International Society for Interferon and Cytokine Research

Selected Abstracts from the Annual MeetingOctober 26–30, 2003

Cairns, Australia

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1ACTIVATION AND INHIBITION OF INTERFERON-RESPON-SIVE STAT-DEPENDENT TRANSCRIPTION IN INNATEANTIVIRAL IMMUNITYC.M. HorvathImmunobiology Center, Mount Sinai School of Medicine, New York, NY.

Interferon stimulation (IFN) produces a broadly effective innate anti-viral response that restricts virus replication. Most of the antiviral ac-tivity is the result of a transcriptional program initiated by IFN that ac-tivates STAT protein transcription factors. Type I IFN accomplishesthis gene regulation in large part through the actions of the ISGF3 tran-scription factor complex, composed of STAT1, STAT2, and IRF9. Thefunction of ISGF3 in transcription has been investigated, and severalclasses of transcriptional coactivating machinery have been found, in-cluding histone modifying enzymes and the pol II mediator complex.Identification of these cofactors has provided insights into the mecha-nisms of antiviral responses, and the development of novel antiviralstrategies. The importance of the IFN antiviral system is underscoredby the numerous examples of viruses that have evolved strategies todestroy elements of this system. One family of viruses, the Paramyx-oviridae, directly target STAT proteins to evade IFN responses. The di-verse mechanisms of STAT-directed IFN evasion used by paramyx-oviruses include STAT ubiquitylation and proteasome degradation,cytoplasmic sequestration, and inhibition of nuclear translocation.These mechanisms share one feature, the induction of high molecularweight STAT-1 containing protein complexes. While they evolved asIFN evasion systems, the virus systems provide unique models for novelSTAT targeting strategies that may have therapeutic value in controlof cancer, inflammation, and other diseases characterized by hyperac-tive STAT signal transduction.

2STAT ACTIVATION DURING RESPONSES TO INTERFER-ONS AND BACTERIALouisa Varinou,1 Silvia Stockinger,1 Katrin Ramsauer,1 BenjaminReutterer,1 Klaus Pfeffer,2 Thomas Kolbe,3 Marina Karaghiosoff,4

Mathias Müller,4 Thomas Decker1

1Max F. Perutz Laboratories, University Departments at the ViennaBiocenter, Department of Microbiology and Genetics, University of Vi-enna; Dr. Bohr-Gasse 9/4; A1030 Vienna, Austria, 2Institute of Med-ical Microbiology, University of Düsseldorf, Universitätsstrasse 1,D40225 Düsseldorf, Germany, 3Department of Biotechnology in Ani-mal Production, IFA Tulln, Tulln, Austria, 4Institute of Animal Breed-ing and Genetics; Veterinary University of Vienna, A-1210 Vienna.

Stat1 is activated in response to interferons (IFN) by both tyrosine andserine phosphorylation. To study the importance of phosphorylating theStat1 transactivation domain at serine 727 (S727) for IFN-dependent im-mune responses we generated mice expressing a Stat1 S727A mutant.Cells from such animals showed reduced IFN-g dependent gene expres-sion and antiviral or antibacterial responses. Moreover, Stat1S727Amice showed increased susceptibility to Listeria monocytogenes (L.monocytogenes) infection and an increased resistance to Lipopolysac-charide. Together our data show an important contribution of Stat1 ser-ine phosphorylation to IFN-g dependent innate immunity. Followingup on L. monocytogenes we observed that infected macrophages areinduced to secrete IFN-b in an IRF-3-dependent manner. Stat1 is acti-vated an its target genes cause a sensitization of macrophages to L.monocytogenes-induced death. Findings will be presented suggestingthat macrophage IFN-b-synthesis and infection-induced death are trig-gered by L. monocytogenes from within the cytoplasm, and do not re-quire the presence of toll-like receptors.

3MULTIPLE DISTINCT MECHANISMS UNDERLIE THE ROLES OFSTAT3 IN MALIGNANT TRANSFORMATIONDavid E. Levy, Karni Schlessinger, Alicia T. Corlett, Marta Sabbadini, GiorgioInghiramiDepartments of Pathology and Microbiology and NYU Cancer Institute, New YorkUniversity School of Medicine, New York NY 10016 USA.

Multiple biological functions have been ascribed to STAT3, including roles incell proliferation, survival, and differentiation. In addition, accumulating evidencesuggests that STAT3 plays a pivotal role in malignant cell transformation. Usinggenetically modified cells and animals, we found that STAT3 is required for thefull transforming functions of several oncoproteins, including v-src, NPM-ALK,and H-RasV12. While proliferation, survival, and growth factor responses of non-transformed cells were largely unaffected by the absence of STAT3, we foundsignificant deficits in cell transformation following ectopic oncoprotein expres-sion. When v-Src, NPM-ALK, or H-RasV12 were expressed in STAT3 null cells,transformation was attenuated, as measured by growth in soft agar and tumor for-mation in nude mice, and STAT3 was required for anchorage-independent growthand metastasis. However, the mechanisms by which STAT3 was required fortransformation by these oncoproteins were fundamentally different. In the caseof v-Src or NPM-ALK, STAT3 was phosphorylated on tyrosine, causing activa-tion of its transcriptional function and induction of downstream target genes.Specifically, STAT3 was required for mediating loss-of-adhesion induced by v-Src through activation of Focal Adhesion Kinase, and it mediated induction ofanti-apoptotic genes in cells expressing NPM-ALK. In cells expressing H-RasV12, STAT3 was not phosphorylated on tyrosine, and a transcriptionally in-competent STAT3 mutant was capable of rescuing the transformation defect. Wefound that STAT3 mediated activation of RalA, a Ras effector required for trans-formation, specifically for metastasis formation. We confirmed this requirementfor STAT3 by metastasis assays in mice, showing that STAT3-null cells wereunable to metastasize, while cells reconstituted with transcriptionally incompe-tent STAT3 mutants formed metastases at high efficiency, in a Ral-dependentmanner. STAT3 was also required in vivo for transformation of lymphoid cellsby NPM-ALK through transcriptional mechanisms affecting cell survival. Thus,STAT3 facilitates malignant transformation through transcriptional and non-tran-scriptional mechanisms necessary for anchorage independent growth, metastasis,and survival of tumor cells.

4REGULATORY CIRCUITS IN INTERFERON PRODUCTION AND ACTIONBryan R. G. Williams, Aristobolo Silva, Mark Whitmore, Carol Sledz, MichelleHolko, Anthony Sadler, Maryam Zamanian-Daryoush, Joao MarquezDepartment of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foun-dation, Cleveland, Ohio USA.

The early events in IFN induction by viruses and dsRNA are being elucidated.These include the activation of Toll like receptor 3 (in cells which express thisTLR), the assembly of a complex including TRAF6, TAK1, TAB1, TAB2, TRIFand Protein kinase R (PKR) at the receptor then translocation to the cytosol, ac-tivation of the IkappaB kinase, IKKepsilon/TBK1 and phosphorylation and acti-vation of IRF3. This critical transcription factor is targeted for inhibition by vac-cinia virus encoded protein E3L and by HCV encoded NS3/4A serine protease,effectively limiting IFN production. PKR is also implicated in the mitogen acti-vated protein kinase (MAPK) pathway where it acts as a signal transducer fordifferent stimuli including dsRNA, and lipopolysaccharide, pathogen associatedmolecular patterns (PAMPS) for viruses and bacteria respectively. PKR interactsin a stimulus dependent manner with MKK6 the upstream kinase required forp38MAPK activation. This interaction is required for the phosphorylation of tran-scription factor ATF2 and the transcription of proinflammatory genes. DsRNA(a viral PAMP) and CpG DNA (a bacterial PAMP recognized by TLR9) com-bine to provide enhanced stimulation of innate immune cells as measured by theproduction of nitric oxide (NO) and IL-12p40. Studies using neutralizing anti-bodies for IFN-beta or IFNAR null mice indicate that synergy is mediated in partby paracrine/autocrine effects of IFN-beta in combination with mechanisms in-dependent of IFN-beta. Synergy for induction of NOS2, IL-12p40, and inter-leukin-6 (IL-6) could be detected at the level of gene induction where a distinctpattern of gene expression is observed. Thus, the combined detection of thesePAMPs may represent a condition of definitive pathogen recognition that directsan enhanced, synergistic immune response. Feedback mechanisms are a featureof IFN regulatory pathways and can be further illustrated by transfection of shortinterfering (si) RNA into cells which results in PKR-dependent activation ofdsRNA-induced genes including IFN and IFN-mediated activation of the Jak-Statpathway and upregulation of ISGs. These results illustrate that si RNAs can havebroad effects beyond selective silencing of target genes.

5REGULATED NUCLEAR TRAFFICKING OF STAT1,STAT2, AND STAT3Gregg Banninger, Ling Liu, Kevin McBride, Nancy C. ReichDepartment of Pathology, Stony Brook University, Stony Brook, New York USA.

There is an inherent elegance in being in the right place at the right time.The STAT1 transcription factor resides primarily in the cytoplasm andfollowing tyrosine phosphorylation, STAT1 dimerizes and this confor-mational change reveals a nuclear localization signal (NLS) that is rec-ognized by a specific importin. In the nucleus the STAT1 dimer dissoci-ates from the importin and binds to DNA target sites in the promoters ofregulated genes. STAT1 is subsequently dephosphorylated in the nucleus,leading to its dissociation from DNA. A nuclear export signal (NES) ofSTAT1 is masked when dimers are bound to DNA, but it becomes ac-cessible to the CRM1 export carrier following dissociation from DNA.CRM1 binds STAT1 and transports the transcription factor back to thecytoplasm. Dimerization via tyrosine phosphorylation serves as an acti-vating switch to stimulate nuclear import and DNA binding, and in thisrespect the behavior of STAT1 serves as a paradigm for other membersof the STAT family. However, our studies indicate that specific aspectsof STAT cellular distribution distinguish family members. We find un-phosphorylated STAT3 exhibits chiefly nuclear presence and has a con-stitutive NLS that is not dependent on tyrosine phosphorylation within itsaminoterminus. Constitutive shuttling in and out of the nucleus allowsSTAT3 to be a substrate for both cytoplasmic and nuclear tyrosine ki-nases. Another STAT family member, STAT2, is clearly cytoplasmic inan unphosphorylated state and localizes to the nucleus in response to ty-rosine phosphorylation and dimerization with STAT1. STAT2 is uniqueamong the STATs in that it is associated constitutively with a non-STATprotein, interferon regulatory factor-9 (IRF-9) that possesses an NLS. Ourstudies demonstrate that unphosphorylated STAT2-IRF-9 complexes aretransported to the nucleus via IRF-9 but returned to the cytoplasm due toa dominant NES located in the carboxyl terminus of STAT2. The STATfactors therefore have evolved distinct features that regulate their nucleartrafficking.

6THERAPEUTIC POTENTIAL OF INTERFERON ALFACON1PLUS CORTICOSTEROIDS IN SARSM.R. Loutfy,1 L. Blatt,2 E.K.Y. Lai,1 E.N. Fish3

1North York General Hospital, Toronto, Canada; 2Intermune Corp.,Brisbane, CA, USA; 3Toronto General Research Inst. & University ofToronto, Toronto, Canada.

SARS is a new clinical entity for which no effective therapeutic strat-egy has been developed. The current study was undertaken for the pur-pose of evaluating Interferon alfacon-1 (Infergen®) for safety, tolera-bility and therapeutic efficacy in SARS patients. Cases and controlswere 22 patients diagnosed with probable SARS and treated with ei-ther corticosteroids alone (n 5 13), or cortcosteroids plus subcutaneousInfergen® (n 5 9) at North York General Hospital, Toronto, betweenApril 19 and May 30, 2003. All study subjects were evaluated for clin-ical parameters including oxygen saturation and requirement, hemato-logical and biochemical parameters and serial chest radiography. In-fergen was well tolerated clinically and transient neutropenia andelevation of serum transaminase levels observed in some Infergen-treated patients were of no clinical consequence and resolved upon drugcessation. Resolution of fever and lymphopenia were identical betweenthe two treatment groups, but Infergen-treated patients showed signif-icantly less increase in creatine kinase (CK) levels and faster resolu-tion of lactate dehydrogenase (LDH) levels than controls. Infergen treat-ment also significantly hastened the time to 50% resolution of chestradiographic abnormalities (median time 4 versus 9 days; p 5 0.001)and was associated with significantly better oxygen saturation. As mor-bidity and mortality in SARS are directly related to pulmonary spaceinfiltration and LDH and CK levels are thought to represent indicatorsof lung parenchymal damage and poor prognosis, respectively, thesefindings provide compelling evidence that Infergen therapy is of ben-efit in the treatment of SARS and its use for this purpose merits fur-ther evaluation in a prospective clinical trial.

