Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.033290
ISSN1083-351X
AutoresCristina Aresté, Mohamed Mutocheluh, David J. Blackbourn,
Tópico(s)Inflammasome and immune disorders
ResumoUpon virus infection, the cell mounts an innate type I interferon (IFN) response to limit the spread. This response is orchestrated by the constitutively expressed IFN regulatory factor (IRF)-3 protein, which becomes post-translationally activated. Although the activation events are understood in detail, the negative regulation of this innate response is less well understood. Many viruses, including Kaposi sarcoma-associated herpesvirus (KSHV), have evolved defense strategies against this IFN response. Thus, KSHV encodes a viral IRF (vIRF)-2 protein, sharing homology with cellular IRFs and is a known inhibitor of the innate IFN response. Here, we show that vIRF-2 mediates IRF-3 inactivation by a mechanism involving caspase-3, although vIRF-2 itself is not pro-apoptotic. Importantly, we also show that caspase-3 participates in normal IRF-3 turnover in the absence of vIRF-2, during the antiviral response induced by poly(I:C) transfection. These data provide unprecedented insight into negative regulation of IRF-3 following activation of the type I IFN antiviral response and the mechanism by which KSHV vIRF-2 inhibits this innate response. Upon virus infection, the cell mounts an innate type I interferon (IFN) response to limit the spread. This response is orchestrated by the constitutively expressed IFN regulatory factor (IRF)-3 protein, which becomes post-translationally activated. Although the activation events are understood in detail, the negative regulation of this innate response is less well understood. Many viruses, including Kaposi sarcoma-associated herpesvirus (KSHV), have evolved defense strategies against this IFN response. Thus, KSHV encodes a viral IRF (vIRF)-2 protein, sharing homology with cellular IRFs and is a known inhibitor of the innate IFN response. Here, we show that vIRF-2 mediates IRF-3 inactivation by a mechanism involving caspase-3, although vIRF-2 itself is not pro-apoptotic. Importantly, we also show that caspase-3 participates in normal IRF-3 turnover in the absence of vIRF-2, during the antiviral response induced by poly(I:C) transfection. These data provide unprecedented insight into negative regulation of IRF-3 following activation of the type I IFN antiviral response and the mechanism by which KSHV vIRF-2 inhibits this innate response. The earliest response at the cellular level to virus infection is the establishment of the antiviral state that results from induction of type I interferon (IFN) 2The abbreviations used are: IFNinterferonIRFIFN regulatory factorKSHVKaposi sarcoma-associated herpesvirusKSKaposi sarcomavIRFviral IRFPARPpoly(adenosine diphosphate-ribose) polymeraseZbenzyloxycarbonylFMKfluoromethyl ketoneEVempty vectorsiRNAsmall interference RNAELISAenzyme-linked immunosorbent assay. expression. The objective of this antiviral state is containment of the virus infection and elimination of the infected cell. It operates in multiple ways, including inhibiting cell growth by blocking proliferation and modulating apoptosis, and augmenting adaptive immunological surveillance and responses (see Ref. 1.Randall R.E. Goodbourn S. J. Gen. Virol. 2008; 89: 1-47Crossref PubMed Scopus (1229) Google Scholar). Sensing of the virus infection to initiate the antiviral response occurs in different ways, in part depending upon whether the virus enters the cell by endocytosis or by fusion with the plasma membrane. One of the most important components transducing these virus-sensing signals is IFN regulatory factor (IRF)-3. It participates in transcribing genes that contribute to establishing the antiviral state. interferon IFN regulatory factor Kaposi sarcoma-associated herpesvirus Kaposi sarcoma viral IRF poly(adenosine diphosphate-ribose) polymerase benzyloxycarbonyl fluoromethyl ketone empty vector small interference RNA enzyme-linked immunosorbent assay. Upon virus infection, IRF-3 is post-translationally modified by C-terminal phosphorylation by a “virus-activated kinase” (2.Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 3.Smith E.J. Marié I. Prakash A. García-Sastre A. Levy D.E. J. Biol. Chem. 2001; 276: 8951-8957Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) that promotes translocation of the protein from the cytoplasm to the nucleus. There, it is assimilated into the IFN-β enhancesome, a multiprotein complex that facilitates transcription of IFN and IFN-responsive genes. This enhancesome, whose structure is well characterized (4.Panne D. Maniatis T. Harrison S.C. Cell. 2007; 129: 1111-1123Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 5.Dragan A.I. Carrillo R. Gerasimova T.I. Privalov P.L. J. Mol. Biol. 2008; 384: 335-348Crossref PubMed Scopus (18) Google Scholar), represents the paradigm for understanding the molecular basis behind regulation of gene transactivation in response to virus infection. The components of virus-activated kinase that phosphorylate IRF-3 include IκB kinase-ϵ and TANK-binding kinase-1 (6.Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1366) Google Scholar, 7.Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2085) Google Scholar). Depending on the pathway leading to IRF-3 activation, other kinases may also participate, including phosphatidylinositol 3-kinase (8.Sarkar S.N. Peters K.L. Elco C.P. Sakamoto S. Pal S. Sen G.C. Nat. Struct. Mol. Biol. 2004; 11: 1060-1067Crossref PubMed Scopus (309) Google Scholar). Although post-translational activation of IRF-3 is understood in detail, less is known of its deactivation that negatively regulates the type I IFN response. Until now, only phosphorylation-dependent ubiquitination of IRF-3, leading to its proteasomal degradation has been recognized. Hiscott and colleagues showed that C-terminal phosphorylation of IRF-3 is necessary for degradation and is followed by Cullin1 interaction, ubiquitination, and proteasomal degradation (9.Bibeau-Poirier A. Gravel S.P. Clément J.F. Rolland S. Rodier G. Coulombe P. Hiscott J. Grandvaux N. Meloche S. Servant M.J. J. Immunol. 2006; 177: 5059-5067Crossref PubMed Scopus (77) Google Scholar; see Ref. 10.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Poly-ubiquitination and concomitant degradation of IRF-3 are regulated by the peptidylprolyl isomerase Pin-1 (11.Saitoh T. Tun-Kyi A. Ryo A. Yamamoto M. Finn G. Fujita T. Akira S. Yamamoto N. Lu K.P. Yamaoka S. Nat. Immunol. 2006; 7: 598-605Crossref PubMed Scopus (254) Google Scholar). Inhibiting the IFN antiviral response is an important component of the biology of many viruses (1.Randall R.E. Goodbourn S. J. Gen. Virol. 2008; 89: 1-47Crossref PubMed Scopus (1229) Google Scholar). Studying the molecular interactions of viruses with the immune system, including their strategies of evasion, has provided deeper understanding of its operation. Recent examples set a precedent in the context of the innate immune system. First, the study of the human immunodeficiency virus Vif protein identified a new innate immune response to retroviruses (12.Sheehy A.M. Gaddis N.C. Choi J.D. Malim M.H. Nature. 2002; 418: 646-650Crossref PubMed Scopus (1914) Google Scholar) mediated by the cellular protein, CEM15 or APOBEC3G, a DNA deaminase, which destroys or mutates the virus genome (13.Harris R.S. Bishop K.N. Sheehy A.M. Craig H.M. Petersen-Mahrt S.K. Watt I.N. Neuberger M.S. Malim M.H. Cell. 2003; 113: 803-809Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar). Second, the recognition through its binding to paramyxovirus V proteins that mda-5 is a central player in the signal transduction cascade leading to IFN-β expression (14.Andrejeva J. Childs K.S. Young D.F. Carlos T.S. Stock N. Goodbourn S. Randall R.E. