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• W2075812596 abstract "Kaposi sarcoma-associated herpesvirus is associated with two lymphoproliferative disorders, primary effusion lymphoma (PEL) and Castleman disease. In PEL, Kaposi sarcoma-associated herpesvirus is present in a latent form expressing only few viral genes. Among them is a viral homologue of cellular interferon regulatory factors, vIRF-3. To study the role of vIRF-3 in PEL lymphomagenesis, we analyzed the interaction of vIRF-3 with cellular proteins. Using yeast two-hybrid screen, we detected the association between vIRF-3 and c-Myc suppressor, MM-1α. The vIRF-3 and MM-1α interaction was also demonstrated by glutathione S-transferase pulldown assay and coimmunoprecipitation of endogenous vIRF-3 and MM-1α in PEL-derived cell lines. Overexpression of vIRF-3 enhanced the c-Myc-dependent transcription of the gene cdk4. Addressing the molecular mechanism of the vIRF-3-mediated stimulation, we demonstrated that the association between MM-1α and c-Myc was inhibited by vIRF-3. Furthermore, the recruitment of vIRF-3 to the cdk4 promoter and the elevated levels of the histone H3 acetylation suggest the direct involvement of vIRF-3 in the activation of c-Myc-mediated transcription. These findings indicate that vIRF-3 can effectively stimulate c-Myc function in PEL cells and consequently contribute to de-regulation of B-cell growth and differentiation. Kaposi sarcoma-associated herpesvirus is associated with two lymphoproliferative disorders, primary effusion lymphoma (PEL) and Castleman disease. In PEL, Kaposi sarcoma-associated herpesvirus is present in a latent form expressing only few viral genes. Among them is a viral homologue of cellular interferon regulatory factors, vIRF-3. To study the role of vIRF-3 in PEL lymphomagenesis, we analyzed the interaction of vIRF-3 with cellular proteins. Using yeast two-hybrid screen, we detected the association between vIRF-3 and c-Myc suppressor, MM-1α. The vIRF-3 and MM-1α interaction was also demonstrated by glutathione S-transferase pulldown assay and coimmunoprecipitation of endogenous vIRF-3 and MM-1α in PEL-derived cell lines. Overexpression of vIRF-3 enhanced the c-Myc-dependent transcription of the gene cdk4. Addressing the molecular mechanism of the vIRF-3-mediated stimulation, we demonstrated that the association between MM-1α and c-Myc was inhibited by vIRF-3. Furthermore, the recruitment of vIRF-3 to the cdk4 promoter and the elevated levels of the histone H3 acetylation suggest the direct involvement of vIRF-3 in the activation of c-Myc-mediated transcription. These findings indicate that vIRF-3 can effectively stimulate c-Myc function in PEL cells and consequently contribute to de-regulation of B-cell growth and differentiation. Kaposi sarcoma-associated herpes virus (KSHV) 2The abbreviations used are: KSHV, Kaposi sarcoma-associated herpesvirus; vIRF-3, viral interferon regulatory factor-3; LANA2, latency-associated nuclear antigen 2; MM-1, Myc modulator-1; HEK293, human embryonic kidney cells 293; GST, glutathione S-transferase; FL, full length; MBS, c-Myc binding site; ORF, open reading frame; IRF, interferon regulatory factor; PEL, primary effusion lymphoma; CREB, cAMP-response element-binding protein; aa, amino acid; HA, hemagglutinin; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pRb, retinoblastoma protein; X-α-gal, 5-bromo-4-chloro-3-indolyl α-d-galactopyranoside. is a member of the γ herpes virus family that is genetically similar to Epstein-Barr virus and monkey herpes virus Saimiri (1Moore P.S. Kim S.J. Dominguez G. Cesarman E. Lungu O. Knowles D.M. Garber R. Pellett P.E. McGeoch D.J. Chang Y. J. Virol. 