Molecular Basis-Paramyxovirus-induced STAT Degradation

描述（由申请方提供）：I型干扰素（IFN）是主要的抗病毒细胞因子，通过阻断病毒复制直接作用于靶细胞。IFN信号转导产生转录复合物ISGF 3，其由DNA结合亚基与来自信号转导子和转录激活子（STAT）家族的两种蛋白质STAT 1和STAT 2结合组成。ISGF3是细胞IFN应答的主要效应子。IFN信号在抗病毒反应中的重要性通过病毒进化以逃避IFN作用的各种策略来强调。病毒逃避机制通常涉及对抗病毒酶，其代表治疗干预和合理药物设计的重要潜在靶点。 负链RNA病毒的副粘病毒科家族包括几种众所周知的人类病原体，如麻疹、腮腺炎、呼吸道合胞病毒和人副流感病毒。PI自己的实验室和其他实验室的最新发现表明，副粘病毒的一个子集可以通过靶向ISGF3的STAT蛋白组分进行蛋白水解降解来逃避IFN抗病毒应答。这种STAT蛋白降解是由编码V蛋白的单个病毒基因的表达介导的。两种不同的副粘病毒，猴病毒5型（SV5）和人副流感病毒2型（HPIV 2），通过靶向ISGF 3转录复合物来逃避IFN，但SV5 V蛋白介导STAT 1的破坏，HPIV 2 V蛋白介导STAT 2的破坏。将研究V蛋白诱导的STAT识别和降解的特异性由离散蛋白片段介导以及V蛋白必须争取细胞蛋白水解机制以靶向特定STAT蛋白进行蛋白水解的假设。嵌合V蛋白和STAT蛋白将用于降解试验，以确定V蛋白介导的STAT靶向中选择性和特异性的分子基础。将在哺乳动物细胞和体外泛素化测定中直接检查V蛋白诱导的自身修饰和STAT靶标通过泛素和类似配体的修饰。GST融合蛋白亲和层析和免疫亲和纯化策略将用于定义参与该反应的细胞机制。总之，这些实验将揭示副粘病毒蛋白靶向和降解STAT蛋白并逃避IFN作用的机制和细胞装置。

DESCRIPTION (provided by applicant): 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.