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17264-17269Crossref PubMed Scopus (818) Google Scholar). Through studying modulation of the IFN response by Kaposi sarcoma-associated herpesvirus (KSHV), we now demonstrate a unique cellular mechanism inhibiting IRF-3 function by a caspase-3-dependent process. KSHV is the etiologic agent of the most common malignancy affecting AIDS patients, Kaposi sarcoma (KS), which is also the most common tumor of men in certain African countries (15.Parkin D.M. Int. J. Cancer. 2006; 118: 3030-3044Crossref PubMed Scopus (2240) Google Scholar). Approximately one quarter of the KSHV genome encodes proteins with either demonstrated or putative immunomodulatory activity (16.Aresté C. Blackbourn D.J. Trends Microbiol. 2009; 17: 119-129Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and one of these viral genes encodes the vIRF-2 protein (17.Cunningham C. Barnard S. Blackbourn D.J. Davison A.J. J. Gen. Virol. 2003; 84: 1471-1483Crossref PubMed Scopus (67) Google Scholar), which inhibits the type I IFN response to viral infection (18.Burysek L. Yeow W.S. Pitha P.M. J. Hum. Virol. 1999; 2: 19-32PubMed Google Scholar, 19.Burýsek L. Pitha P.M. J. Virol. 2001; 75: 2345-2352Crossref PubMed Scopus (136) Google Scholar, 20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). Here, using a model system of activation of the antiviral response by transfection of synthetic double-stranded RNA, we have identified a novel caspase-3-dependent turnover of cellular IRF-3 that is involved in the normal negative feedback loop to help terminate the antiviral response. This caspase-3-dependent mechanism is targeted by vIRF-2 to accelerate cellular IRF-3 turnover and thereby down-modulate the antiviral response. Poly(I:C), MG132, and etoposide were obtained from Sigma-Aldrich, and Z-VAD-FMK was from Calbiochem. SYTO® 16 green-fluorescent nucleic acid stain was a generous gift of Gemma Kelly. HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, penicillin, and streptomycin and 10% heat-inactivated fetal bovine serum. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with penicillin and streptomycin and 10% fetal bovine serum. Full-length KSHV vIRF-2 cDNA was subcloned into the pcDNA4/HisMax expression vector as previously described (20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). The resulting vIRF-2 protein contains contiguous N-terminal polyhistidine and XpressTM epitope tags and is referred to as Xpress-tagged vIRF-2. The caspase-3 (pcDNA3/caspase-3) and FLAG epitope-tagged IRF-3 wild-type (IRF-3WT) (21.Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar) expression plasmids were kindly provided by Reiner U. Jänicke and John Hiscott, respectively. The p125-luc luciferase plasmid contains the full-length IFN-β promoter upstream of the firefly luciferase gene and was generously provided by Takashi Fujita (22.Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). Firefly luciferase levels were normalized against Renilla luciferase constitutively expressed from the co-transfected plasmid pRLSV40 (Stratagene). To mutate the serine and threonine residues in the C-terminal region of IRF-3 to alanine, site-directed mutagenesis was performed with the QuikChangeTM system (Stratagene), according to the manufacturer's instructions. Thus, to generate the 2A(S385/386A), 5A(ST396/398/402/404/405AA), and 2A5A constructs, PCR was performed with the following primers: 2A(S385/386A), 5′-GGGTGCCGCCGCCCTGGAG-3′ and 5′-CTCCAGGGCGGCGGCACCC-3′; 5A(ST396/398/402/404/405AA), 5′-CATTGCCAACGCCCACCCACTCGCCCTCGCCGCCGACC-3′ and 5′-GGTCGGCGGCGAGGGCGAGTGGGTGGGCGTTGGCAATG-3′. The 2A5A mutant was derived with the 2A PCR primers on the 5A mutant. All constructs were verified by sequencing. Transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions on 75% confluent HEK293 or MCF-7 cells grown in 6-well plates. Plasmid quantities were: FLAG-tagged IRF-3WT or mutant expression plasmids (300 ng/well), pcDNA3/caspase-3 expression plasmid (300 ng/well), pcDNA4/vIRF-2 expression plasmid (500 ng/well), p125-luc (100 ng/well), and pRLSV40 (5 ng/well). The corresponding empty vectors were added to equalize DNA concentration (see Ref. 20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). MG132 (10 μm) or Z-VAD-FMK (10 