 medium containing 10% FBS. After incubating the cells with IFN-α (Cosmo Bio), IFN-γ (R&D Systems), or dsRNA (poly(dI·dC), Amersham Biosciences) for 3 days, cell viability was determined using AlamarBlue reagent according to the manufacturer's instructions (BIOSOURCE). Transfection of siRNA, Quantitative Real Time PCR, and Virus Infection Assay—HeLa cells (1 × 105 cells) were grown in semiconfluent monolayers on a 24-well plate (collagen type I-coated, Asahi Techno Glass) in minimal essential medium (MEM) supplemented with 10% heat-inactivated FBS in humidified 5% CO2 at 37 °C. Transfection of siRNA (20 pmol) into HeLa cells was accomplished using LipofectAMINE 2000 (Invitrogen), and the cells were cultured for 24 h. siRNAs for the human 2′-PDE (5′-GUACAAGGUGGAGCGCAACdTdT-3′ and 5′-GUUGCGCUCCACCUUGUACdTdT-3′) and unrelated protein (mouse carboxyl esterase 3, 5′-GGUGCUCUCAGAGCUCUUCdTdG-3′ and 5′-GAAGAGCUCUGAGAGCACCdTdG-3′) were obtained from Dharmacon. After replacing the medium with 250 μl of MEM containing 2.5% FBS, the cells were cultured for 24 h with or without 300 units/well IFN-α (EMD Biosciences). After changing the medium to fresh MEM containing 2.5% FBS, the cells were infected or noninfected with vaccinia virus (WR strain, multiplicity of infection = 0.001), cultured for 24 h, and subjected to analysis of the 2′-PDE transcript and viral replication. For quantification of 2′-PDE mRNA in HeLa cells, total RNA was prepared using RNeasy Mini Kit (Qiagen), and cDNA was prepared from 1 μg of total RNA using oligo(dT) and Superscript II RNase H reverse transcriptase in a final volume of 20 μl. Real time PCR amplifications were performed from 5 μl of cDNA diluted 1:125 by using gene-specific primer sets as follows: 2′-PDE forward, 5′-GTCATCAATGGCAGCATTCCAGAG-3′; 2′-PDE reverse, 5′-CTATTTCCATTTTAAATCACATACAAGTGC-3′; β-actin forward, 5′-CATTGCTCCTCCTGAGCGCAA-3′; β-actin reverse, 5′-CTGCGCAAGTTAGGTTTTGTC-3′. Each primer set was used at a concentration of 300 nm in a final volume of 50 μl using the QuantiTect SYBR Green PCR Kit (Qiagen). All PCRs were performed on a sequence detection system (ABI Prism 7900HT, Applied Biosystems), and quantification of a given gene was calculated after normalization to β-actin. The specificity of the PCR amplification was verified by melt curve analysis of the final products directly in the sequence detector and by agarose gel electrophoresis. To determine viral replication, the cells were washed with phosphate-buffered saline, fixed with absolute ethanol at 25 °C for 10 min, and dried. The procedure for visualization of infected cells by peroxidase-antiperoxidase staining was described previously (36Okuno Y. Tanaka K. Baba K. Maeda A. Kunita N. Ueda S. J. Clin. Microbiol. 1990; 28: 1308-1313Crossref PubMed Google Scholar) and used with some modifications. The fixed cells were treated successively with rabbit anti-vaccinia virus antibody (1:500, Virostat, Portland, ME), goat anti-rabbit immunoglobulin (1:1,000, DakoCytomation), and peroxidase-rabbit antiperoxidase complex (1:1,000, DakoCytomation) for 60 min each. The peroxidase reaction was conducted for 5 min using a liquid DAB Substrate-Chromogen System (DakoCytomation). The plaque numbers were counted visually, and the antiviral effects were normalized using the plaque numbers of virus infection only as 100%. Effect of 2′-PDE Inhibitor on Virus Infection Assay—The effect of a 2′-PDE inhibitor on the virus infection assay was determined in a manner similar to that of siRNA. Semiconfluent HeLa cells on a 24-well plate grown in MEM with 2.5% FBS were treated with or without 300 units/well IFN-α in MEM with 2.5% FBS for 24 h. The medium was changed with fresh MEM containing 2.5% FBS and an inhibitor, and the cells were infected with vaccinia virus (WR strain, multiplicity of infection = 0.001). After 24 h of culture, the degree of viral infection was determined using peroxidase-antiperoxidase staining as described above. Statistical Analysis—Comparison of two groups was analyzed by the two-tailed unpaired Student t test and the dose effects were evaluated by the Dunnett test. Purification and Identification of 2′-PDE—We developed two assays for detecting 2′-PDE activity, an NH3 assay and an HPLC assay. In the NH3 assay, the pr