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PS2-60 Biological characterization of feline interferon-NU: a new member of the Type I interferon family
Abstracts / Cytokine 52 (2010) 50–67 for MCPyV in plasma was measured by an in-house EIA; 10 subjects were seropositive and 10 seronegative (Chen et al, submitted). Results: Formation of icosahedral capsids was demonstrated by electron microscopy. Significantly increased MCVPy-specific interferon-gamma secretion and proliferation responses were detected from peripheral blood mononuclear cell cultures of seropositive healthy adults as compared to seronegative. Conclusions: Initial findings suggest that MCVPy VLPs are the potent inducer of cellular immunity and may serve as an important tool for the evaluation of CD4+ T-cell immune responses to this virus. Frequent detection of IFN-c among seropositive subjects also indicates that VLPs may be a vaccine candidate. However further investigations are needed in order to explore the other markers of adaptive immunity for this newly discovered virus. doi:10.1016/j.cyto.2010.07.264
PS2-60 Biological characterization of feline interferon-NU: A new member of the Type I interferon family Steven Carbone, Sidney Pestka, Ronald G. Jubin, PBL InterferonSource, Piscataway, NJ, USA Type I interferons are a family of related proteins, including interferon (IFN) alpha, beta, delta, epsilon, kappa, omega and tau, where all utilize a common receptor complex. Functionally, each of these proteins exhibits antiviral activity and a range of other bioactivities. Recent genomic sequence analysis identified an undiscovered putative IFN denoted IFN-nu. While the gene was shown to be conserved in several vertebrate species including humans, chimpanzee, dogs and cats, most of the IFNnu genes contained an internal stop codon rendering them pseudogenes. Only in the feline was IFN-nu maintained as a full-length gene. To assess the bioactivity of this gene, an antiviral assay was established using Vesicular Stomatitis Virus (VSV) challenge of feline lung AK-D cells. Using the species-promiscuous human IFN protein fusion termed Universal Interferon as a positive control, direct viral challenge of AK-D cells transfected with IFN-nu expression plasmid exhibited viral protection compared to cells transfected with the vector control. In addition, de novo expression of IFN-nu was observed in VSV-infected AK-D cells. In conclusion, the coding sequence for feline IFN-nu yielded antiviral activity when expressed in feline cells providing the first biological evidence that IFN-nu exhibits the proper hallmark bioactivity. doi:10.1016/j.cyto.2010.07.265
PS2-61 Transcriptional regulation of miR-155 by IRFs in antiviral immunity and viral tumors Ling Wang, Shunbin Ning, Department of Medicine, Miller School of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida 33136, USA miR-155, processed from the B cell integration cluster (BIC), is one of the few well studied miRNAs that is known to be involved in both innate immunity and tumorigenesis. This miRNA is induced by distinct signaling pathways triggered by Toll-like receptors, interferons, TNFa, B-cell receptor engagement, as well as by the Epstein-Barr virus (EBV) principal oncoprotein LMP1. However, very little is known on the underlying mechanisms of its induction or regulation. Many of these signaling pathways have been shown to activate specific interferon regulatory factors (IRFs). Thus, IRFs may be involved in the regulation of miR-155 expression. In support of this hypothesis, we have identified two potential IRF-binding/ IFN-responsive motifs (ISRE and IRFE) in the human miR-155 promoter. EMSA results have shown that IRFs can indeed bind to the synthesized ISRE and IRFE elements derived from the miR-155 promoter. Promoter-reporter assay results show that IRF1 and 3 are two potent transactivators which may be responsible for induction of miR-155 in antiviral responses. More interestingly, IRF4 and 7, two oncogenic IRFs, also significantly transactivate the miR-155 promoter but with much lower capacities compared with IRF1 and 3. Correspondingly, expression of IRF4 and 7 are significantly correlated with BIC transcript in EBV-transformed cells, and knockdown of IRF4 by RNA interference in type III EBVinfected KR4 lymphoblastic cell line decreases the endogenous level of BIC mRNA. These data suggest that IRF4 and 7 contribute to miR-155 overexpression in EBVtransformed cells. Since miR-155 is a potent oncogenic miRNA, modest induction of miR-155 by oncogenic IRFs in the tumor context may help to avoid excessive cell transformation. Further study will focus on the regulation of miR-155 by IRF4 and 7 in EBV- and HTLV1-associated lymphomas, and by IRF1 and 3 in antiviral immunity. Findings from this paramount study will provide evidence, for the first time, for a novel molecular mechanism underlying the IRFs/miR-155 pathway in both viral oncogenesis and innate immune responses. It may provide the basis for identification of molecular targets for therapeutic interventions for virus-associated tumors and diseases. (Supported by Florida Bankhead-Coley Cancer Research Program. Grant 1BN07-34186). doi:10.1016/j.cyto.2010.07.266