μm) treatment (30 min, 37 °C) was performed 24 h following plasmid transfection. Negative controls were treated with the equivalent volume of DMSO. Next, without changing the medium, the cells were transfected with poly(I:C) (10 μg/ml). Caspase-3 knockdown was performed with caspase-3 ShortCut® siRNA (25 nm, New England Biolabs); the corresponding negative control was the nonspecific Lit28i Polylinker ShortCut® siRNA mix (25 nm, New England Biolabs). Plasmid transfection was performed at the same time, as required. Twenty-four hours later, transfection with poly(I:C) (10 μg/ml) was performed as necessary. Cell pellets were suspended in lysis buffer E (100 mm Tris-HCl, pH 8, 100 mm NaCl, 2 mm EDTA, 2 mm EGTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.5 mm phenylmethylsulfonyl fluoride, mini protease inhibitor mixture (Roche Diagnostic)). After incubation (10 min, 4 °C), insoluble material was removed by centrifugation (16,000 × g, 5 min, 4 °C). The protein concentration was determined by the Bradford dye-binding procedure (Bio-Rad Laboratories), and 35 μg of total protein was separated by SDS-PAGE on 12% acrylamide gels. Proteins were transferred to Immobilon-P transfer membrane (Millipore) that was then blocked in Tris-buffered saline containing 0.02% Tween-20 (TBS-T) and nonfat milk (5%) and probed with primary antibodies diluted in TBS-T containing 5% nonfat milk as described below. Bands were visualized by probing with appropriate secondary antibodies conjugated to horseradish peroxidase (DAKO) and performing enhanced chemiluminescence. Native PAGE was performed essentially according to the protocol of a previous study (23.Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Crossref PubMed Scopus (236) Google Scholar). Primary antibodies were used at the following dilutions and incubation conditions: anti-Pro-caspase-3 polyclonal antibody (Cell Signaling Technologies) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-poly(ADP-ribose) polymerase monoclonal antibody (Sigma-Aldrich) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-phospho-IRF-3 (Ser396) polyclonal antibody (Upstate) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-FLAG® M2 monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-XpressTM monoclonal antibody (Invitrogen) diluted 1:5,000 and incubated for 1 h at 4 °C; anti-β-actin monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-IRF-3 monoclonal antibody (Abcam) diluted 1:100 and incubated for 2 h at 4 °C; and for denaturing gels IRF-3 was detected with anti-IRF-3(FL-425) polyclonal antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated 1 h at 4 °C. Cell lysates were prepared in buffer E, as described above. They were pre-cleared by incubation with protein G-Sepharose 4B fast flow (Sigma-Aldrich) or protein A-Sepharose 4B fast flow (Sigma-Aldrich), and supernatants were collected by centrifugation (16,000 × g, 1 min, 4 °C). They were then incubated with either 1 μg of anti-Xpress (90 min) or 1 μl of anti-procaspase-3 antibodies (16 h) at 4 °C. Immunoprecipitation was performed with either protein G- or protein A-Sepharose beads, and proteins were analyzed by Western blot. Negative control immunoprecipitations were performed on cells transfected with the plasmid pCEP4-SM (1 μg) expressing the FLAG-tagged Epstein-Barr virus protein BSLF2/BMLF, a generous gift of Martin Rowe. Dual luciferase assays were performed according to a previous study (20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). Apoptosis was quantified by flow cytometry on cells stained with Syto-16 and propidium iodide according to the method of a previous study (24.Kelly G.L. Milner A.E. Tierney R.J. Croom-Carter D.S. Altmann M. Hammerschmidt W. Bell A.I. Rickinson A.B. J. Virol. 