1996; 70: 549-558Crossref PubMed Google Scholar). Sequence analysis of KSHV genome revealed the presence of ∼80 open reading frames (ORFs) of which a number of ORFs shows homology to cellular genes that regulate cell growth, immune functions, inflammation, and apoptosis (2Bais C. Kim B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar, 3Boshoff C. Kim Y. Collins P.D. Takeuchi Y. Reeves J.D. Schweickart V.L. Siani M.A. Sasaki T. Williams T.J. Gray P.W. Moore P.S. Chang Y. Weiss R.A. Science. 1997; 278: 290-294Crossref PubMed Scopus (443) Google Scholar, 4Molden J. Kim Y. You Y. Moore P.S. Goldsmith M.A. J. Biol. Chem. 1997; 272: 19625-19631Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 5Thome M. Kim P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J.L. Schroter M. Scaffidi C. Krammer P.H. Peter M.E. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1146) Google Scholar). These include a cluster of four ORFs with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family (6Dittmer D.P. Cancer Res. 2003; 63: 2010-2015PubMed Google Scholar, 7Offermann M.K. Curr. Top. Microbiol. Immunol. 2007; 312: 185-209PubMed Google Scholar). One of them, viral interferon regulatory factor-3 (vIRF-3, also referred to as LANA2) (8Lubyova B. Kim P.M. J. Virol. 2000; 74: 8194-8201Crossref PubMed Scopus (152) Google Scholar, 9Lubyova B. Kim M.J. Frisancho A.J. Pitha P.M. J. Biol. Chem. 2004; 279: 7643-7654Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 10Rivas C. Kim A.E. Parravicini C. Moore P.S. Chang Y. J. Virol. 2001; 75: 429-438Crossref PubMed Scopus (256) Google Scholar), is a multifunctional nuclear protein constitutively expressed in KSHV-positive primary effusion lymphoma (PEL) cells and Castleman disease tumors, whereas it is not detected in Kaposi sarcoma spindle cells (8Lubyova B. Kim P.M. J. Virol. 2000; 74: 8194-8201Crossref PubMed Scopus (152) Google Scholar, 10Rivas C. Kim A.E. Parravicini C. Moore P.S. Chang Y. J. Virol. 2001; 75: 429-438Crossref PubMed Scopus (256) Google Scholar). The vIRF-3 protein binds to cellular IRF-3 and IRF-7 and to the transcription co-activators, CREB-binding protein/p300, and modulates IRF-3/IRF-7-mediated transcription of Type I interferon genes (9Lubyova B. Kim M.J. Frisancho A.J. Pitha P.M. J. Biol. Chem. 2004; 279: 7643-7654Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Furthermore, interaction of vIRF-3 with p53 results in inhibition of p53 transcriptional activation and p53-induced apoptosis (10Rivas C. Kim A.E. Parravicini C. Moore P.S. Chang Y. J. Virol. 2001; 75: 429-438Crossref PubMed Scopus (256) Google Scholar). Inhibition of the IκB kinase kinase activity, and down-modulation of the NFκB-dependent transcription by vIRF-3 have also been reported (11Seo T. Kim J. Lim C. Choe J. Oncogene. 2004; 23: 6146-6155Crossref PubMed Scopus (51) Google Scholar). Recently, vIRF-3 was shown to interact with 14-3-3 proteins and inhibit FOXO3a transcription factor, which may contribute to cell cycle de-regulation (12Munoz-Fontela C. Kim L. Gallego P. Arroyo J. Da Costa M. Pomeranz K.M. Lam E.W. Rivas C. J. Virol. 2007; 81: 1511-1516Crossref PubMed Scopus (39) Google Scholar). KSHV-positive PEL is an aggressive B-cell lymphoma typically growing as lymphomatous effusions in the body cavities without a contiguous tumor mass. In addition to KSHV, some PEL cells also carry the Epstein-Barr virus genome, but are devoid of genetic lesions of c-myc, bcl-2, and p53 (13Carbone A. Kim A.M. Gloghini A. Capello D. Todesco M. Quattrone S. Volpe R. Gaidano G. Br. J. Haematol. 