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PS2-62 Differential STAT targeting by paramyxovirus V proteins Andy Schroeder, Curt Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA Cells detect viral invaders by recognizing any of several pathogen-associated molecular patterns, such as dsRNA. Recognition of a viral attack induces production of type I interferons (IFNs). These cytokines are released and upon binding to nearby cells, induce Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascades that lead to expression of antiviral genes. The paramyxovirus family encodes an immune-evasion V protein that allows the virus to suppress the innate immune response. This is often accomplished by degrading STATs via the formation of an E3 ubiquitin ligase known as a V-dependent degradation complex (VDC). Different paramyxoviruses target distinct STATs to suppress the immune response. PIV5 specifically targets STAT1, while HPIV2 degrades STAT2. The V protein of mumps virus is unique for its ability to target both STAT1 and STAT3 by assembling two distinct VDC. Recently, a mutant version of the mumps V protein was designed with an aspartic acid-to-glutamic acid substitution at residue 95. This mutation impairs the V protein’s ability to degrade STAT3, though STAT1 degradation is intact. This newly engineered virus shows that degradation of STAT1 and STAT3 can be separated and studied independently. A critical question remains as to what cellular components are required for and allow for the differential targeting of STAT1 and STAT3. By elucidating the constituents of the mumps VDC in comparison with VDC assembled by other STAT-degrading V proteins we will be able to further understand the interaction between invading paramyxoviruses and the host immune response. doi:10.1016/j.cyto.2010.07.267
PS2-63 A shared interface mediates paramyxovirus interference with antiviral RNA helicases MDA5 and LGP2 Jean-Patrick Parisien 1, Darja Bamming 1, Akihiko Komuro 1, Aparna Ramachandran 1, Jason J. Rodriguez 1, Glen Barber 2, Robert D. Wojahn 1, Curt M. Horvath 1, 1 Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA, 2 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida USA Diverse members of the Paramyxovirus family of negative-strand RNA viruses effectively suppress host innate immune responses through the actions of their V proteins. The V protein mediates interference with the interferon regulatory RNA helicase MDA5 to avoid cellular antiviral responses. Analysis of the interaction interface revealed the MDA5 helicase C domain as necessary and sufficient for association with V proteins from human parainfluenza virus type 2, parainfluenza virus type 5, measles virus, mumps virus, Hendra virus, and Nipah virus. The identified approximately 130-residue region is highly homologous between MDA5 and the related antiviral helicase LGP2, but not RIG-I. Results indicate that the paramyxovirus V proteins can also associate with LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins. doi:10.1016/j.cyto.2010.07.268
PS2-64 Phosphorylations of the NFAR proteins constitute a novel, conserved mechanism of translational regulation and cellular defense Ai Harashima, Toumy Guettouche, Glen N. Barber, Department of Medicine and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA We have isolated and characterized two proteins nuclear factors associated with dsRNA1 and 2 (NFAR1 and NFAR2), which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. Two such products were found to be related, spliced variants encoding major products of 90 and 110 kDa, respectively. These were transcribed from a single human gene on chromosome 19. Studies have demonstrated that loss of the NFARs leads to an increase in protein synthesis rates, through enhancing mRNP nuclear export, indicating that the NFARs may be negative regulators of this process. RNAi knockdown of NFARs renders MEFs susceptible to VSV (vesicular stomatitis virus) infection, suggesting a putative role for the NFARs in cellular host defense by mechanisms that remain to be clarified. Here, we demonstrate that NFAR1 and NFAR2 are bona fide substrates for the interferon-inducible dsRNA activated protein kinase, PKR which are phosphorylated on two residues, in vitro and in vivo, in response to virus infection, an event found to be evolutionarily conserved in Xenopus laevis. Phosphorylation of NFARs was specific for PKR since no phosphorylation was observed in PKR-/- cell or mice. NFAR phosphorylation caused