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7THE NOVEL INTERFERON-LIKE CYTOKINES, IL28, IL29:EXPRESSION, BIOLOGICAL ACTIVITY AND SIGNALINGCOMPARED TO TYPE I INTERFERONWayne Kindsvogel,1 Kevin Klucher,2 Steven D. Levin,2 WenFeng Xu,2

Paul Sheppard,3 Katherine Henderson,1 Stacy Schlutsmeyer,2 MarylandRosenfeld-Franklin,4 Ken Bannink,4 Dennis Dong,5 Pallavur Sivakumar,1

Monica Anderson,1 Heidi Brunell,2 Chris Clegg,1 Don Foster2

Departments of Immunology,1 Cytokine Biology,2 Bioinformatics,3 Pre-Clinical Development,4 Protein Biochemistry,5 ZymoGenetics, Inc.,Seattle, WA.

We have recently identified a family of human cytokine genes that aredistantly related to type I interferons. These new cytokine genes, IL28A,IL28B and IL29, are distinct from the known type I interferon genesin several respects including their chromosomal localization and genestructure. Unlike type I interferons, IL-28 and IL-29 do not interact withthe type I interferon receptor but instead interact with a novel het-erodimeric class II cytokine receptor consisting of IL-10 receptor betaand an orphan class II receptor chain, designated IL-28 receptor alpha(IL28Ra). Semi-quantitative RT-PCR analysis of IL-28Ra expressionindicates a more limited expression pattern than that seen with the typeI interferon receptor. However gene expression analysis indicates thatlike interferon alpha, IL-28 and IL-29 are inducible by viral infectionand double-stranded RNA. In addition, exposure of a variety of celltypes to purified recombinant IL-28 or IL-29 leads to induction of in-terferon-stimulated genes resulting in an antiviral state. Yet repeatedattempts to demonstrate antiproliferative activity in vitro with IL28,IL29 have failed, even though signal transduction studies indicate thatthe IL-28R signals through the JAK/STAT pathway in a manner sim-ilar to type I interferons. The in vitro and in vivo biological activitiesof IL-28 and IL-29 suggest they may serve as an alternative to type Iinterferons in providing immunity to viral infection with distinct tissuespecificity and distinct secondary activities.

8ROLE OF ADAPTER MOLECULES IN TOLL-LIKE RECEP-TOR SIGNALINGKate A. FitzgeraldUniversity of Massachusetts Medical School, Worcester, MA 01605,USA.

The TLR superfamily is the essential recognition and signaling com-ponent of mammalian host defense. TLRs recognize microbial prod-ucts derived from all the major classes of pathogens including bacte-ria, virus, fungi and yeast. Toll signaling originates from the conservedToll-IL-1-Resistance (TIR) domain, which recruits the TIR domain-containing adapter molecule, MyD88 (myeloid differentiation factor88). Recruitment of MyD88 activates a signaling pathway resulting inNF-kB activation via the IKKa/b/g complex and induction of pro-in-flammatory cytokines. TLR3 and TLR4, which recognize doublestranded RNA (dsRNA) and lipopolysaccharide (LPS) respectively, areunique in their ability to signal in MyD882/2 macrophages. Gene ex-pression profiling of LPS-stimulated MyD881/1 and 2/2 macro-phages shows that MyD88-independent signaling accounts for 76% ofthe gene expression profile. Activation of interferon regulatory factor-3 (IRF-3), induction of type I interferons and interferon stimulatedgenes is one key component of MyD88-independent signaling. Threeadditional TIR domain containing adapter molecules also participate inTLR4 signaling, namely Mal (MyD88-adapter like, also known asTIRAP), TRIF, (TIR-containing adapter inducing interferon-b, alsocalled TICAM-1) and TRAM (TRIF-related adapter molecule, alsocalled TIRP). Mal cooperates with MyD88 to activate NF-kB but doesnot participate in MyD88-independent signaling. In contrast, TRIFfunctions downstream of both the TLR-3 and TLR-4 signaling path-ways leading to IRF-3 activation, while the function of TRAM is re-stricted to the TLR-4 pathway. Moreover, TRIF and TRAM activateIRF-3 via the non-canonical IKK-related kinases TBK1 (TANK bind-ing kinase-1) and IKKe, which directly phosphorylates and activatesIRF-3. Thus, MyD88-independent signaling is mediated by TRAM,TRIF, TBK1/IKKe and IRF-3 during the innate immune response tolipopolysaccharide.

9TRIGGERING THE INTERFERON ANTIVIRAL RESPONSETHROUGH AN IKK-RELATED PATHWAYJohn Hiscott, Sonia Sharma, Benjamin tenOever, Nathalie Grandvaux,Rongtuan LinLady Davis Institute-Jewish General Hospital, Departments of Medicine andMicrobiology, McGill University, Montreal, Canada.

Virus infection of susceptible cells activates multiple signaling pathways(IKK-NF-kB/JNK/PI-3K-AKT) that orchestrates the induction of cytokinesinvolved in the antiviral and innate immune response. Virus infection alsostimulates a virus-activated kinase (VAK) responsible for the C-terminalphosphorylation and activation of interferon regulatory factor 3 (IRF-3) andthe closely related IRF-7, both of which are central to IFN production andactivation of the antiviral cascade. Recognition of bacterial infection medi-ated through lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR-4) anddouble stranded RNA by TLR-3 also induces IRF-3/IRF-7. The C-terminalend of IRF-3 and IRF-7 contains several potential phosphoacceptor sites thatare targeted by the VAK activity. Identification of VAK activity is criticalto an understanding of the signaling pathways involved in linking the hostresponse to pathogens with the establishment of the antiviral state. We nowdemonstrate that the IKK-related kinases—IKKepsilon/TBK1—are compo-nents of VAK and specifically phosphorylate the C-terminal residues of IRF-3 and IRF-7. Expression of IKKepsilon or TBK1 was sufficient to inducecytoplasmic to nuclear translocation of 90-95% of IRF-7 and about 40% ofIRF-3. Nuclear extracts of IKKepsilon-transfected cells are specifically rec-ognized in immunoblot analysis using an IRF-3 phosphospecific Ser396 an-tibody. Co-transfection of IKKepsilon alone stimulates reporter gene activ-ities corresponding to RANTES, IFNb, IFNa1 and IFNa4; the combinationof IRF-7 and IKKepsilon resulted in a 2000-fold stimulation of the IRF-7-regulated IFNa4 promoter. IKKepsilon mediated activation of the cytokinereporter genes was blocked by co-expression of dominant negative mutantsof IKKepsilon or TBK1 and by siRNA targeting. Furthermore, IKKepsilonstimulates expression of the endogenous IRF-3-dependent gene ISG56 andmounts an antiviral response that inhibits VSV multiplication. These stud-ies constitute the first biochemical evidence linking the NF-kB and IRFpathways in the response to pathogens, specifically through a distinct IKK-related pathway.

10TYPE 1 IFN FUNCTIONS AND THEIR REGULATION DURINGVIRAL INFECTIONSChristine A. BironDepartment of Molecular Microbiology and Immunology, Division ofBiology and Medicine, Brown University, Providence, RI 02912, USA.

The type 1 interferons, i.e. interferons alpha and beta (IFN-a/b), in-duce a wide range of biological effects and certain of these are para-doxical. The understanding of how they are accessed is still limited.Our group has been investigating the endogenous type 1 IFN effects inthe context of infections of mice with lymphocytic choriomeningitisvirus (LCMV) or murine cytomegalovirus (MCMV). The work hashelped demonstrate a number of positive and negative immunoregula-tory functions. These include the enhancement of a cluster of innate re-sponses including promotion of IFN-a/b production, accumulation ofmature plasmacytoid dendritic cells, activation of natural killer cell cy-totoxicity, and induction of interleukin 15 (IL-15). All of these are de-pendent on a functional IFN-a/b receptor (IFN-a/bR) and the signaltransducer and activator of transcription (STAT) 1. In the absence ofSTAT1, however, there is not only a block in these positive effects buta substitution of different responses. The best characterized is induc-tion of IFN-gamma (g). Studies suggest that the natural regulation ofSTAT1 is in place to shape the downstream consequences of type 1IFN exposure. Early during infection, STAT1 is induced to elevatedlevels and is required for the cluster of innate responses elicited by type1 IFNs. However, expression declines as the immune response pro-gresses to times of overlapping innate and adaptive responses. Thischange correlates with the acquisition of a different IFN-a/b function,i.e. enhancement of CD8 T cell IFN-g production through a STAT4-dependent mechanism. The picture emerging is one whereby regula-tion of access to signaling pathways is an important mechanism forchanging the biological consequences of type 1 IFNs as needed. (Thiswork is supported by grants from the National Institutes of HealthCA041268 and AI055677.)

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11IRF-8/ICSBP AND IRF-4/PIP DIRECT DEVELOPMENT OFDENDRITIC CELL SUBSETS AND REGULATE TOLL-LIKERECEPTOR MEDIATED CYTOKINE INDUCTIONKeiko Ozato, Tomohiko Tamura, Hideki Tsujimura, Hee Jeong Kong,Praful TailorLaboratory of Molecular Growth Regulation, National Institute of ChildHealth and Human Development, National Institutes of Health,Bethesda, MD, USA.

IRF-8/ICSBP and IRF-4/Pip are immune system specific members ofthe IRF family that play a key role in the development of myeloid andlymphoid cells. We found that these proteins are also critical for thegeneration of dendritic cells (DCs) and that the two members have com-plementary roles in directing different DC subsets. We have shown thatIRF-8 is essential for the development of plasmacytoid DC (pDCs) andCD8a1 DC subsets.1,2 pDCs produce a large amount of type I IFNs(IFNa/b). IRF-82/2 DCs are defective in the production of IFNa/b andIL-12p40 in response to viral infection and toll-like receptor (TLR) sig-naling.3 IRF-82/2 DCs were also unresponsive to CpG to induce TNFaand IL-6. We have studied the development of DC subsets from bonemarrow progenitors in the Flt3L based culture system, and found thatneither pDCs nor CD8a1 DCs develop from 2/2 progenitors. In ad-dition, in vitro generated IRF-82/2 DCs failed to induce IFNa and IL-12p40, and were unresponsive to CpG. However, transfer of retroviralIRF-8 rescued the development of pDC and CD8a1 DCs. In accor-dance, TLR mediated induction of IFNa and IL-12p40 as well as CpGresponse were fully restored upon IRF-8 transfer. These results indi-cate that IRF-8 regulates the generation of DC subsets responsible forproduction of critical cytokines for establishing innate immunity. Morerecently we found that IRF-4 also has an important role for the devel-opment of DC subsets. The phenotype of double KO mice is consis-tent with the separate and overlapping roles of IRF-4 and IRF-8 in thedevelopment of the DC subsets.1. Tsujimura, H. et al. (2003) Blood 101, 961–969.2. Tsujimura, H. et al. (2003). J. Immunol, Cutting Edge. 170, 1131–1135.3. Ozato, K. (2002). BioTechniques (Suppl), 66–73.

12IRFS: A WELL ARTICULATED FAMILYP.M. Pitha,1 B.J. Barnes,1 K.A. Fitzgerald,2 B. Lubyova,1 P. Fitzgerald- Bocarsly,3

L. Baretta4

1The Johns Hopkins University, Baltimore, MD, 2University of Massachusetts,Worcester, MA, 3UMDNJ–New Jersey Medical School, Newark, N.J., 4University ofMichigan, Ann Arbor, MI.

Three IRFs play a critical role in the innate immune response to infectious agents.Viral infection or binding of a ligand to Toll like receptors (TLR) coordinately ac-tivates IRF and NFkB pathways. IRF-3 co-operates with IRF-5 or IRF-7 in the in-duction of Type I IFN genes in infected cells and IRF-3:IRF-7 or IRF-3:IRF-5 het-erodimers are components of an enhanceosome assembled on IFN promoters ininfected cells. In contrast the interaction between IRF-5 and IRF-7 results in re-pression of IFNA genes transcription, suggesting that IRF-5 can act both as an ac-tivator and suppressor. IRF-3 is expressed constitutively in cell lines and primarycells, while the expression of IRF-5 and IRF-7 is very low in fibroblasts and can bestimulated by Type I IFNs. However in primary lymphoid cells, such as PBMC,monocytes and dendritic cells, IRF-5 and IRF-7 are expressed constitutively and thehigh IFN producing cells, pDC2 express significantly higher levels of IRF-7 thenthe other cell types suggesting that high constitutive level of IRF-7 may contributeto the high levels of interferon synthesis. TLR l 3 and 4 activate IRF-3 and IRF-7in response to dsRNA and LPS respectively, to induce IFNA/B; two adaptor mole-cules TRIF and TRAM activate IRF-3 and IRF-7 as well as NFkB. The function ofTRAM is specific for LPS activation of TLR4, while TRIF appears to function inboth TLR3 and TLR4 pathways.

While both IRF-5 and IRF-7 can rescue expression of IFNA genes in infectedcells the profile of IFNA subtypes is distinct. Also the function of these two IRFsuninfected cells is not redundant. IRF-7 induces differentiation of monocytes to mac-rophages while IRF-5 induces p21waf, apoptosis and cell cycle arrest. The gene ar-ray analysis has shown that over expression of IRF-5 and IRF-7 in B cells modu-lates expression of a large group of distinct and overlapping genes both in infectedand uninfected cells. However the signature of IRF-5 in infected cells was a strongimmune response, while IRF-7 up regulated expression of chaperons and genes mod-ulating RNA processing.