2005; 79: 10709-10717Crossref PubMed Scopus (86) Google Scholar). Data for ∼25,000 cells were collected for each treatment condition. Caspase activity was measured with the Caspase-Glo®-3/7 assay kit (Promega) that makes use of the luminogenic caspase-3/-7 substrate Z-DEVD-aminoluciferin. Measurements were performed according to the manufacturer's instructions. MCF-7 cells were plated on coverslips in 24-well plates. The next day, they were transfected with expression vectors for FLAG-tagged IRF-3 wild-type (IRF-3WT), or Xpress-tagged vIRF-2 (vIRF-2) or caspase-3, as required. The vIRF-2 protein is dually tagged with both Xpress and His epitopes. Twenty-four hours later, cells were transfected with poly(I:C) (10 μg/ml) for 16 h, as required. The cells were fixed with a final concentration of 2% of formaldehyde solution (methanol-free, Pierce) added directly to the medium for 20 min at room temperature. After fixation, the cells were washed three times (PBS, 15 min, room temperature) and were then permeabilized (2% bovine serum albumin, 0.5% Triton X-100 in PBS, 10 min, at room temperature) and blocked (blocking buffer: 2% bovine serum albumin, 0.1% Triton X-100 in PBS, 60 min at room temperature). Primary antibodies were mouse anti-His (Sigma-Aldrich, 1/100) to detect vIRF-2, goat anti-IRF-3 (RandD, 10 μg/ml), and rabbit anti-caspase-3 (AbCam) diluted 1/50. All primary antibody incubations were performed in blocking buffer overnight at 4 °C. After primary antibody incubation, cells were washed (PBS, four times for 15 min, at room temperature) and incubated with appropriate secondary antibodies: anti-mouse AlexaFluor®-555 (Invitrogen, 1/500 dilution, kindly provided by Dr. Claire Shannon-Lowe), anti-goat AlexaFluor®-633 (Invitrogen, diluted 1/100), anti-rabbit-fluorescein isothiocyanate (Sigma-Aldrich, at 1/100 dilution). All secondary antibody incubations were performed in blocking buffer for 1 h at room temperature. Cells were then washed (PBS, four times for 15 min, at room temperature) and incubated with 4′,6-diamidino-2-phenylindole (1/1000 in PBS, 5 min, at room temperature). The coverslips were mounted with ProLong® Gold Antifade reagent (Molecular Probes, Invitrogen). The vIRF-2 gene was subcloned in-frame into the doxycycline-inducible expression vector pTRE2-pur-Myc (Clontech), creating a 5′-Myc-labeled vIRF-2 derivative. This plasmid was transfected into HEK293-Tet-On cells (Clontech) that stably express the tetracycline-regulated transactivator rtTA. From these cells, a clonal puromycin-resistant cell line was derived with minimal basal vIRF-2 expression; it is referred to as “clone #3–9.” A negative control cell line (“Empty Vector” (EV)), isogenic to clone 3–9 cells except that it lacked vIRF-2, was established in the same way. Active endogenous IRF-3 was measured by TransAMTM IRF-3 transcription factor ELISA (Active Motif) that was performed according to the manufacturer's directions. Nuclear extracts for this assay were prepared with the Nuclear Extract Kit (Active Motif). Ectopic expression studies of vIRF-2 were performed to investigate the function of the vIRF-2 protein without confounding its activity by the presence of other KSHV immunomodulatory proteins. Thus, the cDNA encoding the spliced vIRF-2 gene was cloned into the pcDNA4/HisMax vector (Invitrogen) to generate pcDNA/vIRF-2 expressing the full-length vIRF-2 protein containing contiguous N-terminal polyhistidine and Xpress epitope tags (20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). When measuring the activity of the full-length IFN-β promoter of the transiently co-transfected reporter plasmid p125-luc, vIRF-2 inhibited IFN-β promoter transactivation by wild-type FLAG-tagged IRF-3 (IRF-3WT), by up to 70% (Fig. 1A, upper panel). In this model system, the antiviral response and IRF-3 phosphorylation are activated by transfection of synthetic double-stranded RNA (poly(I:C)), as opposed to using the constitutively active phosphomimetic IRF-3 mutant, IRF-3(5D) (21.Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar). These data are consistent with our previous findings on vIRF-2 inhibition of IRF-3(5D) function (20.Fuld S. Cunningham C. Klucher K. Davison A.J. Blackbourn D.J. J. Virol. 