1998; 102: 1081-1089Crossref PubMed Scopus (73) Google Scholar). Cell lines established from PEL continuously express only six viral genes, vFLIP (ORF71), vCYC (ORF72), latency-associated nuclear antigen-1, LANA (ORF73), Kaposin (K12) (14Zhong W. Kim H. Herndier B. Ganem D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6641-6646Crossref PubMed Scopus (412) Google Scholar, 15Sarid R. Kim O. Bohenzky R.A. Chang Y. Moore P.S. J. Virol. 1998; 72: 1005-1012Crossref PubMed Google Scholar, 16Dittmer D. Kim M. Renne R. Staskus K. Haase A. Ganem D. J. Virol. 1998; 72: 8309-8315Crossref PubMed Google Scholar, 17Talbot S.J. Kim R.A. Kellam P. Boshoff C. Virology. 1999; 257: 84-94Crossref PubMed Scopus (141) Google Scholar), vIRF-3/LANA2, and short ORF K11.1 encoding vIRF-2 (8Lubyova B. Kim P.M. J. Virol. 2000; 74: 8194-8201Crossref PubMed Scopus (152) Google Scholar, 10Rivas C. Kim A.E. Parravicini C. Moore P.S. Chang Y. J. Virol. 2001; 75: 429-438Crossref PubMed Scopus (256) Google Scholar, 18Burysek L. Kim W.S. Pitha P.M. J. Hum. Virol. 1999; 2: 19-32PubMed Google Scholar). How KSHV contributes to the establishment of the KSHV-associated neoplasia remains to be determined. Proliferation of PEL cells in culture was found to depend on the autocrine production of KSHV-encoded vIL-6, expressed during lytic infection (19Jones K.D. Kim Y. Chang Y. Moore P.S. Yarchoan R. Tosato G. Blood. 1999; 94: 2871-2879Crossref PubMed Google Scholar). However, because the expression of KSHV in PEL cells is latent, it is assumed that latent genes rather than the genes expressed during the lytic infection may contribute to leukemogenesis. Two of these latent genes, vFLIP and Kaposin, have transforming potential in vitro (20Djerbi M. Kim V. Catrina A.I. Bogen B. Biberfeld P. Grandien A. J. Exp. Med. 1999; 190: 1025-1032Crossref PubMed Scopus (386) Google Scholar, 21Muralidhar S. Kim A.M. Hassani M. Sadaie M.R. Kishishita M. Brady J.N. Doniger J. Medveczky P. Rosenthal L.J. J. Virol. 1998; 72: 4980-4988Crossref PubMed Google Scholar); however, they were unable to induce tumors in vivo in transgenic mice. The expression of vGPCR was sufficient to induce endothelial tumors resembling Kaposi sarcoma (22Montaner S. Kim A. Molinolo A. Bugge T.H. Sawai E.T. He Y. Li Y. Ray P.E. Gutkind J.S. Cancer Cell. 2003; 3: 23-36Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 23Guo H.G. Kim M. Reid W. Tschachler E. Hayward G. Reitz M. J. Virol. 2003; 77: 2631-2639Crossref PubMed Scopus (133) Google Scholar) but did not induce B-cell lymphomas. Although it has been accepted that lymphomagenesis is a multistep transformation process, a number of genetic changes and infection agents contribute to B-cell lymphoproliferative disorders. c-myc is a proto-oncogene that has been implicated in controlling cellular growth, proliferation, and cell survival (24Grandori C. Kim S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1040) Google Scholar). It also plays a critical role in the development of B-cell lymphomas (25Keller U.B. Kim J.B. Dorsey F.C. Nilsson J.A. Nilsson L. MacLean K.H. Chung L. Yang C. Spruck C. Boyd K. Reed S.I. Cleveland J.L. EMBO J. 2007; 26: 2562-2574Crossref PubMed Scopus (79) Google Scholar, 26Janz S. DNA Repair (Amst.). 2006; 5: 1213-1224Crossref PubMed Scopus (83) Google Scholar, 27Rui L. Kim C.C. Curr. Mol. Med. 2006; 6: 291-308Crossref PubMed Scopus (32) Google Scholar). Overexpression of c-myc, as a consequence of a reciprocal chromosomal exchange involving the immunoglobulin loci, can be seen in Epstein-Barr virus-positive B-cell lymphomas. Expression of c-myc in transgenic mice results in the formation of pre-B-cell lymphoma (28Adams J.M. Kim A.W. Pinkert C.A. Corcoran L.M. Alexander W.S. Cory S. Palmiter R.D. Brinster R.L. Nature. 