To overcome the innate antiviral response viruses target the function of IRF-3and IRF-7. The KSHV encodes four homologues of cellular IRFs; vIRF-1 expressedduring the lytic cycle targets the transcriptional activity of IRF-3 and inhibits his-tone acetylation. vIRF-3 is latently expressed nuclear antigen in B cell lymphomas(PEL) that associates both with IRF-3 and IRF-7 and is assembled in the IFNA en-hanceosome.The transcription profile induced by vIRF-1 and vIRF-3 in B cells isdistinct, suggesting that these two vIRFs target expression of distinct genes,. Thusthe virus uses vIRF-1 to overcome the innate antiviral response, while vIRF-3 mayplay a role in KSHV latency and B cells lymphogenesis.

13X-RAY CRYSTAL STRUCTURE OF IRF-3 AND ITS FUNC-TIONAL IMPLICATIONSTakashi Fujita,1 Kiyohiro Takahasi,2 Nobuo N. Suzuki,3 Masataka Ho-riuchi,2,3 Mitsuaki Mori,1 Wakako Suhara,1 Yasutaka Okabe,1 YukikoFukuhara,1 Hiroaki Terasawa,4 Fuyuhiko Inagaki2,3

1Department of Tumor Cell Biology, Tokyo Metropolitan Institute ofMedical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613,Japan. 2Graduate School of Pharmaceutical Sciences, Hokkaido Uni-versity, Sapporo 060-0812, Japan. 3CREST/JST, Kawaguchi, Japan.4De-partment of Molecular Physiology, Tokyo Metropolitan Institute ofMedical Science, Tokyo 113-8613, Japan.

Transcription factor IRF-3 is post translationally activated by signalingtriggered by virus infection or pathogen-associated molecules throughthe Toll-like receptor (TLR) and plays critical roles in the regulationof innate immunity. We found that a fragment of IRF-3 175-427 (IRF-3 175C) is specifically phosphorylated and form a dimer in cells. Weproduced IRF-3 175C in E. coli and purified in large scale and deter-mined its X-ray crystal structure at 2.3 A° resolution. The obtainedstructure suggested a mode of IRF-3 activation through its specificphosphorylation. We generated mutants according to the structure andanalyzed their phenotype in cells. The results strongly supported a newmodel of IRF-3 activation: phosphorylation-induced dimerization,rather than de-repression from autoinhibition. Furthermore we deter-mined an interface for the interaction with p300/CBP. We will also dis-cuss the evolutional origin of IRF-3.

14ROLE OF TYPE I INTERFERON PRODUCING CELLS (PLASMACY-TOID DENDRITIC CELLS) IN THE ANTI-VIRAL RESPONSEFrancine BrièreSchering-Plough, Laboratory for Immunological Research, Dardilly, France.

Differentiation of dendritic cells (DC) into particular subsets may act to shape in-nate and adaptive immune responses. Mouse plasmacytoid DC (PDC) have beenrecently identified which share most characteristics of their human counterpartand are major producers of interferon (IFN)-a/b in response to certain viruses.The generation of an antibody specific for mouse PDC demonstrates strain- andorgan-specific differences in the frequency of PDC and other DC subsets. PDCare located in T cell area of the spleen lymph nodes and Peyer’s patches. 129Svmice have a much higher frequency of PDC, together with a lower frequency ofCD8a1CD11chi DC, compared to C57BL/6 mice, both in spleen and blood. Thehigher ability of 129Sv mice to produce IFN-a in vivo is related to a higher num-ber of PDC, but also to a higher ability of PDC from 129Sv mice to produce IFN-a in vitro in response to viral stimulation. The responsiveness of DC subsets toToll-like receptors (TLR) ligands correlates with their differential TLR profiles.Using IFNa/bR2/2 mice, CpG-induced activation of all DC subsets appearsIFNa/bR dependent. Depletion of PDC shows that PDC is the major subset re-sponsible for IFNa/b secretion in response to TLR9 ligand in vivo while IFNa/bin response to TLR7 ligand is produced by an unidentified non-PDC population.In addition, PDC have been demonstrated to be the major producer of cytokinesduring murine cytomegalovirus (MCMV) but not lymphocytic choriomeningitisvirus (LCMV) infections. TLR play a crucial role in innate recognition of bacte-rial or fungal pathogens. However, interplay between TLR and viruses remainspoorly characterized. In response to intraperitoneal MCMV challenge, MyD88adapter molecule of TLR/Interleukin-1 receptor signalling and TLR9 deficientBL/6 mice presented a striking default in immune responses. MyD882/2 and toa lower extent TLR92/2 mice were more susceptible to MCMV infection thanwild type mice in terms of morbidity and mortality. This loss of resistance wascorrelated with dramatically impaired cytokine production in the serum as wellas an increase of viral load in total spleen cells. Thus, PDC is an important playerin the establishment of an anti-viral response and TLRs expressed by DC are crit-ically required in the process of MCMV detection and immune alarming.

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15THE INTERFERON IFN-b TRANSGENE IS SUCCESSFUL INANTAGONIZING HERPES SIMPLEX VIRUS TYPE 1 ACUTEAND LATENT INFECTION IN THE EYE AND NERVOUSSYSTEMDaniel J.J. CarrDepartment of Ophthalmology, University of Oklahoma Health Sci-ences Center, Oklahoma City, OK, USA.

Herpes simplex virus type 1 (HSV-1) is a highly successful neurotropicvirus that has evolved multiple means of evading the host immune re-sponse. We previously evaluated a battery of type I IFN transgenes ap-plied to the scarified cornea and chose one efficacious (IFN-b) and onenon-efficacious (IFN-a6) transgene to further define differences in theanti-viral action of these type I IFN species. Whereas both type I IFNssuppressed viral replication in the eye, only the IFN-b transgene sup-pressed viral replication in the trigeminal ganglion (TG). In a similarmanner, both type I IFN transgenes augmented 29-59 oligoadenylatesynthetase (OAS)2a in the eye but not double-stranded RNA-depen-dent protein kinase (PKR) 24 hr post transfection. However, the appli-cation of the IFN-b transgene to the cornea also elevated OAS2a in theTG whereas such an occurrence was not found following IFN-a6 trans-gene application. In evaluating the Jak1/Stat1 signaling cascade in thecornea and TG of HSV-1 infected mice over time, there were no no-ticeable difference between mice treated with the type I IFN transgenesmeasuring total or phosphorylated Jak1 or Stat1 with the exception oftotal Stat1 which was consistently elevated in the cornea and TG ofIFN-b transgene-treated mice 5 days post infection. In determining theefficacy of type I IFN in hindering HSV-1 reactivation, latently infectedTG explant cultures transduced with an adenoviral vector containingthe IFN-b transgene were found to generate significantly less virus incomparison to TG explant cultures transduced with the null adenoviralvector or recombinant IFN-b. Collectively, the results demonstrate thepotential application of the IFN-b transgene against ocular HSV-1 dis-ease. Support: USPHS AI053108 and RPB Stein Professorship.

16STAT1 OR STAT2 DEFICIENCY REVEAL NOVEL INTER-FERON SIGNALING MECHANISMS, ACTIONS AND DISEASEIN THE LIVING CENTRAL NERVOUS SYSTEMIain L. CampbellThe Scripps Research Institute, La Jolla, CA, USA.

Type I interferons (IFNa/b), crucial in anti-viral defense and im-munoregulation, signal via the Jak/STAT pathway with activation ofSTAT1 and STAT2. Transgenic mice (termed GIFN) with CNS-pro-duction of IFN-a, while resistant to CNS viral infection, develop a late-onset progressive neurodegenerative disease with inflammation, calci-fication, increased expression of IFN-regulated genes and activation ofSTAT1 and STAT2. We explored the role of STAT1 or STAT2 in me-diating the actions of IFN-a by generating GIFN mice null for theseSTAT genes. Surprisingly, these animals developed either more severeand accelerated neurodegeneration with calcification and inflammation(GIFN/STAT1 null) or severe inflammation and medulloblastoma(GIFN/STAT2 null). In the brain of GIFN/STAT2 null mice, IFN-ggene expression by CD31 T-cells and the activation of the STAT1,STAT3, STAT4 and STAT5 molecules.and the induction of GAS-dri-ven (e.g. CIITA and IRF-1) gene expression was present. Expressionof the Sonic hedgehog (Shh) and the downstream transcriptional fac-tor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, wasfound to be significantly increased and co-transcribed in cerebellargranule neurons of the GIFN/STAT2 null mice. IFN-g, but not IFN-a,induced STAT1-dependent expression of the Shh gene in cultured cere-bellar granule neurons. In GIFN/STAT1 null CNS, expression of a num-ber of genes was increased, some of which (e.g. ISG12) suggest in-volvement of the novel STAT2/Brg-1 signaling pathway. Thus, 1)despite the absence of STAT1 or STAT2, IFN-a causes more severeand distinct neurologic diseases, 2) STAT1 and STAT2 have commonand unique roles following type I IFN receptor activation, 3) alterna-tive novel IFN-a signaling pathways are potent disease inducers in theabsence of STAT1 or STAT2, and 4) a hitherto unknown role is indi-cated for the immune system in tumorigenesis in the CNS and perhapsother organs, through dysregulated Shh signaling mediated by IFN-g.This study was supported by National Institutes of Health grants MH62231 and MH 62261.

17MODE OF ACTIONS OF P56 AND PACTGanes C. SenDepartment of Molecular Biology, Cleveland Clinic Foundation, Cleve-land, Ohio, USA.

The P56 family of genes is strongly induced by interferons, dsRNA andvirus infection. The encoded proteins contain multiple TPR motifs thatmediate interactions with other cellular and viral proteins. Human P56interacted with HPV E1 protein and translocated it from the nucleus tothe cytoplasm. In a cell-free replication system, addition of recombi-nant P56 strongly inhibited E1-dependent HPV DNA synthesis indi-cating that P56 may mediate IFN’s action against HPV in vivo. Bothhuman and mouse P56 could bind to the translation initiation factoreIF3 and inhibit protein synthesis. However, they interacted with twodifferent subunits of eIF3 and consequently blocked its two differentfunctions in initiating translation. Thus, different P56 proteins have sim-ilar but distinct functions.

PACT is the protein-activator of PKR that can mediate PKR acti-vation and consequent apoptosis of cells in response to a variety ofstresses. Alanine scanning mutagenesis of PACT domain 3, that is suf-ficient for PKR activation, identified two specific serine residues thatwere the targets of phosphorylation. Constitutive phosphorylation ofthe first serine was a prerequisite for stress-induced phosphorylation ofthe second. Substitution of these residues with the phosphoserinemimetic, aspartic acid, produced a PACT mutant that could cause cel-lular apoptosis without any extracellular stress. These results led us topropose a model for PKR activation by PACT in vivo.

18INTERFERON-INDUCIBLE P202: REGULATION AND ROLEDivaker Choubey and Hong XinDepartment of Radiation Oncology, Stritch School of Medicine, Loy-ola University Chicago, Maywood, IL 60151, USA.

The Ifi200-gene family includes interferon-activatable and structurallyrelated murine and human genes. The murine genes (Ifi202a, Ifi202b,Ifi203, Ifi204 etc.) form a cluster, which is located between the Spna-1 and the Sap locus on Chromosome 1. The human genes (IFI 16,MNDA and AIM2) are located in the 1q22 region. Proteins (the p200-proteins) encoded by the Ifi200-genes share at least one partially con-served repeat of 200-amino acids. Although, the p200-proteins are notknown to have any enzymatic activity, they are thought to act as a scaf-fold to assemble protein complexes that regulate gene transcription.Consistent with this idea, p202a (referred as p202) binds to pRb andother ‘pocket’ family proteins, and transcription factors (for example,E2Fs, p53, c-Myc, and AP-1). Increased expression of p202 in cell linesand in cells of the immune system correlates with retardation of cellproliferation and a decrease in the susceptibility to apoptosis. In con-trast, reduced expression of p202 in cell lines increases the suscepti-bility to apoptosis. The ability of p202 to regulate cell growth may de-pend on its binding to pRb and various transcription factors. Of note,pRb, p53 and E2Fs regulate expression of p202. Generation of micecongenic for the Nba2 locus (derived from the NZB strain of mice) onthe C57BL/6 genetic background, coupled with gene expression analy-ses, has identified p202 as a candidate for lupus susceptibility. Thesestudies also revealed that increased expression of p202 in splenic cells(derived from congenic B6.Nba2 mice) correlates with autoantibodyproduction, splenomegaly, and defects in apoptosis. Furthermore, ourrecent studies support the idea that the mouse strain-specific increasedexpression of p202 as well as its nucleocytoplasmic distribution maycontribute to lupus susceptibility.