2006; 80: 3092-3097Crossref PubMed Scopus (84) Google Scholar). Because these studies (Fig. 1A, upper panel) were performed by activating IRF-3WT with transfection of poly(I:C), the influence of IRF-3 phosphorylation on the control of transactivation by vIRF-2 was investigated. IRF-3 phosphorylation occurs predominantly within two C-terminal domains, sites 1 and 2, containing serine/threonine-rich tracts (Fig. 1A, lower panel) and is essential for the activity of the protein. Although some debate continues as to the precise function of phosphorylation of specific residues within these sites (see Refs. 10.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 25.Panne D. McWhirter S.M. Maniatis T. Harrison S.C. J. Biol. Chem. 2007; 282: 22816-22822Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), a detailed understanding of the significance of phosphorylation of sites 1 and 2 en masse exists. Thus, IRF-3 autoinhibition is repressed by phosphorylation of the seven serine and threonine residues in these sites that induces a structural change releasing the N terminus and allowing the protein to homodimerize, interact with the transcriptional coactivator CBP/p300, and participate in assembly of the enhancesome (21.Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 26.Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). Specifically, phosphorylation within IRF-3 site 2 represses autoinhibition, permitting interaction with CBP/p300, and facilitating phosphorylation within site 1 that then enables homodimerization (25.Panne D. McWhirter S.M. Maniatis T. Harrison S.C. J. Biol. Chem. 2007; 282: 22816-22822Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Sites 1 and 2 were mutated separately and together, creating two previously described IRF-3 mutants (21.Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 27.Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) 2A and 5A and the double mutant 2A5A (Fig. 1A, lower panel). As expected, the inherent transactivation capacity of the 5A mutant, IRF-3(5A), was reduced by ∼50% compared with that of IRF-3WT (Fig. 1A, upper panel), consistent with its reduced dimerization capability (27.Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). vIRF-2 reproducibly reduced IRF-3(5A) transactivation of the IFN-β promoter slightly, but not by a statistically significant amount (Fig. 1A, upper panel; p > 0.05, Student's t test). These data suggest vIRF-2 inhibition of IRF-3 transactivation depends on cognate phosphorylation of residues at IRF-3 C-terminal site 2. Neither the 2A mutant, IRF-3(2A), nor the double mutant, IRF-3(2A5A), possess significant transactivation activity (Fig. 1A, upper panel), consistent with their inability to dimerize (27.Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and therefore the impact of vIRF-2 on their function is indeterminable. Quantifying the temporal amounts of phosphorylated IRF-3 revealed they were reduced in the presence of vIRF-2. Thus, 18 h after activating the antiviral response by poly(I:C) transfection, phosphorylated IRF-3 levels were reduced in the presence of vIRF-2 (Fig. 1B, upper panel, compare the level of phospho-IRF-3 in the presence of vIRF-2, lanes 9 and 10, with that in the absence of vIRF-2, lanes 4 and 5). Total IRF-3 levels, detected with anti-FLAG antibody, were likewise also reduced to some extent, and this finding was confirmed by non-denaturing gel analyses that revealed vIRF-2 reduced both dimeric (phosphorylated) and monomeric IRF-3 levels (Fig. 1C, upper panel). Repeating these studies up to 48 h post-activation of the antiviral response with poly(I:C) transfection provided further evidence that vIRF-2 reduced IRF-3 levels (data not shown). Thus, these data correlate vIRF-2-mediated inhibition of IRF-3 transactivation of the IFN-β promoter (Fig. 1A) with reduced amounts of phosphorylated IRF-3, following poly(I:C) activation of the antiviral response. Given these findings, the physical interaction of vIRF-2 and IRF-3 was investigated. vIRF-2 was immunoprecipitated, and the products were analyzed for the presence of IRF-3 by Western