1985; 318: 533-538Crossref PubMed Scopus (1337) Google Scholar) and immature CD4+ and CD8+ T-cell lymphoma (29Felsher D.W. Kim J.M. Mol. Cell. 1999; 4: 199-207Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Thus, overexpression of c-myc may have an important role in lymphoid cell neoplasia. The ability of c-Myc to promote proliferation through cell cycle re-entry seems critical to its oncogenic function. Constitutive expression of c-myc reduces the growth factor requirement, prevents growth arrest, and blocks cellular differentiation (30Alexandrow M.G. Kim M. Aakre M. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3239-3243Crossref PubMed Scopus (118) Google Scholar, 31Hermeking H. Kim J.O. Reichert M. Ellwart J.W. Eick D. Oncogene. 1995; 11: 1409-1415PubMed Google Scholar). The c-myc gene encodes a transcription factor of the basic-helix-loop-helix-leucine zipper family. c-Myc dimerization with another basic-helix-loop-helix-leucine zipper protein, Max, is required for its binding to the specific DNA sequence CACGTG (E-box) and activation of transcription (32Amati B. Kim K. Vlach J. Front. Biosci. 1998; 3: d250-d268Crossref PubMed Scopus (329) Google Scholar, 33Henriksson M. Kim B. Adv. Cancer Res. 1996; 68: 109-182Crossref PubMed Google Scholar). Two domains of c-Myc are crucial for its biological activities. The N-terminal domain consists of the transcriptional activation domain, whereas the C-terminal domain mediates DNA binding to promoters of target genes. In recent years, many new C-terminal domain- and N-terminal domain-interacting proteins have been identified (34Sakamuro D. Kim G.C. Oncogene. 1999; 18: 2942-2954Crossref PubMed Scopus (153) Google Scholar, 35Blackwood E.M. Kim B. Kretzner L. Eisenman R.N. Cold Spring Harb. Symp. Quant. Biol. 1991; 56: 109-117Crossref PubMed Scopus (29) Google Scholar). The transcriptional activity of Myc can be repressed by FoxO proteins (36Bouchard C. Kim J. Bras A. Medema R.H. Eilers M. EMBO J. 2004; 23: 2830-2840Crossref PubMed Scopus (172) Google Scholar), and by a novel c-Myc repressor, Myc modulator-1 (MM-1). MM-1 binds to the N-terminal domain of c-Myc and suppresses its E-box-dependent transcriptional activity (37Satou A. Kim T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 46562-46567Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A mutation of Ala to Arg at amino acid 157 in MM-1, which is frequently observed in patients with leukemia and lymphoma, abrogated the MM-1 suppression activity of c-Myc (38Fujioka Y. Kim T. Maeda Y. Tanaka S. Nishihara H. Iguchi-Ariga S.M. Nagashima K. Ariga H. J. Biol. Chem. 2001; 276: 45137-45144Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). MM-1 cDNA was originally identified as a fusion gene derived from sequences on chromosomes 14 and 12 (39Hagio Y. Kim Y. Taira T. Fujioka Y. Iguchi-Ariga S.M. Ariga H. J. Cell. Biochem. 2006; 97: 145-155Crossref PubMed Scopus (12) Google Scholar). Of additional isolated MM-1 isoforms (MM-1α, -β, -γ, and -δ), the ubiquitously expressed MM-1α is the most predominant with the strongest suppression activity toward c-Myc (39Hagio Y. Kim Y. Taira T. Fujioka Y. Iguchi-Ariga S.M. Ariga H. J. Cell. Biochem. 2006; 97: 145-155Crossref PubMed Scopus (12) Google Scholar). The aim of this study was to analyze a possible role of vIRF-3 in KSHV-associated PEL lymphomas. Using yeast two-hybrid screening, we identified MM-1α as the vIRF-3-interacting protein, and showed that MM-1α is associated with vIRF-3 in PEL cells. The vIRF-3-MM-1α interaction resulted in the release of c-Myc from MM-1α-mediated transcriptional repression. Furthermore, vIRF-3 was tethered to cdk4 and the synthetic E-box-containing promoters through its association with c-Myc. These results suggest a novel mechanism by which KSHV targets the c-Myc-regulated pathways that may inadvertently result in uncontrolled cell growth and transformation. Cell Lines and Culture Conditions—BCBL-1, BC-3, and Daudi cells were grown in RPMI 1640 supplemented with 10% or 20% (for BC-3 cells) fetal bovine serum. The RAT1 fibroblast subclone TGR-1 (c-myc+/+) and the c-myc–/– derivative, HO15 (kindly provided by Dr. Chi V. Dang, Johns Hopkins University, Baltimore, MD), have been previously described (40Mateyak M.K. Kim A.J. Adachi S. Sedivy J.M. Cell Growth & Differ. 