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19INTERFERON-STIMULATED GENE ISG60 MIMICS TRUNCATEDCDC16 BY DISRUPTING THE ANAPHASE PROMOTING COM-PLEX ACTIVITY IN THE CELL CYCLEHelena Y. Sim,4 Michael J. de Veer,3 Rodney J. Devenish,2 Stephen J.Ralph1

1School of Health Science, Griffith University, Gold Coast Campus, South-port, Queensland, Australia. 2Department of Biochemistry and MolecularBiology, Monash University, Clayton, Victoria, Australia. 3Walter and ElizaHall Institute, Parkville, Victoria, Australia. 4Prince Henry’s Institute,Monash Medical Centre, Clayton, Victoria, Australia.

Increasingly evidence is accumulating that the interferons play importantroles in regulating the cell cycle and growth of mammalian cells. One ofthe four members of the ISG54 family, ISG56 has been shown to bind toeIF-3 and inhibit protein synthesis in cells1. We have examined the role ofanother member of the ISG54 family, ISG60 that resembles a truncated formof the Cdc16, Cdc23 or Cdc27 components of the anaphase-promoting com-plex (APC) important in regulating cell cycle progression through mitosis.ISG60 protein concentrates to centrosomes in mitotic cells together with theAPC proteins. Cellular expression of ISG60 or a related truncated Cdc16protein resulted in accumulation of giant multinucleated cells that progres-sively died after 23 weeks in culture. Multiple centrosomes and mitotic spin-dles present in individual giant cells indicated repeated nuclear division inthe absence of cytokinesis. When early passages of cells expressing ISG60or truncated Cdc16 protein were synchronised and released from mitotic ar-rest, there was no significant change in levels of cyclin B protein, unlikethe control cells in which cyclin B was extensively degraded. Cells ex-pressing ISG60 or truncated Cdc16 protein contained greatly reduced Cdc27protein levels, indicating disrupted APC function. A model is proposedwhereby these two related proteins disrupt the functional integrity of theAPC and mitotic exit resulting in cellular multinucleation. The evidence im-plicates a role for the interferons in regulating the mammalian APC/C andcyclin B1/cdk, affecting signalling to activate cytokinesis in late stage mi-tosis.1. Guo, J., Hui, D.J., Merrick, W.C. and Sen, G.C. (2000). A new pathway

of translational regulation mediated by eukaryotic initiation factor 3.Embo. J. 19, 6891–6899.

20ROLE OF THE INTERFERON-REGULATED RNASE L IN THEBIOLOGY OF PROSTATE CANCERRobert H. Silverman, Ying Xiang, and Malathi KrishnamurthyDepartment of Cancer Biology, Lerner Research Institute, ClevelandClinic Foundation, Cleveland, Ohio.

The RNASEL gene, a strong candidate for the hereditary prostate can-cer 1 (HPC1) allele, encodes a single-stranded specific endoribonucle-ase, 2-5A-dependent RNase (RNase L), involved in the anti-viral ac-tions of interferons. RNase L is enzymatically activated after bindingto unusual 59-phosphorylated, 29 to 59 linked oligoadenylates (2-5A).2-5A treatment of the late-stage, metastatic human prostate cancer celllines DU145, PC3 and LNCaP activated RNase L and caused apopto-sis. However, DU145 and PC3 cells were more sensitive to 2-5A thanLNCaP cells which are heterozygous for an inactivating deletion mu-tation in RNase L. The impact on apoptosis of specific mutations andpolymorphisms in RNase L observed in HPC1 families was studied.The R462Q missense mutation, implicated in 13% of unselectedprostate cancer cases, was deficient in RNase L activity and in the abil-ity to induce apoptosis due to a defect in enzyme dimerization. To in-vestigate the role of RNase L in prostate cancer, we decreased levelsof RNase L several-fold in the DU145 human prostate cancer cell lineby stable expression of an siRNA against RNase L mRNA. Controlcells expressed an siRNA with three mismatched nucleotides to theRNase L sequence. Cells deficient in RNase L, but not the control cells,were highly resistant to apoptosis by 2-5A treatment. Surprisingly, theRNase L deficient cells were also highly resistant to apoptosis by camp-tothecin and TRAIL or anti-fas. In contrast, cells expressing an siRNAto the RNase L inhibitor (RLI) showed enhanced apoptosis in responseto these agents. Our finding support the notion that RNASEL muta-tions and variants allow prostate tumor cells to escape a potent apop-totic pathway.

21A NOVEL INTER-RELATIONSHIP BETWEEN INTERFERON-a/b SIGNALLING AND P53Akinori Takaoka,1 Sumio Hayakawa,1 Hideyuki Yanai,1 DagmarStoiber,1 Hideo Negishi,1 Hideaki Kikuchi,1 Shigeru Sasaki,2 KohzohImai,2 Tsukasa Shibue,1 Kenya Honda,2 Tadatsugu Taniguchi21Department of Immunology, Faculty of Medicine and Graduate Schoolof Medicine, University of Tokyo, Tokyo, Japan. 2First Department ofInternal Medicine, Sapporo Medical University, Sapporo, Japan.

Interferon-a and -b (IFN-a/b) are inducible cytokines, which are pro-duced by a variety of cells in response to viral infections. It has beenso far reported that these cytokines show versatile functions in host de-fense systems such as immune system and tumour suppression. The tu-mour suppressor p53 is known to be one of the key factors for induc-tion of apoptosis in tumour cells, while IFN-a/b are critically involvedin apoptotic responses during viral infections. In this study, we show anovel inter-relationship between IFN-a/b signalling and p53. IFN-a/binduce transcription of the p53 gene and eventually enhance p53 pro-tein level. The p53 gene induction by IFN-a/b is mediated through theactivation of two ISREs (interferon-stimulated response elements),which were found to be within the promoter and first-intron regions ofthe p53 gene. IFN-b itself is not able to induce the serine phosphory-lation of p53 and the induction of the p53 target genes, however, theupregulation of p53 by IFN-b contributes to boosting p53 responses tostress signals. In this regard, we show that IFN-b treatment resulted insuppression of oncogene-induced cell transformation as well as en-hancement of an apoptotic response of tumour cells in response to DNAdamaging agents. On the other hand, we also found that p53 is phos-phorylated and its target genes are induced in virally infected cells. Ev-idence is further provided that the activation of p53 plays an importantrole to evoke an apoptotic response during virus infection and that p53-deficient mice are more susceptible to VSV (vesicular stomatitis virus)infection than the wild-type mice, with a higher titer of VSV in sera ofthe deficient mice. Thus, this study reveals a novel linkage betweenIFN-a/b and p53 in tumour suppression and antiviral defense.

22EOSINOPHILS, A NEW SOURCE OF HELP TO NK CELLSTHROUGH RESPONSE TO CPGSJeremy Perkins,1,3 Kelly Warfield,2 Howard A. Young1

1Laboratory of Experimental Immunology, Cancer for Cancer Re-search, NCI-Frederick, Frederick, MD. 2United States Army ResearchInstitute of Infectious Diseases, Fort Detrick, Frederick, MD. 3WalterReed Army Medical Center, Washington, DC.

Natural killer cells (NK) are an important part of the innate immunesystem providing early protection from viral infection, contributing toadaptive immune responses, providing tumor surveillance, and killingof malignant cells. CpG motifs are being investigated as a tool to en-hance vaccination as well as to augment the anti-cancer host responsein cancer treatment. CpGs combined with cytokines induced synergis-tic cytokine responses from fresh murine liver NK, but not with NKcell lines. Cytotoxicity of NK was enhanced in the presence of CpGsalone and in combination with cytokines. RPA analysis showed syner-gistic IFN-g mRNA production, which was entirely blocked by the ad-dition of cyclohexamide. Blocking antibodies against IL-12 blocked syn-ergistic IFN-g responses seen with CpG 1/2 IL-18; however, NK fromIL12p35 knockout mice were able to secrete IFN-g in response to CpGand cytokines. Depletion or addition of DC did not alter IFN-g secretion,whereas depletion or addition of eosinophils to sorted NK1.1 cells re-stored synergistic IFN-g secretion. Our work, along with others, showthat NK do not directly respond to CpG. We found that synergistic NKIFN-g secretion and cytotoxicity are seen in response to CpG in combi-nation with cytokines. Additionally, eosinophils, not previously recog-nized as responding to CpGs, are sufficient and necessary for fresh liverNK responses to CpGs most likely via eosinophils-production of IL-12.

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23(1) MULTIPLE PATHWAYS ACTIVATED BY INTERFERONg(2) SIGNALING IN RESPONSE TO UNPHOSPHORYLATEDSTAT 3Nywana Sizemore, Yulan Qing, David Shultz, Jinbo Yang, and GeorgeR. StarkLerner Research Institute, Cleveland Clinic Foundation, Cleveland,Ohio, USA.

(1) In addition to activating Stat 1, the IFNg receptor generates im-portant additional signals. In mouse embryo fibroblasts (MEFs) lack-ing both the a and b subunits of IkB kinase (IKK), many Stat 1-de-pendent genes are activated normally by IFNg, but many are not. Thereis no defect in the phosphorylation or function of Stat 1. Therefore, asubset of the genes that use Stat 1 dimers to respond to IFNg also re-quire an additional signal that is mediated by the IFN-dependent acti-vation of IKK. Several lines of evidence indicate that this novel func-tion of IKK does not involve NFkB. In MEFs, Y419 of the receptorsubunit IFNGR1 is required for robust activation of Stat 1 and modestactivation of Stat 3 in response to IFNg. If Stat 1 is absent, the activa-tion of Stat 3 is greatly increased and prolonged. The SOCS 3 gene hasa GAS element that drives its expression in response to homodimers ofStat 1 or Stat 3 and thus it responds well to IFNg in Stat 1-null cells.However, in contrast to Stat 1, the tyrosine phosphorylation of Stat 3by the IFNg receptor requires src-family kinases, activated by IFNg.The IFNg receptor also generates additional signals that activate a smallnumber of genes independently of any Stat. (2) As previously shownfor Stat 1, the overexpression of unphosphorylated Stat 3 drives thetranscription of many genes. In tumors, overexpressed unphosphory-lated Stat 3 drives the transcription of several oncogenes that do not re-spond to Stat 3 homodimers. Since Stat 3 gene expression is increasedin response to Stat 3 dimers, the signal-dependent activation of Stat 3-responsive genes occurs in two phases: Immediate expression of genesthat respond to Stat 3 dimers directly and secondary, slower responseof genes that respond to increased levels of unphosphorylated Stat 3.

24REGULATORY EFFECTS OF NON-STAT PATHWAYS IN IN-TERFERON SIGNALINGLeonidas C. PlataniasRobert H. Lurie Comprehensive Cancer Center, Northwestern UniversityMedical School, Chicago, Illinois, USA.

There is accumulating evidence that non-Stat pathways are engaged by theType I IFN receptor and play important roles in the generation of the ef-fects of interferons. Recent studies have demonstrated that IFNa activatesthe small G-protein Rac1 and the p38 Map kinase, resulting in downstreamengagement of other kinases, including MapKapK-2, MapKapK-3, andMsk1. The activation of the p38-cacade is required for IFN-dependent genetranscription via ISRE and GAS elements, and plays a critical role in thegeneration of the growth inhibitory effects of IFNa on normal and malig-nant hematopoietic cells. In recent studies using p38a knockout cells, wehave established that the regulatory effects of this pathway on IFN-depen-dent transcriptional activation are not mediated via effects on the Stat-path-way, and apparently involve distinct mechanisms. Thus, an emerging modelis that non-Stat pathways cooperate with Stat-pathways at the nucleus, foroptimal induction of IFN-dependent gene transcription. Beyond gene-tran-scription, it appears that non-Stat pathways play critical roles in the regula-tion of mRNA translation in response to Type I IFNs. The FKBP12-ra-pamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR)is activated in a Type I IFN-dependent manner downstream of the insulinreceptor substrate (IRS)-PI 39-kinase pathway. mTOR subsequently regu-lates activation of the p70 S6 kinase, which in turn mediates phosphoryla-tion of the S6 ribosomal protein. In addition, IFN-dependent activation ofthe PI39K/mTOR cascade, ultimately results in phosphorylation of the 4E-BP1 repressor of mRNA translation. Such phosphorylation of 4E-BP1 re-sults in the dissociation of the protein from the eukaryotic initiation factor-4E (eIF4E) complex, an event required for initiation of mRNA translation.Thus, the IRS/PI39kinase/mTOR signaling cascade regulates mRNA trans-lation via two distinct mechanisms, one involving activation of the p70 S6kinase and phosphorylation of the ribosomal S6 protein, and the other in-volving de-activation of 4E-BP1. Altogether, the emerging picture is thatType I IFN-dependent non-Stat pathways are required both for IFN-depen-dent gene regulation and for the initiation of mRNA translation for proteinproducts that mediate the biological properties of Type I IFNs.

25ANTIVIRAL ACTION OF HUMAN MXA GTPASE AGAINSTBUNYAVIRUSES INVOLVES REMODELING OF INTRACEL-LULAR MEMBRANESMike Reichelt, Silke Stertz, Georg Kochs, and Otto HallerDepartment of Virology, University of Freiburg, Freiburg, Germany.

