Type I interferons (IFN) are the principal antiviral cytokines and function directly on target cells by blocking virus replication. IFN signal transduction produces a transcriptional complex, ISGF3, that is composed of a DNA binding subunit in association with two proteins from the signal transducer and activator of transcription (STAT) family, STAT1 and STAT2. ISGF3 is the main effector of cellular IFN responses. The importance of IFN signaling in antiviral responses is underscored by the wide variety of strategies that viruses have evolved to evade IFN actions. The viral evasion mechanisms typically involve antagonism of antiviral enzymes that represent important potential targets for therapeutic intervention and rational drug design. ? ? The Paramyxoviridae family of negative-strand RNA viruses includes several well known human pathogens like measles, mumps, respiratory syncytial, and human parainfluenza viruses. Recent findings from the PI's own lab and others indicate that a subset of paramyxoviruses can evade IFN antiviral responses by targeting the STAT protein components of ISGF3 for proteolytic degradation. This STAT protein degradation is mediated by expression of a single viral gene coding for the V protein. Two different paramyxoviruses, simian virus 5 (SV5) and human parainfluenza virus type 2 (HPIV2), evade IFN by targeting the ISGF3 transcription complex, but while the SV5 V protein mediates destruction of STAT1, the HPIV2 V protein mediates destruction of STAT2. The hypothesis that the specificity of V protein-induced STAT recognition and degradation is mediated by discrete protein segments and that V proteins must enlist cellular proteolytic machinery to target specific STAT proteins for proteolysis will be investigated. Chimeric V proteins and STAT proteins will be used in degradation assays to determine the molecular basis for selectivity and specificity in the V protein mediated STAT targeting. V protein-induced modifications of themselves and the STAT targets by ubiquitin and similar ligands will be directly examined in mammalian cells and in vitro ubiquitination assays. GST fusion protein affinity chromatography and immuno-affinity purification strategies will be used to define the cellular machinery involved in this reaction. Together, these experiments will reveal the mechanisms and cellular apparatus used by the paramyxovirus proteins to target and degrade STAT proteins and evade IFN actions.
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