1997; 8: 1039-1048PubMed Google Scholar). TGR-1, HO15, and HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Plasmids and Antibodies—For the yeast-two hybrid screening, the vIRF-3-C′/pAS2 was cloned by inserting the C-terminal part of vIRF-3 (aa 254–566) between the EcoRI and BamHI sites of pAS2–1 (Clontech Laboratories, Inc., Palo Alto, CA). Full-length vIRF-3 (vIRF-3-FL; aa 1–566), vIRF-3-N′ (aa 1–254), vIRF-3-C′ (aa 254–566) and vIRF-3-GST were described previously (8Lubyova B. Kim P.M. J. Virol. 2000; 74: 8194-8201Crossref PubMed Scopus (152) Google Scholar). Expression plasmid c-myc-HA and cdk4 reporter constructs, wtMBS1–4, mutMBS1–4, and mut-MBS3 + 4, were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The 4XE-box-luc reporter plasmid was kindly provided by Dr. Hiroyoshi Ariga (Hokkaido University, Sapporo, Japan). The T7-MM-1α expression plasmid was cloned by reverse transcription-PCR amplification of MM-1α cDNA using RNA isolated from BCBL-1 cells. The 5′ primer carried a T7 tag sequence that was in-frame with the MM-1α open reading frame. The PCR product was subcloned into pcDNA3.1 vector (Invitrogen). GST-MM-1α-FL (aa 1–154), and its deletion constructs GST-MM-1α-(1–125), -(1–93), -(1–62), -(1–30), -(1–62), and -(63–154) were cloned by amplification of the corresponding region of MM-1α cDNA from the T7-MM-1α expression plasmid and subcloned into pGEX-4T vector (Amersham Biosciences). The fidelity of all constructs was verified on the ABI PRISM™ 377 automated DNA sequencer (Applied Biosystems, Foster City, CA). The following antibodies were used: polyclonal antibodies against c-Myc (N 262), p73, IRF-3, actin, and HA (Santa Cruz Biotechnology Inc., Santa Cruz, CA), T7 monoclonal antibodies (Novagen, EMD Chemicals Inc., San Diego, CA), acetylated-histone 3 antibodies (Upstate Millipore, Billerica, MA), and antibodies against GST (Amersham Biosciences). Production and purification of polyclonal antibodies against vIRF-3 was described previously (9Lubyova B. Kim M.J. Frisancho A.J. Pitha P.M. J. Biol. Chem. 2004; 279: 7643-7654Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The MM-1α monoclonal antibodies were kindly provided by Dr. Hiroyoshi Ariga. Yeast Two-hybrid Screen—The MATCHMAKER Two-hybrid System 3 was purchased from Clontech Laboratories, Inc. The bait plasmid, vIRF-3-C′/pAS2, and the human leukocyte cDNA library (Clontech Laboratories, Inc.) were introduced into the Saccharomyces cerevisiae strain AH109 using the lithium acetate/heat shock procedure (41Chien C.T. Kim P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1224) Google Scholar). The transformed yeast cells were plated on SD/-Ade/-His/-Leu/-Trp/X-α-gal medium and incubated for 1 week at 30 °C. The interacting cDNA clones were rescued from positive colonies. The sequences of positive cDNA clones were determined on ABI PRISM™ 377 automated DNA sequencer (Applied Biosystems) and analyzed using the BLAST program. Immunoprecipitation and Western Blot Analysis—HEK293 cells were co-transfected with vIRF-3, c-myc-HA, and T7-MM-1α expression plasmids using the SuperFect transfection reagent (Qiagen). At 24 h post-transfection, the cells were lysed in co-immunoprecipitation