The interferon-induced human MxA protein is a key component of theantiviral response against a number of RNA viruses, including bun-yaviruses, such as Rift valley fever virus and La Crosse virus (LACV).MxA belongs to the dynamin superfamily of large GTPases known tobe involved in vesicle trafficking and membrane remodeling. It accu-mulates in the cytoplasm and is partly associated with the smooth en-doplasmic reticulum. LACV replicates in the cytoplasm where nucle-ocapsid (N) proteins are synthesized and transported to the Golgiapparatus, the site of viral assembly. In MxA-expressing cells, the vi-ral N protein is recognized by MxA and sequestered into large peri-nuclear complexes. We used green fluorescent protein-tagged MxA andconfocal time-lapse imaging to study the dynamics of this process. Fivehours after infection, MxA/N complexes became visible as small dotsthat were scattered throughout the cytoplasm. Later, these dots fusedwith each other forming increasingly larger complexes that eventuallymoved to the perinuclear area. This process occurred unhindered in cy-tochalasin D or nocodazole-treated cells, indicating that an intact mi-crotubule or actin cytoskeleton was not required. However, the smoothendoplasmic reticulum was reorganized, as demonstrated by the pres-ence of Syntaxin17 in the growing perinuclear MxA/N complexes.These data suggest that the antiviral action of MxA against LACV in-volves remodeling of specific intracellular membrane compartments,leading to trapping and missorting of a major viral component.

26THE INNATE CELLULAR RESPONSE TO VIRUS PARTICLEENTRY REQUIRES IRF3 BUT NOT VIRUS REPLICATIONSusan E. Collins,1 Ryan S. Noyce,2 Karen L. Mossman1,2

Departments of 1Pathology and Molecular Medicine and 2Biochem-istry, Centre for Gene Therapeutics, McMaster University, Hamilton,Ontario, Canada, L8N 3Z5.

Mammalian cells respond to virus infection by eliciting both innate andadaptive immune responses. One of the most effective innate antiviralresponses is the production of type I interferons, a family of pleiotropiccytokines that induce the expression of proteins which collectively func-tion to limit virus replication and spread. We reported previously thatherpes simplex virus particles induce an antiviral state in fibroblasts inan interferon-independent fashion in the absence of virus gene expres-sion. Here, we show that the cellular transcription factor IRF3 is es-sential for the direct induction of interferon-stimulated genes follow-ing treatment with dsRNA or infection with viruses representing diversevirus families. Although virus replication of RNA viruses leads to thehyperphosphorylation of IRF3, this modification is not required for anti-viral state induction. We propose a model in which incoming virus par-ticles initiate an innate antiviral response that is dependent on IRF3.Subsequent virus replication modifies this response in a variety of ways,including post-translational modification of the IRF3 protein. In addi-tion, we demonstrate that herpesviruses and poxviruses block IRF3-me-diated accumulation of interferon-stimulated mRNAs whereas the rhab-dovirus, vesicular stomatitis virus, induces a post-transcriptional block.Furthermore, we provide evidence that incoming virus particles, viralnucleic acid and replicating virus serve as distinct forms of stimuliwithin an infected cell.

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27THE MOLECULAR BASIS FOR INHIBITON OF INTERFERONPRODUCTION AND SIGNALLING BY PARAMYXOVIRUSESAND THE PRACTICAL APPLICATION OF SUCH STUDIESD.F. Young,1 B. Precious, L. Andrejeva,1 S. Goodbourn,2 R.A. Lamb,3

R.E. Randall11School of Biology, University of St. Andrews, Fife, Scotland. 2Depart-ment of Biochemistry and Immunology, St. George’s Hospital MedicalSchool, University of London, UK. 3Department of Biochemistry, Mo-lecular Biology and Cell Biology, Northwestern University, Illinois, USA.

We originally showed that many paramyxoviruses block both interferon(IFN) signaling and IFN production. In the case of Simian Virus 5(SV5), the virus blocks IFN signaling by targeting STAT1 for protea-some-mediated degradation. To further define the molecular interac-tions involved in this process we have recently developed an in vitroubiquitination and degradation assay. Central to this process, is the Vprotein of SV5, as well as the cellular proteins STAT2, the p127 sub-unit of the DNA damage binding protein (DDB1) and Cullin 4a. Theseproteins form part of a multiprotein complex which is involved in theubiquitination of STAT1. As well as blocking IFN signaling, the V pro-tein of SV5 also inhibits IFN production. The functional domains onV involved in blocking IFN production overlap but are different fromthose required to target STAT1 for degradation. With regards inhibi-tion of IFN production, the transcription factors NF-kB and IRF3 arenot activated in cells infected with wild type SV5, but are activated incells infected with a mutant virus (SV5VDC) that makes a truncatedversion of V and induces the production of large amounts of IFN byinfected cells. Furthermore, the V protein of SV5 can act in trans toblock dsRNA and SV5VDC induction of IFN. On a practical side, wehave used the information generated from these studies to engineer cell-lines which are non-responsive to IFN. The practical applications of us-ing such cells in vaccine development and manufacture, virus diag-nostics and isolation of newly emerging viruses, and studies on hostcell tropism and pathogenesis will briefly be discussed.

28ACTIVATION OF THE INTERFERON SYSTEM BY SHORT IN-TERFERING RNASC.A. Sledz,1,2 M. Holko,1,2 M.J. deVeer,1 R.H. Silverman,1,2 B.R.G.Williams1,2,3

1Department of Cancer Biology, Lerner Research Institute, ClevelandClinic Foundation, Cleveland, Ohio, USA. 2Molecular Virology Pro-gram and 3Department of Genetics, Case Western Reserve University,Cleveland, Ohio, USA.

RNA interference (RNAi) is a powerful tool used to manipulate geneexpression or determine gene function. Many methods of expressingthe short double stranded (ds) RNA intermediates required for inter-ference in mammalian systems are being developed. One technique thatis not only effective at inducing the silencing of endogenous gene ex-pression, but is also currently being investigated for its ability to si-lence the expression of exogenous viral genes, is the introduction ofshort interfering (si) RNAs. While RNAi relies upon a high degree ofspecificity, little attention has been given to the potential non-specificeffects that may be induced. We found that transfection of siRNAs re-sults in an interferon (IFN) mediated activation of the Jak-Stat path-way and global upregulation of IFN-stimulated genes (ISGs). This ef-fect is mediated by the dsRNA dependent protein kinase PKR, as thiskinase is activated by the 21 base pair (bp) siRNA and is required forIFNb up-regulation in response to the siRNAs. Thus, siRNAs havebroad and potentially complicating effects beyond the selective silenc-ing of target genes when introduced into cells. This is of critical im-portance as siRNAs are currently being explored for their potential ther-apeutic use against viral replication.

29A ROLE FOR TYPE I INTERFERONS IN THE REGULATIONOF CLASS II MAJOR HISTOCOMPATIBILITY COMPLEXEXPRESSIONEdward Cha and Christian SchindlerIntegrated Program in Cellular, Molecular, and Biophysical Studies,Medical Scientist Training Program, Departments of Medicine and Mi-crobiology, Columbia University, New York City, New York, USA.

The type I interferon (IFN-I) family of cytokines regulates both innateand acquired immunity but so far has few roles prescribed to it re-garding class II major histocompatibility complex expression. We pres-ent evidence that in response to IFN-I, macrophages derived from micedevoid of signal transducer and activator of transcription 2 (STAT2),a transcription factor that regulates gene expression driven by IFN-I,are now able to induce MHC II surface expression and that this regu-lation is mediated by the class II transcriptional coactivator (CIITA).Northern analyses and RNAse protection assays reveal that in the ab-sence of STAT2, not only are CIITA transcripts expressed in responseto IFN-I but also are present in unstimulated STAT2-null macrophagesand that expression is significantly derived from the dendritic cell-spe-cific promoter (pI) of CIITA. Our analyses do not reveal a role for theB-cell specific isoform (pIII). Chromatin immunoprecipitation assaysreveal that although IFN-I does not stimulate CIITA and MHC II ex-pression in wild type macrophages, signal transducer and activator oftranscription 1 (STAT1) still binds to the IFN-gamma-inducible pro-moter (pIV) in response to IFN-I. Together, these data suggest that eventhough IFN-I sufficiently activates STAT1, it also suppresses CIITAexpression at the transcriptional level in a STAT2-dependent manner,thereby preventing subsequent MHC II expression.

30INTERFERON REGULATORY FACTOR-2 REGULATESCELL GROWTH THROUGH ITS ACETYLATION Atsuko Masumi,1 Yoshio Yamakawa,1 Hidesuke Fukazawa,1 KeikoOzato,2 Katsutoshi Komuro1

1National Institute of Infectious Diseases, Tokyo, Japan. 2Laboratoryof Molecular Growth and Regulation, National Institute of Child Healthand Human Development, National Institutes of Health, Bethesda, MD,20892, USA.

We have previously shown that interferon regulatory factor-2 (IRF-2)is acetylated by p300 and PCAF in vivo and in vitro. In this study weidentified, by mass spectrometry, two lysine residues in the DNA bind-ing domain (DBD), K75 and K78, to be the major acetylation sites inIRF-2. Although acetylation of IRF-2 did not alter DNA binding ac-tivity in vitro, mutation of K75 diminished IRF-2 dependent activationof histone H4 promoter activity. Acetylation of IRF-2 and IRF-2-stim-ulated H4 promoter activity were inhibited by the adenovirus E1A, in-dicating the involvement of p300/CBP. Mutation of K78, a residue con-served throughout the IRF family members, led to the abrogation ofDNA binding activity independently of acetylation. H4 is transcribedonly in rapidly growing cells and its promoter activity is dependent oncell growth. Consistent with a role for acetylated IRF-2 in cell growthcontrol, IRF-2 was acetylated only in growing NIH 3T3 cells, but notin growth-arrested counterparts. Chromatin immunoprecipitation assaysshowed that IRF-2 interacted with p300 and bound to the endogenousH4 promoter only in growing cells, although the levels of total IRF-2were comparable in both growing and growth-arrested cells. These re-sults indicate that IRF-2 is acetylated in a cell growth-dependent man-ner, which enables it to contribute to transcription of cell growth reg-ulated promoters.

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31CONTROL OF THE IRF-3 PATHWAY BY THE HEPATITIS CVIRUS NS3/4A PROTEASEEileen Foy,1 Kui Li,2 Chunfu Wang,1 Rhea Sumpter,1 Stanley Lemon,2

Michael Gale, Jr.11Department of Microbiology, University of Texas Southwestern MedicalCenter, Dallas, Texas, USA, and 2Department of Microbiology and Immu-nology, University of Texas Medical Branch, Galveston, Texas, USA.

Over 200 million people are infected with Hepatitis C Virus (HCV) withmost individuals developing a chronic infection. We hypothesize that HCVpersistence is modulated, in part, through disruption of IRF-3 function, akey regulator of the innate intracellular antiviral response. We used tetra-cycline-regulated cell lines and both genomic and subgenomic HCV repli-cons to examine the impact of HCV protein expression and viral RNA repli-cation on IRF-3 function. We found that the replication of HCV RNA inhuman hepatoma cells inhibits the IRF-3 pathway and prevents activationof IRF-3 upon further stimulation with Sendai virus. Our studies revealedthat expression of the NS3/4A viral protein complex was sufficient to blockvirus-induced hyperphosphorylation and translocation of IRF-3 thereby im-pairing IRF-3 dependent gene expression. Regulation of IRF-3 by HCVNS3/4A was mapped to the complex’s protease activity through mutationalanalysis and the use of an NS3/4A specific protease inhibitor. Analysis ofcells harboring genetically distinct HCV RNA replicon quasispecies has re-vealed that activation of IRF-3 correlates with induction of the innate anti-viral response and inefficient viral RNA replication, whereas disruption ofIRF-3 activation confers increased viral fitness and efficient replication. Fur-thermore, in cells harboring replicating HCV RNA, restoration of the IRF-3 response through forced expression of a phosphomimetic IRF-3 mutant,or the upstream signaling components TRIF, TBK1 and IKKe drasticallyreduced HCV replication concomitant with induction of IRF-3 target geneexpression. Thus IRF-3 performs a fundamental role by which the host re-sponse controls HCV RNA replication. Our results suggest that control ofthe IRF-3 signaling pathway is critical for HCV to establish and maintainpersistent infection. Finally, strategies that restore the host response to HCVmay represent potent therapeutic approaches to controlling HCV infection.

32AN IFNg-INDUCED HETERODIMER OF IRF-1 AND C/EBPbTRANSLOCATES TO THE NUCLEUS AND PROMOTES GENE EX-PRESSIONVladimir Hurgin, Ana Hernandez, Daniela Novick, and Menachem Rubin-steinDepartment of Molecular Genetics, The Weizmann Institute of Science, Re-hovot, Israel.