PS2-60 Biological characterization of feline interferon-NU: A new member of the Type I interferon family Steven Carbone, Sidney Pestka, Ronald G. Jubin, PBL InterferonSource, Piscataway, NJ, USA Type I interferons are a family of related proteins, including interferon (IFN) alpha, beta, delta, epsilon, kappa, omega and tau, where all utilize a common receptor complex. Functionally, each of these proteins exhibits antiviral activity and a range of other bioactivities. Recent genomic sequence analysis identified an undiscovered putative IFN denoted IFN-nu. While the gene was shown to be conserved in several vertebrate species including humans, chimpanzee, dogs and cats, most of the IFNnu genes contained an internal stop codon rendering them pseudogenes. Only in the feline was IFN-nu maintained as a full-length gene. To assess the bioactivity of this gene, an antiviral assay was established using Vesicular Stomatitis Virus (VSV) challenge of feline lung AK-D cells. Using the species-promiscuous human IFN protein fusion termed Universal Interferon as a positive control, direct viral challenge of AK-D cells transfected with IFN-nu expression plasmid exhibited viral protection compared to cells transfected with the vector control. In addition, de novo expression of IFN-nu was observed in VSV-infected AK-D cells. In conclusion, the coding sequence for feline IFN-nu yielded antiviral activity when expressed in feline cells providing the first biological evidence that IFN-nu exhibits the proper hallmark bioactivity. doi:10.1016/j.cyto.2010.07.265
PS2-61 Transcriptional regulation of miR-155 by IRFs in antiviral immunity and viral tumors Ling Wang, Shunbin Ning, Department of Medicine, Miller School of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida 33136, USA miR-155, processed from the B cell integration cluster (BIC), is one of the few well studied miRNAs that is known to be involved in both innate immunity and tumorigenesis. This miRNA is induced by distinct signaling pathways triggered by Toll-like receptors, interferons, TNFa, B-cell receptor engagement, as well as by the Epstein-Barr virus (EBV) principal oncoprotein LMP1. However, very little is known on the underlying mechanisms of its induction or regulation. Many of these signaling pathways have been shown to activate specific interferon regulatory factors (IRFs). Thus, IRFs may be involved in the regulation of miR-155 expression. In support of this hypothesis, we have identified two potential IRF-binding/ IFN-responsive motifs (ISRE and IRFE) in the human miR-155 promoter. EMSA results have shown that IRFs can indeed bind to the synthesized ISRE and IRFE elements derived from the miR-155 promoter. Promoter-reporter assay results show that IRF1 and 3 are two potent transactivators which may be responsible for induction of miR-155 in antiviral responses. More interestingly, IRF4 and 7, two oncogenic IRFs, also significantly transactivate the miR-155 promoter but with much lower capacities compared with IRF1 and 3. Correspondingly, expression of IRF4 and 7 are significantly correlated with BIC transcript in EBV-transformed cells, and knockdown of IRF4 by RNA interference in type III EBVinfected KR4 lymphoblastic cell line decreases the endogenous level of BIC mRNA. These data suggest that IRF4 and 7 contribute to miR-155 overexpression in EBVtransformed cells. Since miR-155 is a potent oncogenic miRNA, modest induction of miR-155 by oncogenic IRFs in the tumor context may help to avoid excessive cell transformation. Further study will focus on the regulation of miR-155 by IRF4 and 7 in EBV- and HTLV1-associated lymphomas, and by IRF1 and 3 in antiviral immunity. Findings from this paramount study will provide evidence, for the first time, for a novel molecular mechanism underlying the IRFs/miR-155 pathway in both viral oncogenesis and innate immune responses. It may provide the basis for identification of molecular targets for therapeutic interventions for virus-associated tumors and diseases. (Supported by Florida Bankhead-Coley Cancer Research Program. Grant 1BN07-34186). doi:10.1016/j.cyto.2010.07.266