blot. Only IRF-3WT and IRF-3(5A) co-precipitated with vIRF-2 to any demonstrable extent (Fig. 1D). Indeed, the amount of IRF-3WT co-precipitating was always less than that of IRF-3(5A). Furthermore, activation of the antiviral response by poly(I:C) transfection consistently reduced the amount of IRF-3WT co-precipitating with vIRF-2 (Fig. 1D, top panel, compare lanes 1 and 2), whereas the level of co-precipitating IRF-3(5A) was unaffected or even increased following poly(I:C) treatment (Fig. 1D, top panel, compare lanes 5 and 6). The faint band appearing in the presence of IRF-3(2A) (Fig. 1D, top panel, lane 4) is a nonspecific protein of lower molecular size than IRF-3. Like IRF-3WT (Fig. 1B, upper panel, lanes 4 and 5), IRF-3(5A) gradually accumulated after poly(I:C) transfection in the absence of vIRF-2 (Fig. 1E, upper panel, lanes 1–4), most likely as a consequence of phosphorylation within C-terminal site 1. The absolute levels are lower than for IRF-3WT, because the mutant protein is expressed less efficiently than the wild type (compare “input levels” detected by anti-FLAG antibody in Fig. 1D, bottom panel, lanes 5 and 6 for IRF-3(5A) and lanes 1 and 2 for IRF3WT). However, this mutant, whose transactivation of the IFN-β promoter is not significantly inhibited by vIRF-2, but which binds this viral protein, is not depleted from the cell in the presence of the viral protein, unlike IRF-3WT. Rather, IRF-3(5A) accumulates in the presence of vIRF-2 (Fig. 1E, upper panel, compare the IRF-3(5A) level in the presence of vIRF-2, lanes 5–8, with that in its absence, lanes 1–4). How might vIRF-2 cause the selective turnover of IRF-3WT? Multiple processes, enzymes, and subcellular components drive cellular protein turnover. They include calpain, autophagy, and the proteasome. The stability of IRF-3 in the presence of vIRF-2 was not affected by treatment with a specific inhibitor of calpain (calpain inhibitor I, data not shown), nor by an activator of autophagy (rapamycin, data not shown). The proteasome degrades poly-ubiquitinated proteins. Indeed, as a strategy to evade induction of the innate antiviral response, viruses as diverse as the flavivirus bovine viral diarrhea virus (28.Hilton L. Moganeradj K. Zhang G. Chen Y.H. Randall R.E. McCauley J.W. Goodbourn S. J. Virol. 2006; 80: 11723-11732Crossref PubMed Scopus (202) Google Scholar), having an RNA genome, and bovine herpesvirus 1 (29.Saira K. Zhou Y. Jones C. J. Virol. 2007; 81: 3077-3086Crossref PubMed Scopus (92) Google Scholar) with a DNA genome, can accelerate the decay of IRF-3 by augmenting its poly-ubiquitination and proteasomal degradation (see Ref. 1.Randall R.E. Goodbourn S. J. Gen. Virol. 2008; 89: 1-47Crossref PubMed Scopus (1229) Google Scholar). Proteasome degradation is inhibited by treatment with MG132. As expected, treating cells with MG132 stabilized IRF-3 following poly(I:C) transfection (Fig. 2A, top panel, compare the amount of IRF-3 in lanes 4–6 in the presence of MG132 with that in its absence in lanes 1–3; see also supplemental Figs. S1 and supplemental Fig. S2A(i)). These data confirm that IRF-3 can be the target of a proteasome-dependent degradative process, as previously reported (9.Bibeau-Poirier A. Gravel S.P. Clément J.F. Rolland S. Rodier G. Coulombe P. Hiscott J. Grandvaux N. Meloche S. Servant M.J. J. Immunol. 2006; 177: 5059-5067Crossref PubMed Scopus (77) Google Scholar). Consistent with the previous data (Fig. 1, B–E), the level of IRF-3 was reduced in the presence of vIRF-2 (Fig. 2A, top panel, compare the amount of IRF-3 in lanes 1–3 in the presence of DMSO and absence of vIRF-2 with that in the presence of both DMSO and vIRF-2 at the same times in lanes 8–10; see also supplemental Fig. S2A(ii)). However, importantly and remarkably, IRF-3 turnover was dramatically accelerated in the presence of vIRF-2 and MG132 compared with the absence of vIRF-2 (Fig. 2A, upper panels, compare the amount of IRF-3 in lanes 12–15 in the presence of MG132
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