IL-18 binding protein (IL-18BP) is an IFNg-induced inhibitor of the pro-inflammatory cytokine IL-18, whose expression level profoundly affectsTh1 and Th2 responses. The IL-18BP promoter includes within its proxi-mal region (bases 21 to 2122) a gamma-activated sequence (GAS) fol-lowed by an IRF-1 response element (IRF-E) that are essential for basal andIFNg-induced promoter activity. We found that IFNg induced the associa-tion of IRF-1 and C/EBPb, forming a heterodimer that translocated fromthe cytoplasm to the nucleus. Electrophoretic mobility shift assays (EMSA)revealed that this heterodimer binds to the proximal GAS-IRF-E pair. Fur-ther characterization of the promoter revealed a clockwork of silencers andenhancers that mediated responses to various cytokines. In addition to theGAS-IRF-E pair, the proximal region (bases 21 to 2450) contained twoAP-1 sites, acting as silencers, two additional C/EBP-Es and two NF-kB-Es, all acting as enhancers. Mutation of any one of these elements affectedboth basal and IFNg-induced activity by orders of magnitude. The NF-kB-E probably mediated responses to IL-1b and TNFa, as these cytokines syn-ergized with IFNg in inducing IL-18BP. Indeed, SN50, a specific NF-kBinhibitor, significantly reduced IL-18BP mRNA induction. Furthermore, wefound that IFNg induced a complex between NF-kB and the proximal AP-1-E, suggesting a possible mechanism by which IFNg may overcome theAP-1-mediated repression of IL-18BP gene expression. Further analysis hasidentified a distal silencer/enhancer pair within bases 21081 to 21272, thatwas physically associated with the proximal IRF-1. Chromatin immuno-precipitation confirmed the presence of these separate proximal and distalpromoter regions, which merged into a single transcription-activation com-plex following induction by IFNg. Expression profiling revealed several lateIFNg-induced genes whose proximal promoter regions contain adjacent IRF-E and C/EBP-E. Indeed, EMSA demonstrated binding of the IRF-1-C/EBPbheterodimer to these elements, suggesting that this heterodimer is a generaltranscription-activator of many IRF-1-dependent IFNg-induced genes.

33A NOVEL ANTI-ONCOGENIC INHIBITOR OF STAT3Dhan V. Kalvakolanu,1 Jun Zhang,1 Jiadi Hu,1 Jinbo Yang,2 Sanjit K.Roy,1 Silvia Tininini,4 Jacqueline F. Bromberg,3 Valeria Poli,4 andGeorge R. Stark2

1Greenebaum Cancer Center, Department of Microbiology and Immu-nology, University of Maryland School of Medicine, Baltimore, MD21201. 2Department of Molecular Biology, Lerner Research Institute,Cleveland Clinic Foundation, Cleveland, OH. 3Sloan Kettering Can-cer Center, New York, NY, 4University of Torino, Torino, Italy.

The IFN-family of cytokines regulates cell growth by promoting sometranscriptional processes and opposing others. The full spectrum ofthese actions and the critical players involved in them are only begin-ning to be understood. Previously we have described the isolation ofGRIM-19, a novel cell death activator, using a genetic screen. It codesfor a ,16 kDa protein that induces apoptosis in a number of cell lines.Antisense ablation of GRIM-19 caused resistance to cell death inducedby interferon plus retinoic acid and conferred a growth advantage toseveral cancer cells. To understand the molecular bases for its cell deathregulatory activity, we employed a yeast-2-hybrid screen and identifiedthat the transcription factor STAT3 binds to GRIM-19. GRIM-19 in-hibits transcription driven by activation of STAT3, but not STAT1. Itneither inhibits the ligand-induced activation of STAT3 nor blocks itsability to bind to DNA. Thus, GRIM-19 differs from the previously de-scribed STAT3-inhibitors: PIAS3 and SOCS proteins. Mutational anal-ysis indicates that the transactivation domain of STAT3, especiallyresidue S727, is required for GRIM-19 binding. Since GRIM-19 doesnot bind significantly to other STATs, our studies identify a novel spe-cific inhibitor of STAT3. Because constitutively active STAT3 upreg-ulates anti-apoptotic genes to promote tumor survival, its inhibition byGRIM-19 also demonstrates a novel anti-oncogenic effect exerted bybiological therapeutics. We will also describe inactivation of GRIM-19-induced apoptosis by viral oncogenes and its inactivation in humantumors.

34IRF-5, A NOVEL MEDIATOR OF CELL-CYCLE ARREST ANDCELL DEATH B.J. Barnes, M. Mancl, K. Pinder, and P. PithaSidney Kimmel Comprehensive Cancer Center, Johns Hopkins Schoolof Medicine, Baltimore, MD 21231.

Transcription factors of the interferon regulatory factor (IRF) familyplay essential roles in the regulation of genes induced by viral infec-tion, immuno-stimulation, as well as cell growth regulation. The tran-scription factor IRF-5 was originally identified as a regulator of type Iinterferon (IFN) gene expression, however more recent studies have re-vealed that it plays a role in many aspects of host defense including in-duction of cytokines and chemokines involved in the recruitment of T-lymphocytes. In addition, current data suggests a tight regulatory systemcontrolling IRF-5 and IRF-7 protein levels for the repression and in-duction of IFNA gene expression post-virus infection. While the con-stitutive expression of IRF-5 is limited to lymphoid organs, dendriticcells and PBL, we have recently determined that IRF-5 transcripts areabsent in primary hematologic malignancies (ALL, AML, CLL). In aneffort to gain further insight into the possible role of IRF-5 in oncoge-nesis, we have analyzed the effect of IRF-5 on cell-cycle, tumor growthand apoptosis. IRF-5 inhibits both in vitro and in vivo B-cell lymphomatumor growth. The molecular mechanism(s) of IRF-5–(mediated growthinhibition is associated with a G2/M cell-cycle arrest and modulationof growth regulatory and pro-apoptotic genes. While IRF-5 is a directtarget of p53, its tumor suppressor functions are independent of p53.Yet, these data clearly indicate that IRF-5 is a downstream componentof the p53-signaling pathway. Finally, we shall present data indicatingthat viral proteins involved in oncogenesis may inhibit IRF-5 cell-cy-cle regulatory functions leading to uncontrolled cell proliferation.

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35IFN-gR2 EXTRACELLULAR DOMAIN AND JAK1 ARE ESSENTIALFOR PREASSEMBLY AND STAT1 FOR LIGAND-INDUCED MOVE-MENT OF THE CHAINS OF THE IFN-g RECEPTOR COMPLEXChristopher D. Krause,1 Natasha Lavnikova,1 Junxia Xie,1 Erwen Mei,2 Olga V.Mirochnitchenko,1 Yiwei Jia,3 Robin M. Hochstrasser,2 Sidney Pestka1

1Department of Molecular Genetics, Microbiology, and Immunology, The Uni-versity of Medicine and Dentistry of New Jersey—Robert Wood Johnson Med-ical School, Piscataway, NJ 08854 USA, 2Regional Laser Biotechnology Labo-ratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA19103 USA, 3Olympus America, Inc., SEG, Melville, NY 11747 USA.

We previously used fluorescence resonance energy transfer (FRET) in intact cellsto show that the interferon-gamma (IFN-g) receptor complex is preformed, andthat this preformed complex undergoes a conformational change upon the treat-ment of cells with IFN-g. Here we mutated various elements of the IFN-g re-ceptor complex known to affect its biological activity to delineate the determi-nants mediating receptor preassembly and ligand-mediated changes of the receptorcomplex. We observed that nearly all the ligand-mediated decrease in FRET ef-ficiency was eliminated when Tyr-457 on IFN-gR1 was mutated to phenylala-nine. Exchange of the extracellular domain of Hu-IFN-gR1 with its murine ho-molog only weakly perturbed interactions with other receptor chains, but exchangeof the extracellular domain of Hu-IFN-gR2 with its murine homolog inhibitedinteractions with both Hu-IFN-gR1 and Hu-IFN-gR2. Therefore, the extracellu-lar domain of IFN-gR2 plays a significant role in its interaction with the IFN-gR1 chain and mediates its interaction with another IFN-gR2 chain. Mutation ofIFN-gR1 to disrupt its interaction with Jak1 strongly inhibited its ability to in-teract with IFN-gR2 but did not affect its ability to interact with another IFN-gR1 chain. However, mutation of IFN-gR2 so that its ability to interact with Jak2was blocked did not affect its ability to interact with either IFN-gR1 or IFN-gR2.Thus Jak1 but not Jak2 plays a role in IFN-gR1: IFN-gR2 interactions. The dataindicate that (i) interactions between IFN-gR2 chains are mediated almost en-tirely by species-specific residues in their extracellular domains, (ii) interactionsbetween IFN-gR1 and IFN-gR2 are mediated partially by species-specificresidues in the IFN-gR2 extracellular domains but principally by Jak1, (iii) in-teractions between two IFN-gR1 molecules are mediated neither by species-spe-cific residues in their extracellular domains nor by Jak1, and (iv) large confor-mational changes in the IFN-g receptor complex require that IFN-gR1 be able toassociate with Stat1.

36SOCS1 REGULATION OF TYPE I INTERFERON RE-SPONSESJ.E. Fenner,1 R. Starr,2 A. Cornish,2 D. Metcalf,2 W. Alexan-der,2 D.J. Hilton,2 P.J. Hertzog1

1Centre for Functional Genomics and Human Disease, MonashInstitute of Reproduction and Development, Monash Univer-sity, Melbourne, Australia, 2Walter and Eliza Hall Institute ofMedical Research, Melbourne, Australia.

In vitro studies have demonstrated that Socs1 expression is in-duced by both type I and II interferons (IFNs), as well as theprotein being able to suppress the actions of both type I and IIIFNs. The Socs12 /2 mice suffer from pathologies resulting inneonatal death that has been found to be due to a hypersensi-tivity to IFNg. Subsequently the importance of SOCS1 in IFNgsignaling was elucidated, however the role of SOCS1 in thetype I IFN signaling system was thus far unknown. We haveinvestigated molecular signaling mechanisms, susceptibility toinfections and genetic crosses of Socs1, Ifnar1 and Ifnar2 genetargeted mice to demonstrate a role for Socs1 in mediating typeI IFN responses in vivo. Socs12 /2 and Socs12/2 Ifng2/2 miceshow enhanced type I IFN responses. Furthermore, the If-nar12/2 Socs12/2 mice survive past weaning unlike theSocs12/2 but die between 46 weeks of a pathology different tothat of the Socs12/2 mice. The Ifnar2 crosses to the Socs12 /2

demonstrate a difference between the two Ifnar2 /2 mice, asthese mice die during the neonatal period. Currently we are ex-amining the molecular mechanisms of this signaling pathway.An understanding of both the inhibition and activation of sig-naling pathways is important in order to understand the bene-ficial and harmful effects of cytokines.

37DSRNA-DEPENDENT TLR3 TYROSINE PHOSPORYLATION-MEDIATED ACTIVATION OF PI3 KINASE IS ESSENTIALFOR P56 GENE INDUCTION BY IRF-3Saumendra N. Sarkar, Kristi L. Peters, Christopher P. Elco, Ganes C. SenDepartment of Molecular Biology, The Lerner Research Institute,Cleveland Clinic Foundation, Cleveland, OHIO, USA.

Double-stranded (ds) RNA, a common product of viral infection, caninduce transcription of many cellular genes, including the 561 (ISG 56)gene that encodes a regulator of protein synthesis. The signaling path-ways activated by dsRNA initiate from Toll-like Receptor 3 (TLR3)and use the transcription factors IRF-3, NFkB and others. We observedthat ligand-dependent tyrosine phosphorylation in the cytoplasmic do-main of TLR3 and concomitant recruitment of IRF-3 to the receptorcomplex are required for dsRNA-signaling. We have identified the spe-cific TLR3 tyrosine residues that are targets of phosphorylation and ini-tiate the two-step signaling process. In the first step, IRF-3 phospho-rylation leads to its dimerization and nuclear translocation and in thesecond step it acquires the ability to activate gene transcription. Thesecond step requires activation of the PI3-kinase pathway that isachieved by the recruitment PI3K in the dsRNA-receptor complex uponphosphorylation of a specific Tyr residue of TLR3. The above resultsprovide the first demonstration of the needs for TLR3 tyrosine phos-phorylation and the resultant recruitment and activation of PI3 kinasein IRF3-mediated gene induction by dsRNA.

38TOLL-LIKE RECEPTOR 3-MEDIATED ACTIVATION OFNFkB AND IRF3 DIVERGE AT TRIFZhengfan Jiang,1 Ganes Sen,2 and Xiaoxia Li11Departments of Immunology and 2Molecular Biology, Lerner ResearchInstitute, Cleveland Clinic Foundation, Cleveland, OH 44195.