buffer (20 mm HEPES (pH 7.9), 50 mm NaCl, 5 mm EDTA, 2 mm EGTA, 0.1% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 0.2 mm protease inhibitor mixture (Sigma Co., St. Louis, MO)). The protein extracts (400 μg) were then incubated with the respective antibodies at 4 °C for 1 h, and then 30 μlof protein A/G-Sepharose beads (Santa Cruz Biotechnology Inc.) was added, followed by overnight incubation at 4 °C. Immune complexes were extensively washed with co-immunoprecipitation buffer, and precipitated proteins were resolved by SDS-PAGE and analyzed by Western blot. Luciferase Assays—For luciferase assays with HEK293 cells, the cells were seeded in 6-well tissue culture plates 12 h before transfection. Sub-confluent cells were transfected with equal amounts (1 μg) of luciferase reporter and vIRF-3, c-Myc-HA, or T7-MM-1α expression plasmids together with control plasmid pRL-SV40 (0.1 μg, Promega, Madison, WI) using SuperFect transfection reagent (Qiagen). Forty-eight hours after transfection the cells were lysed with the Reporter Lysis Buffer (Promega), and luciferase activity was measured in 20 μl of the lysate using Dual Luciferase Reporter Assay Kit (Promega) as recommended by the manufacturer. Each experiment was repeated three times. The Renilla luciferase activity levels were used to normalize the differences in the transfection efficiency. For luciferase assays with BCBL-1 cells, the procedure was identical except that transfections were done using the Nucleofector transfection device (Amaxa Inc., Gaithersburg, MD). GST Pulldown Assay—In vitro translated proteins were synthesized using the coupled TnT T7 transcription-translation system (Promega) according to the manufacturer’s instruction. GST fusion proteins (0.5 μg) bound to glutathione-Sepharose beads were incubated with 10 μl of the reaction mixture consisting of in vitro translated proteins in 250 μl of binding buffer (10 mm Tris (pH 7.6), 100 mm NaCl, 0.1 mm EDTA (pH 8.0), 1 mm dithiothreitol, 5 mm MgCl2, 0.05% Nonidet P-40, 8% glycerol, 0.2 mm protease inhibitor mixture (Sigma)) at 4 °C for 90 min. After three 10-min washes with binding buffer, the proteins bound to the beads were resolved by SDS-PAGE and detected by Western blotting with specific antibodies. For the control of quality and equal loading of GST fusion proteins, the Western blot was re-hybridized with GST antibodies (Amersham Biosciences). Oligonucleotide Pulldown Assay—The DNA pulldown assay was done as previously described (42Au W.C. Kim P.M. J. Biol. Chem. 2001; 276: 41629-41637Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, double-stranded oligomers corresponding to the MBS4 in the human cdk4 promoter region (5′-CCCTCAGCGCATGGGTGGCGGTCACGTGCCCAGAACGTCCGG-3′), and tetramerized E-box sequences, 4XE-box (5′-CCCTCAGCGCATGGGTGGCGGTCACGTGCACGTGCACGTGCACGTGCCCAGAACG-3′; E-boxes are underlined), were synthesized and biotin-labeled at the 5′ end of the sense strand and coupled with streptavidin magnetic beads (Dynal, Invitrogen). Whole cell lysates (350 μg) were then incubated with the DNA bound to magnetic beads for 3 h at 4 °C. After extensive washing, the bound proteins were resolved by SDS-PAGE and analyzed by Western blot. Chromatin Immunoprecipitation—The assay was performed using a chromatin immunoprecipitation assay kit (Upstate Millipore) following the manufacturer’s instructions. Briefly, for the endogenous cdk4 promoter studies, BCBL-1 or HEK293 cells (107) were transfected with an empty vector, pcDNA3.1 (control), c-myc-HA-, or vIRF-3 expression plasmids. At 24 h post-transfection, the proteins bound to DNA were cross-linked in the presence of 1% formaldehyde; cells were resuspended in the SDS lysis