63
PS2-62 Differential STAT targeting by paramyxovirus V proteins Andy Schroeder, Curt Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA Cells detect viral invaders by recognizing any of several pathogen-associated molecular patterns, such as dsRNA. Recognition of a viral attack induces production of type I interferons (IFNs). These cytokines are released and upon binding to nearby cells, induce Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascades that lead to expression of antiviral genes. The paramyxovirus family encodes an immune-evasion V protein that allows the virus to suppress the innate immune response. This is often accomplished by degrading STATs via the formation of an E3 ubiquitin ligase known as a V-dependent degradation complex (VDC). Different paramyxoviruses target distinct STATs to suppress the immune response. PIV5 specifically targets STAT1, while HPIV2 degrades STAT2. The V protein of mumps virus is unique for its ability to target both STAT1 and STAT3 by assembling two distinct VDC. Recently, a mutant version of the mumps V protein was designed with an aspartic acid-to-glutamic acid substitution at residue 95. This mutation impairs the V protein’s ability to degrade STAT3, though STAT1 degradation is intact. This newly engineered virus shows that degradation of STAT1 and STAT3 can be separated and studied independently. A critical question remains as to what cellular components are required for and allow for the differential targeting of STAT1 and STAT3. By elucidating the constituents of the mumps VDC in comparison with VDC assembled by other STAT-degrading V proteins we will be able to further understand the interaction between invading paramyxoviruses and the host immune response. doi:10.1016/j.cyto.2010.07.267
PS2-63 A shared interface mediates paramyxovirus interference with antiviral RNA helicases MDA5 and LGP2 Jean-Patrick Parisien 1, Darja Bamming 1, Akihiko Komuro 1, Aparna Ramachandran 1, Jason J. Rodriguez 1, Glen Barber 2, Robert D. Wojahn 1, Curt M. Horvath 1, 1 Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA, 2 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida USA Diverse members of the Paramyxovirus family of negative-strand RNA viruses effectively suppress host innate immune responses through the actions of their V proteins. The V protein mediates interference with the interferon regulatory RNA helicase MDA5 to avoid cellular antiviral responses. Analysis of the interaction interface revealed the MDA5 helicase C domain as necessary and sufficient for association with V proteins from human parainfluenza virus type 2, parainfluenza virus type 5, measles virus, mumps virus, Hendra virus, and Nipah virus. The identified approximately 130-residue region is highly homologous between MDA5 and the related antiviral helicase LGP2, but not RIG-I. Results indicate that the paramyxovirus V proteins can also associate with LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins. doi:10.1016/j.cyto.2010.07.268
PS2-64 Phosphorylations of the NFAR proteins constitute a novel, conserved mechanism of translational regulation and cellular defense Ai Harashima, Toumy Guettouche, Glen N. Barber, Department of Medicine and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA We have isolated and characterized two proteins nuclear factors associated with dsRNA1 and 2 (NFAR1 and NFAR2), which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. Two such products were found to be related, spliced variants encoding major products of 90 and 110 kDa, respectively. These were transcribed from a single human gene on chromosome 19. Studies have demonstrated that loss of the NFARs leads to an increase in protein synthesis rates, through enhancing mRNP nuclear export, indicating that the NFARs may be negative regulators of this process. RNAi knockdown of NFARs renders MEFs susceptible to VSV (vesicular stomatitis virus) infection, suggesting a putative role for the NFARs in cellular host defense by mechanisms that remain to be clarified. Here, we demonstrate that NFAR1 and NFAR2 are bona fide substrates for the interferon-inducible dsRNA activated protein kinase, PKR which are phosphorylated on two residues, in vitro and in vivo, in response to virus infection, an event found to be evolutionarily conserved in Xenopus laevis. Phosphorylation of NFARs was specific for PKR since no phosphorylation was observed in PKR-/- cell or mice. NFAR phosphorylation caused