We have previously shown that double-stranded RNA (dsRNA)-trig-gered, Toll-like receptor 3 (TLR3)-mediated signaling is independentof MyD88, IRAK4 and IRAK. Instead, TRAF6, TAK1 and TAB2 arerecruited to TLR3 upon Poly I:C stimulation. TRAF6-TAK1-TAB2 arethen translocated to the cytosol where TAK1 is phosphorylated and ac-tivated, leading to the activation of IKK and NFkB. The present studyaddressed two important questions: 1) How are TRAF6, TAK1 andTAB2 recruited to TLR3? 2) Are TRAF6, TAK1 and TAB2 also re-quired for TLR3-mediated IRF3 activation? Recently, a novel TIR-con-taining adapter, TRIF, has been implicated in TLR3-mediated activa-tion of NFkB and IRF3. We found that TLR3 recruits TRAF6 viaadapter TRIF through a TRAF6-binding sequence in TRIF (PEEMSW,amino acids 250 to 255). Mutation of this TRAF6-binding sequenceabolishes the interaction of TRIF with TRAF6, but not with TLR3. In-terestingly, mutation of the TRAF6-binding site of TRIF only abolishesits ability to activate NFkB but not IRF3, suggesting that TLR3-medi-ated activation of NFkB and IRF3 might bifurcate at TRIF. In supportof this finding, we showed that DN-TRAF6 and DN-TAK1 blockedPoly IC-induced NFkB but not IRF3 activation. Furthermore, usingTRAF6 deficient MEFs, it was clearly shown that whereas Poly IC-in-duced-NFkB activation is completely abolished in these cells, the sig-nal-induced activation of IRF3 is TRAF6 independent. In conclusion,TRIF recruits TRAF6-TAK1-TAB2 to TLR3 through its TRAF6-bind-ing site, which is required for NFkB but not IRF3 activation. There-fore, dsRNA-induced TLR3/TRIF-mediated NFkB and IRF3 activationdiverge at TRIF.

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39OROMUCOSAL INTERFERON THERAPY: MECHANISM(S)OF ACTIONMichael Tovey, Christophe Lallemand, Jean-Francois Méritet, andChantal MauryUPR 9045 CNRS, Laboratory of Viral Oncology, Villejuif, France.

The use of transgenic mice expressing a green fluorescent protein re-porter gene regulated by an IFN responsive element has shown that IFNactivated cells are present in the peripheral circulation of tumor bear-ing mice as early as 4 hours after the initiation of oromucosal IFN atherapy. The principal cell populations activated by IFN treatment werecharacterized as NK1.1, CD11b1, CD19/CD20 B-cells, and subpopu-lations of T-cells. In addition to green fluorescent cells activated by di-rect contact with IFN in the oral cavity, oromucosal IFN therapy alsoinduced trafficking of cells from both the spleen and peripheral lymphnodes to the site of tumor cell replication. Differential display analysisshowed that numerous IFN responsive genes were induced in the lym-phoid tissue of the oral cavity and spleen together with a number ofgenes not previously shown to be induced by IFN a including Crg2and other chemokines, proteases associated with antigen processing,and genes involved in lymphocyte activation, apoptosis, and proteindegradation. Together these results may explain in part the biologicalactivity of oromucosal IFN therapy.

40DISASSOCIATION OF BIOLOGICAL RESPONSE MODULATORYEFFECTS OF INTERFERON (IFN)a-1B FROM CHRONIC DOSELIMITING SIDE EFFECTS IN PATIENTS WITH MALIGNANCIESE. Borden, P. Masci, B. Jacobs, L. Wood, L. Rybicki, M. Hussein, T.Olencki, R. Bukowski, and K. TongCleveland Clinic Foundation, Ohio 44195 and Ministry of Public Health,Shanghai.

IFN-a1 is one of the IFNs-a produced in largest amounts upon acute viralinfection. Data from prior clinical studies suggested IFN-a1a was signifi-cantly better tolerated than IFN-a2a but production of IFN-a1a was dis-continued two decades ago. IFN-a1b has been safely and effectively usedin China at low doses for therapy of hepatitis B and C virus chronic infec-tions. The objectives of this trial were 1) to confirm the safety, tolerabilityand maximum dose of recombinant human IFN-a1b (specific activity 107

WISH/VSV antiviral units/mg protein) and 2) to measure the effects of IFN-a1b on IFN-stimulated genes (ISGs). Eligible patients had metastatic ma-lignancies refractory to standard treatments. Sequential cohorts (n 5 25 to-tal) received increasing doses of 15 (IFN-a2 equivalence 3 3 106 units), 45,135, and 270 ag/m2 daily administered subcutaneously daily. Granulocytes,1000/mm3 occurred at all dose levels including the lowest. Biologic ef-fects of IFN-a1b were assessed at baseline and on days 2, 8 and 29 throughISG products in serum (b2-microglobulin, GTP-cyclohydrolase, ISG 15) andISGs not previously assessed in vivo (TRAIL, TNF-(a). Increases in ISGproducts occurred by 24 hrs at all doses and were sustained through day 29(p , 0.01). Only for ISG15 and TRAIL was there a significant trend forgreater increase at higher doses. At 270 mg/m2 Grade III fever and uncon-trolled rigors (despite acetoaminophen and narcotics) on the initial treatmentday became a clinical problem. No patient was removed from study for anadverse event secondary to IFN-a1b. Even at 270 mg/m2, no patients hadsignificant weight loss or decline in performance status attributable to IFN-a1b. Chronic side effects usually limit administration of IFN-a2 at .20–25mg/M2 daily. After previous steadily progressive disease, two patients re-mained stable on 45 mg/m2 of IFN-a1b for .1 yr. One patient with renalcarcinoma had a partial response at 270 mg/m2. Results suggest a disasso-ciation of biological response modulatory and clinical activity from doselimiting side effects.

41INTERFERON-ALPHA INDUCED APOPTOSIS IN MALIGNANTCELLS IS MEDIATED THROUGH A PI3K/MTOR DEPENDENT SIG-NALING PATHWAYL. Thyrell,1 L. Hjortsberg,1 V. Arulampalam,2 T. Panaretakis,1 S. Uhles,4

B. Zhivotovsky, 3 I. Leibiger,4 D. Grandér,1 K. Pokrovskaja1

1Department of Oncology and Pathology, Cancer Center Karolinska (CCK),Karolinska Hospital and Institute, Stockholm, Sweden. 2Microciology and Tu-morbiology Centre, Karolinska Institute, Stockholm, Sweden. 3Institute of En-vironmental Medicine, Department of Toxicology, Karolinska Institute, Stock-holm, Sweden. 4The Rolf Luft Center for Diabetes Research, Department ofMolecular Medicine, Karolinska Institutet, Stockholm, Sweden.

Interferon-alpha (IFN) induces apoptosis in malignant cells of different origin.We have recently demonstrated that IFN induced apoptosis is associated withthe activation of caspases and the pro-apoptotic proteins Bak and Bax, loss ofmitochondrial membrane potential and release of cytochrome c (Thyrell L, etal., Oncogene 2002, 21, 1251-62, Panaretakis T, et al., Oncogene 2003, 22,4543-56). Several signaling pathways are activated following binding of IFNto its receptor. In addition to onset of the classical Jak-STAT pathway, IFNalso induces PI3K activity. Here we show that chemical inhibition of the PI3Kactivity by Ly294002 abrogates IFN-induced apoptosis as measured by an-nexinV/PI and TMRE stainings. Activation of Bak and Bax was also inhibiteddemonstrating that apoptosis is disrupted by Ly294002 upstream of mito-chondria. Chemical inhibition of the downstream kinase in the PI3K pathway,the mammalian target of rapamycin (mTOR) by rapamycin, also efficientlyblocked IFN-induced apoptosis in a similar manner to Ly294002. PI3K andmTOR-dependent phosphorylation of two proteins involved in the regulationof translation, p70S6 kinase and 4E-BP1 repressor, was induced by IFN andstrongly inhibited by either Ly294002 or rapamycin. IFN-induced phosphory-lation of STAT proteins, followed by their translocation to the nucleus andbinding to the consensus sequences were not affected by either Ly294002 orrapamycin. Neither was the expression of several IFN target genes altered bythe inhibition of the PI3K/mTOR pathway. Finally, the ability of IFN to pro-tect against viral propagation and virus-induced cell death was not affected byinhibition of the PI3K/mTOR pathway. These data demonstrate that an acti-vation of the PI3K/mTOR pathway is required for IFN-alpha to induce apop-tosis of tumor cells, while the classical Jak-STAT signaling pathway alone isnot sufficient.

42ROLE OF TYPE I INTERFERONS IN LIPOPOLYSACCHA-RIDE-INDUCED RESPONSESS. Noppert,1,2,3 A. Mansell,2,3 S. Samarajiwa,2 J. Fenner,2,3 B. Scott,2,3

P.Hertzog2,3

1School of Applied Sciences, Monash University, Gippsland, Australia,2Centre for Functional Genomics and Human Disease, Monash Insti-tute of Reproduction and Development, Monash University, Clayton,Australia, 3CRC for Chronic Inflammatory Disease.

The innate immune system has evolved to recognize pathogen-associ-ated molecular patterns (PAMPs) and responds by initiating signalingcascades to protect the host. One such PAMP is lipopolysaccharide(LPS), a substance synthesized by gram-negative bacteria as an essen-tial external membrane component.

Toll-like receptor 4 (TLR4), highly expressed in macrophages,monocytes and dendritic cells, has been shown to be a major LPS recog-nition receptor. Activation of TLR4 by LPS triggers signaling pathwaysthat lead to activation of the transcriptions factors NF-kB, AP-1 andIRF3 and the subsequent production of a range of cytokines includingTNFa, IL-6 and interferonb. To determine the role of both interferon-dependent and -independent signaling pathways in LPS-induced re-sponses, gene microarray analysis was performed on bone marrow mac-rophages from IFNAR-deficient mice. Furthermore, a specific role forIFNAR receptors in a model of septic shock is demonstrated in IFNAR-deficient mice. Using an LPS dose lethal to the majority of wildtypemice within 48 hours, IFNAR1-null mice were resistant to the LPSchallenge, with 88% survival after 96 hours. This data, combined withthe microarray analysis of IFNAR-deficient mice demonstrates a clearrole for individual interferon receptors in the pathological response toLPS challenge and the subsequent consequences of septic shock.

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43IDENTIFICATION OF DIFFERENTIAL GENE REGULATIONBY ENGINEERED IFN-aS USING DNA MICROARRAYRenqiu Hu,1 Amy Xiaoyen Yang,2 Raj K. Puri,2 Hana Schmeisser,1

Kathryn Zoon1

1National Cancer Institute, National Institutes of Health, Bethesda MD20892, USA. 2Division of Cellular and Gene Therapies, CBER, FDA,Bethesda, MD 20892, USA.

In our previous studies we showed that the biological effects of engi-neered IFN-as provide evidence for a divergent mechanism by whichIFN-as induce their antiproliferative and antiviral effects. IFN-as mayturn on several signaling pathways to different degrees. Recently, it hasbeen reported that the activation of the JAK-STAT pathway alone isinsufficient for the induction of antiproliferative activity of IFN-a. OurEMSA (Electrophoretic Mobility Shift Assay) and RPA (Rnase Pro-tection Assay) results suggest, that antiproliferative response may notbe directed by activation of STAT1 and STAT2 alone. Therefore, wefocused on a more detailed understanding of the signaling mechanismused to elicit biological responses. The aim of this study was to revealthe activity of critical biological pathways and allow us to identify theunderlying signaling mechanism for mediation of antiproliferative ac-tivity of IFNs. We used oligonucleotide microarray technology to com-pare expression profiles of IFN-a2c and IFN-a21a. Preliminary dataderived from a comparison of expression profiles of those two inter-feron species showed that IFN-a21a up-regulates 49 genes and down-regulates 9 genes by over 3-fold, while IFN-a2c up-regulates 17 genesand down-regulates 8 genes by over 3-fold. Some genes were up-reg-ulated or down-regulated by both interferon species, but to a differentdegree. For example, “IFN-a inducible protein” was up-regulated 30-fold by IFN-a21a but only 15-fold by IFN-a2c. A number of genes arespecifically up-regulated by IFN-a21a but down-regulated by IFN-a2c.An example is the gene IGFBP7 relating to regulation of cell prolifera-tion, that was up-regulated 4-fold by IFN-a21a but down-regulated 1.4-fold by IFN-a2c. Those differences in gene regulation may contribute todifferent biological activities of the studied interferons.

44DEFECTS IN INTERFERON ACTION MEDIATE VIRUS-IN-DUCED ONCOLYSISJason Emmanuel, Marilyn Fernandez, Masatsugu Obuchi, SiddharthBalachandran, and Glen N. BarberDepartment of Microbiology and Immunology and Sylvester Compre-hensive Cancer Center, University of Miami School of Medicine, Mi-ami, Florida, USA, 33136.