buffer, followed by sonication. After pre-clearing with salmon sperm DNA/Protein A-agarose (50% slurry), the protein extracts were subjected to immunoprecipitation with antibodies against c-Myc, vIRF-3, or acetylated H3. Immunoprecipitation with antibodies against p73 or IRF-3 was used as a negative control. Immunocomplexes were extensively washed, and the DNA was recovered by phenol/chloroform extraction and resuspended in 50 μl of 10 mm Tris-HCl (pH 8.5). Serial dilutions (1, 5, 10, and 20 μl) were used as template for PCR amplification to show that the response was in the linear rage. Each experiment was repeated three times. PCR amplification was performed with the following cdk4-specific primers: cdk4-FWD, 5-AGTGAGACAATCCTTCAGCCG-3′; cdk4-REV, 5′-GACGTTCTGGGCACGTGAC-3′. The samples were also amplified with GAPDH primers: GAPDH-FWD, 5′-CCCAACTTTCCCGCCTCTC-3′; GAPDH-REV, 5′-CAGCCGCCTGGTTCAACTG-3′, which were used as controls (43Fulmer-Smentek S.B. Kim U. Hum. Mol. Genet. 2001; 10: 645-652Crossref PubMed Scopus (34) Google Scholar). Isolation of MM-1α as a vIRF-3-binding Protein in Yeast Two-hybrid Screen—In the search for one or more cellular proteins that may interact with vIRF-3, we employed a yeast two-hybrid system to screen a human leukocyte cDNA library. Initially, we used the full-length vIRF-3 (vIRF-3-FL) as a bait, but the control experiments showed that vIRF-3 may have a strong activation domain, because introduction of the vIRF-3-FL/pAS2 construct into Saccharomyces cerevisiae strain AH109 yielded numerous positive colonies on plates with SD/-Ade/-His/-Leu/-Trp/X-α-gal medium. Therefore, we used the plasmid, vIRF-3-C′/pAS2, encoding only the C-terminal half (aa 254–566) of the vIRF-3 protein. Co-transformation of AH109 yeast cells with the vIRF-3-C′/pAS2 bait and the human leukocyte cDNA library resulted in 42 positive colonies that grew on selection medium lacking Trp, Leu, His, and Ade. 22 of 42 His/Ade-positive transformants formed blue colonies. Plasmids rescued from these positive colonies were analyzed using restriction enzymes, and their inserts were sequenced. One clone, termed C19, contained partial cDNA for human MM-1, which has been previously shown to be a c-Myc-binding protein (44Mori K. Kim Y. Kitaura H. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 1998; 273: 29794-29800Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cDNA insert of this plasmid encoded the protein ranging from amino acids 17–167 and 4–167 of MM-1 and MM-1α, respectively (Fig. 1A). Because the C19-encoded protein was almost identical to MM-1α, we cloned a T7-tagged MM-1α cDNA from a KSHV-positive PEL cell line, BCBL-1, into pcDNA3.1 vector and used the construct for the experiments described in this study. The originally isolated MM-1 (44Mori K. Kim Y. Kitaura H. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 1998; 273: 29794-29800Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), which represents a fusion gene derived from sequences on chromosome 14 and 12, and the recently identified isoform MM-1α (39Hagio Y. Kim Y. Taira T. Fujioka Y. Iguchi-Ariga S.M. Ariga H. J. Cell. Biochem. 2006; 97: 145-155Crossref PubMed Scopus (12) Google Scholar), share the same properties and functions. Both of them are nuclear proteins that exhibit a strong inhibitory effect on c-Myc-mediated transcription (39Hagio Y. Kim Y. Taira T. Fujioka Y. Iguchi-Ariga S.M. Ariga H. J. Cell. Biochem. 2006; 97: 145-155Crossref PubMed Scopus (12) Google Scholar). Specificity of the MM-1α-vIRF-3 Interaction in Vitro and in Vivo—To confirm the results of the yeast two-hybrid assay, we first examined the interaction of vIRF-3 with MM-1α in vitro by a GST pulldown assay and then in vivo by co-immunoprecipitation. The results in Fig. 1B showed that in vitro translated MM-1α bound strongly to the full-length GST-vIRF-3 fusion protein, whereas, it did not interact with GST alone. The in vivo interaction between ectopically expressed vIRF-3 and MM-1α in HEK293 cells was examined by co-immunoprecipitation. As shown in Fig. 1C, T7-tagged MM-1α co-precipitated with transfected vIRF-3 protein. In addition, we detected the interaction between endogenously expressed vIRF-3 and MM-1α proteins in two PEL-derived cell lines, BCBL-1 and BC-3 (Fig. 1D). Co-immunoprecipitation in Daudi cells, which are KSHV-negative and do not express vIRF-3, was used as a negative control. Identification of the MM-1α Interaction Domain—To determine which part of MM-1α protein interacted with vIRF-3, we constructed a series of MM-1α deletion mutants, which were expressed as GST fusion proteins (Fig. 2A). The mobility and purity of these recombinant fusion proteins is shown in Fig. 2B. When used in the GST pulldown assay, all GST-MM-1α deletion mutants were able to bind effectively to the in vitro translated full-length vIRF-3, except the shortest MM-1α-(1–30)-GST and GST alone (Fig. 2C). These results suggest that the MM-1α protein interacted with vIRF-3 through the binding domain that was located in the region between amino acids 31 and 62. To confirm the binding of this peptide to vIRF-3, we constructed the MM-1α (aa 31–62) and MM-1α (aa 63–154) fused to GST and analyzed their ability to interact with vIRF-3 in the GST pulldown assay. The results in Fig. 2D showed that in vitro translated vIRF-3 bound with the same intensities to the MM-1α-FL-GST and MM-1α-(31–62)-GST protein, whereas it did not interact with either MM-1α-(63–154)-GST or GST alone. These data suggest that the interaction between vIRF-3 and MM-1α is direct and that the region between amino acids 31 and 62 of MM-1α protein is its primary interaction domain with vIRF-3. Stimulation of c-Myc Transcriptional Activity by vIRF-3—The ability of c-Myc to promote cell cycle re-entry is in part due to its ability to induce transcription of cdk4 (45Hermeking H. Kim C. Schuhmacher M. Li Q. Barrett J.F. Obaya A.J. O'Connell B.C. Mateyak M.K. Tam W. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2229-2234Crossref PubMed Scopus (387) Google Scholar). The cdk4 gene promoter contains four putative c-Myc binding sites (MBS1–4). Mutation analysis of individual MBS elements suggested that MBS3 and MBS4 were particularly important in the transactivation of cdk4 promoter by c-Myc (45Hermeking H. Kim C. Schuhmacher M. Li Q. Barrett J.F. Obaya A.J. O'Connell B.C. Mateyak M.K. Tam W. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2229-2234Crossref PubMed Scopus (387) Google Scholar). To determine the effect of vIRF-3 on transcriptional activity of c-Myc, we transfected HEK293 cells with c-myc, vIRF-3, and MM-1α expression plasmids together with a reporter construct containing the luciferase gene linked to the wild-type cdk4 promoter, wtMBS1–4. As shown in Fig. 3A, both c-Myc and vIRF-3 activated the cdk4 promoter by ∼2-fold, whereas MM-1α suppressed its activity. In addition, co-transfection of vIRF-3 together with c-myc resulted in further stimulation of cdk4 promoter activity to ∼3.3-fold. Additional co-transfection" @default.
• W2075812596 created "2016-06-24" @default.
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• W2075812596 date "2007-11-01" @default.
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• W2075812596 title "Stimulation of c-Myc Transcriptional Activity by vIRF-3 of Kaposi Sarcoma-associated Herpesvirus" @default.
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