Our laboratory has recently shown that vesicular stomatitis virus, VSV,a relatively non-pathogenic, negative-stranded RNA virus, can selec-tively induce the cytolysis of malignant cells, but not normal cells,through the induction of apoptotic cell death. VSV appears able to se-lectively replicate in transformed cells since these hosts exhibit the hall-marks of a flawed interferon (IFN) system, which is essential for pre-venting VSV replication. VSV was found to cause significant tumorregression when administered at sites distal from the tumor, when de-livered intravenously, or against syngeneic tumors in immunocompe-tent hosts. However, the simple genetic constitution of VSV, lack ofany known transforming properties, well studied immunobiology andthe ability to genetically manipulate this virus affords an ideal oppor-tunity to further enhance the oncolytic potential of this generally in-nocuous organism. To examine this possibility, we attempted to con-struct recombinant VSVs that carried either the Type I or II interferonsor cytokines IL-4 and IL-12. Significantly, we determined that suchviruses were not only viable but synthesized their heterologous prod-ucts to extremely high levels. Engineered viruses, especially those ex-pressing murine or human Type I IFN exhibited greatly increased at-tenuation in normal cells and more potent oncolytic activity againstmetastatic disease in immunocompetent animals than the wild-typevirus and were able to stimulate specific anti-tumor CTL responses.Defects in the innate immune system that facilitate VSV-oncolysis aswell as future directions involving the use of these vectors in gene ther-apy will be discussed.

45TYPE I INTERFERON DIFFERENTIAL THERAPY FOR ERY-THROLEUKEMIA: SPECIFICITY OF STAT ACTIVATIONV.S. Cull,1 P.A. Tilbrook,2 E.J. Bartlett,1 N.L. Brekalo,1 C.M. James1

1Division of Health Sciences, Murdoch University, Perth, Australia,2Laboratory for Cancer Medicine, Royal Perth Hospital, University ofWestern Australia, Perth, Australia.

Type I interferons (IFNs), pleiotropic cytokines with antiviral, antipro-liferative, apoptotic and immuno-regulatory functions, are efficaciousin the treatment of malignancies, viral infections and autoimmune dis-eases. Binding of these cytokines to their cognate receptor leads to ac-tivation of the Jak-signal transducers and activators of transcription(STAT) signaling pathway and altered gene expression. This signalpathway has been intensely studied using human IFN-a2 and IFN-b.However, there are over 14 human IFN-a subtypes and over 10 murineIFN-a subtypes, with a single IFN-b subtype in both species. J2E cellsare immortalized at the proerythroblast stage of development and pro-duce a rapid and fatal erythroleukemia in vivo. These cells retain theability to respond to erythropoietin in vitro by proliferating, differenti-ating, and remaining viable in the absence of serum. Here, we showthat J2E cells are also functionally regulated differentially by IFN sub-type treatment in vitro. A novel finding was the selective activation ofSTAT and miogen-activated protein kinase (MAPK) molecules by dif-ferent subtypes binding the IFN receptor. These findings indicate dis-tinct effects for individual type I IFN subtypes, which are able to dif-ferentially activate members of the STAT and MAPK family. Finally,we investigated the efficacy of IFN naked DNA therapy in treating J2E-induced erythroleukemia in athymic nude mice. IFN subtypes differ-entially regulated the onset of erythroleukemia with delayed onset andincreased survival possibly via a reduction in cell viability, and en-hanced anti-proliferative/apoptotic effects observed for IFNA6 andIFNA9 treatment, respectively. Moreover, this data highlights the ne-cessity for best choice of IFN subtype in disease treatment.

46CRYSTAL STRUCTURE OF THE 29-SPECIFIC AND DOUBLE-STRANDED RNA ACTIVATED INTERFERON-INDUCED ANTI-VIRAL PROTEIN 29-59-OLIGOADENYLATE SYNTHETASERune Hartmann,1,2,3 Just Justesen,3 Saumendra N. Sarkar,2 Ganes C. Sen,2

and Vivien C. Yee1

1Department of Molecular Cardiology and 2Department of Molecular Bi-ology, Cleveland Clinic Foundation, Cleveland, Ohio. 3Department of Mo-lecular Biology, University of Aarhus, Aarhus, Denmark.

29-59-oligoadenylate synthetases are interferon-induced, double strandedRNA-activated antiviral enzymes. They are also the only proteins knownto catalyze 29-specific nucleotidyl transfer. This first crystal structure ofa 29-59-oligoadenylate synthetase reveals a protein made of two major do-mains. The N-terminal domain is a classical 5 stranded antiparallel b-sheetdomain and the C-terminal domains most prominent features are a fourhelix bundle and a three-stranded mixed b-sheet. The crystal structure of29-59-oligoadenylate synthetase reveals structural conservation with the39-specific poly(A) polymerase that, coupled with structure-guided muta-genesis, supports a conserved catalytic mechanism for the 29- and 39-spe-cific nucleotidyl transferases. Comparison with structures of other super-family members indicates that the donor substrates are bound by conservedactive site features while the acceptor substrates are oriented by noncon-served regions. The 29-59-oligoadenylate synthetases are activated by vi-ral produced double stranded RNA in infected cells and initiate a cellularresponse by synthesizing 29-59-oligoadenylates, that in turn activate RNaseL. We have used structural guided mutagenesis to identify the RNA bind-ing/activation site of 29-59-oligoadenylate synthetase. The RNA bind-ing/activation site of 29-59-oligoadenylate synthetase is not a defined do-main, but is found at the interface of the two major domains of29-59-oligoadenylate synthetase. Furthermore, this crystal structure sug-gests that activation involves a domain-domain shift and thus providingstructural insight into cellular recognition of virus-produced double-stranded RNA.

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47EXPRESSION PROFILING OF IRF-3 RESPONSIVE GENESDURING VIRAL INFECTION Janet Andersen,1 Sarah Van Scoy,1 Tadatsugu Taniguchi,2 and NancyC. Reich1

1Department of Pathology, Stony Brook University, Stony Brook, NewYork USA, 2Department of Immunology, University of Tokyo, Tokyo,Japan.

Interferon regulatory factor-3 (IRF-3) is a cellular transcription factorthat resides predominantly in the cytoplasm until it is activated by ser-ine phosphorylation during the course of viral infection. Following spe-cific phosphorylation, IRF-3 accumulates in the nucleus in associationwith the histone acetylases CBP or p300. The IRF-3/CBPcomplex bindsdirectly to a subset of interferon-a stimulated response elements (ISRE)found within responsive genes. By evaluating the effects of a constitu-tively active mutation of IRF-3, a subset of responsive genes have beenidentified, and the induction of this set of genes appears to elicit anapoptotic cellular response. In the context of a viral infection the phos-phorylated IRF-3 also associates with other DNA-binding factors in en-hanceosome complexes to effect transcription. A prominent exampleof a gene regulated by concerted effects of IRF-3 with other en-hanceosome factors is the interferon-b gene. Activation of IRF-3 is nec-essary but not sufficient for induction of this set of genes. To identifydirect gene targets of IRF-3/CBP that contain a specific ISRE target aswell as genes that respond to IRF-3 together with other enhanceosomefactors, we compared gene expression profiles of mouse embryo fi-broblasts from IRF-3 knockout animals and from wild type siblings fol-lowing infection with Newcastle Disease Virus. The fibroblasts lack-ing IRF-3 were notably more susceptible to viral infection than the wildtype fibroblasts as measured by viral gene expression and apoptotic re-sponse. Affymetrix DNA microarray profiles were compared with RNAisolated from uninfected cells or cells infected in the presence or ab-sence of cycloheximide or neutralizing interferon antibodies. Thesecomparisons have identified likely candidates for IRF-3 primary re-sponse genes, and ontologies indicate the induced genes are involvedin proliferation, innate immunity, and acquired immunity.

48IRF-3 DETERMINES HOST CELL PERMISSIVENESS TO HCVREPLICATIONRhea Sumpter, Jr., Eileen Foy, and Michael Gale, Jr.University of Texas Southwestern Medical Center, Dallas, TX, USA.

The development of the hepatitis C virus (HCV) subgenomic RNAreplicon system has allowed the study of HCV replication in tissue cul-ture. We show that HCV subgenomic RNA replicons may also be usedto model the early events of HCV infection in which virus-host inter-actions determine the eventual outcome of either viral persistence orclearance. HCV subgenomic RNA replicons rapidly induce the cellu-lar antiviral response upon their introduction into host Huh7 cells. Wefound that attenuation of interferon-b (IFNb) and interferon stimulatedgene (ISG) production in the first 24 hours post-transfection was cor-related with an increased transduction efficiency of a highly adaptedsubgenomic HCV replicon. In the present study, we identified the HCVinternal ribosome entry site as a potent agonist of host cell dsRNA-ac-tivated pathways, suggesting that the host response to viral RNA mayaffect HCV persistence and replication. To test the hypothesis that oneor more components of the host response determines permissiveness topersistent HCV RNA replication, we evaluated virus-host interactionsin a cell line (Huh7.5) that is highly permissive for transduction byHCV replicons. Cellular permissiveness to HCV was linked to a lesionin the IRF-3 response pathway that is normally triggered by viral RNA,while other virus-responsive pathways remained intact. This defect al-lowed the efficient transduction and replication of otherwise poorlyadapted HCV subgenomic replicons. Moreover, in Huh7 cells, whichhave a competent IRF-3 pathway, the ability of HCV subgenomic repli-cons to transduce and replicate was linked to disruption of IRF-3 acti-vation by the viral NS3/4A protease. Our results identify the virus-hostinteractions that control the IRF-3 pathway as critical determinants forHCV persistence.

49ACTIVATION OF IRF7 DURING PATHOGEN INFECTION:KINASES, PHOSPHORYLATION SITES, AND REGULATORYMECHANISMSAlexandre Caillaud,1 Ara G. Hovanessian,1 David E. Levy,2 and IsabelleJ. Marie1,2

1Institut Pasteur, Paris, France, 2NYU School of Medicine, New York, NY,USA.

Interferons (IFN) play a vital role in innate resistance to a wide variety ofinfectious agents. Interferon induction in response to pathogen invasionis tightly regulated. Much of this regulation is at the transcriptional level,and interferon regulatory factors (IRF), in particular IRF3 and IRF7, playan essential role in type I IFN regulation. Activation of IRF proteins pro-vides both qualitative and quantitative control of IFN production throughdifferential induction of distinct IFN isotypes.

IRF7 is activated in response to pathogen infection by phosphoryla-tion on serine residues located in the carboxyl-terminal regulatory domainof the protein, and the non-conventional IkB kinases Ikk-e and TBK1 havebeen implicated in this process. However, the exact sites of phosphoryla-tion have yet to be definitively established, and the possibility of differ-ential activation processes for distinct pathogens remains to be investi-gated.

We undertook a comprehensive approach of the identification of thephosphorylation sites of IRF7 in response to different inducing agents byexamining mutant proteins in which specific serine residues were alteredto alanine or aspartate. The phosphorylation patterns of these mutants wereanalyzed by 2D gels and their activity monitored by reporter assay. Thesepatterns were compared to proteins phosphorylated in vivo in response todifferent pathogens or to individual kinases. Our data show that the ser-ine-rich regulatory domain of IRF7 contains a kinase-recognition regionthat is necessary for distal phosphorylation but is not itself phosphory-lated. The kinase recognition region is followed by multiple sites for phos-phorylation, only some of which are needed for the majority of IRF7 tran-scriptional activity. In addition, the data suggest heterogeneity in theactivation process by distinct pathogen activators.

50CHARACTERIZATION OF THE 29-59-OLIGOADENYLATE SYN-THETASE UBIQUITIN-LIKE FAMILYSigne Eskildsen,1 Just Justesen,1 Mikkel Heide Schierup,2 and Rune Hartmann3

1Department of Molecular Biology, and 2Bioinformatics Research Cen-ter, University of Aarhus, Aarhus, Denmark. 3Department of Molecu-lar Cardiology, Lerner Research Institute, Cleveland Clinic Founda-tion, Cleveland, USA.

The 29-59-oligoadenylate synthetases (OAS) are interferon type I andtype II induced protein family important for the anti-viral activity ofinterferons. The human and murine OAS gene families each containfour genes: OAS1, OAS2, OAS3 and OASL, all having one or moreconserved OAS units composed of five translated exons. The OASLgene has both an OAS unit and a C-terminal of two ubiquitin-like re-peats. In humans a single OASL gene (hOASL) has been identified,whereas in mouse two OASL genes exist (mOasl1 and mOasl2). In thisstudy we demonstrate that murine Oasl1 protein is inactive while murineOasl2 is active as 29-59-oligoadenylate synthetase. Furthermore murineOasl2 requires double-stranded RNA as co-factor like the active humanand chicken proteins. The affinity of mOasl2 for the double-strandedRNA activator is higher than that of human OAS1 (p42 isoform). Wepropose a model for the evolutionary origin of the murine Oasl1 andOasl2 genes. We have identified a human OASL2 orthologue to themurine Oasl2 gene which has not previously been described. This es-tablishes that the OASL gene was duplicated prior to the radiation ofthe rodent and primate groups. Thus we suggest that murine Oasl2which has both enzymatic activity and an ubiquitin-like domain is afunctional intermediate between the active OAS species and the inac-tive human OASL1/murine Oasl1 proteins. In addition, we propose thatmurine Oasl1 appears to have gained a hitherto uncharacterized func-tion independent of 2-5A synthesis.

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