HSV-1 US11 MEDIATED EVASION OF HOST SHUTOFF

指导临床科学家发展奖将为申请人提供扩展分子病毒学强化培训和发展蛋白质生物化学和细胞生物学专业知识的机会。这些技能将使申请人成为一名完全独立的研究科学家，并从事解剖病毒感染和人类疾病的分子和遗传基础的研究。指导这次培训的导师是单纯疱疹病毒学方面的专家。候选人的职业目标是成为一名儿科医生-科学家，为病毒发病机制提供见解，最终将改善病毒感染患者的治疗和管理决策。申请人的研究兴趣集中在病毒发病机制的分子和遗传基础上：具体来说，病毒如何逃避内在和免疫宿主防御系统。本研究探讨了无毒疱疹病毒的第二位点突变如何使后代重新获得其致病潜力，并逃避细胞内宿主防御系统——干扰素诱导蛋白激酶（PKR）。研究已经确定，第二位点突变（US11）旁的HSV基因动力学表达的变化有助于这些病毒的新致病性。这些研究没有解决一个重要的悖论：US11蛋白的早期合成使病毒能够逃避PKR，但与病毒一起携带的预制蛋白是无效的。本提案的工作假设是，虽然这些PKR抑制的功能差异可能反映了预制和合成的US11之间内在的生化差异，但这更可能反映了合成的US11招募附属感染细胞蛋白的相对能力。生化技术（色谱、二维电泳、体内磷酸化）将用于分离和分析病毒粒子相关和合成的US11。随后将使用PKR体外激酶测定法进行功能差异测试。使用生化（免疫沉淀，蛋白质亲和）和遗传方法（酵母双杂交系统）的亲和研究将评估US11或PKR是否招募参与修饰PKR通路的感染细胞蛋白。体外PKR激酶试验将测试鉴定的蛋白质的功能意义。最后，生物学功能将通过创建表达US11和鉴定基因的细胞系与无毒病毒和检查表型变化来评估。

The Mentored Clinical Scientist Development Award will provide the opportunity to extend the applicant's intensive molecular virology training and develop an expertise in protein biochemistry and cellular biology. These skills will enable the applicant to become a fully independent research scientist and to address studies that dissect the molecular and genetic basis of viral infection and human disease. The mentor who will direct this training is an expert in herpes simplex virology. The candidate's career objective is to become a pediatrician- scientist who provides insights into viral pathogenesis which ultimately will improve the therapeutic and management decisions for patients with viral infections. Research interests of the applicant focus on the molecular and genetic basis for viral pathogenesis: specifically, how viruses evade intrinsic and immune host defense systems. This proposal examines how second-site mutations in avirulent herpesviruses enable progeny to reacquire their pathogenic potential and evade an intracellular host defense system, the interferon induced protein kinase (PKR). Studies have identified that a change in the kinetic expression of an HSV gene flanking the second site mutations (US11) contributes to the renewed pathogenicity of these viruses. These studies have not resolved a significant paradox: the early synthesis of US11 protein enables viral evasion of PKR but pre-made protein carried in with the virus is ineffective. The working hypothesis of this proposal is that while these functional differences in PKR inhibition may reflect inherent biochemical differences between the pre-made and synthesized US11, it is more likely that this reflects the relative ability of synthesized US11 to recruit accessory infected cell proteins. Biochemical techniques (chromatography, 2-D electrophoresis, in vivo phosphorylation) will be used to isolate and analyze virion-associated and synthesized US11. This will be followed by tests for functional differences using a PKR in vitro kinase assay. Affinity studies using both biochemical (immunoprecipitation, protein affinity) and genetic methods (yeast two hybrid system) will evaluate if US11 or PKR recruit participating infected cell proteins that modify the PKR pathway. An in vitro PKR kinase assay will test the functional significance of the identified proteins. Finally, biologic function will be evaluated by creating a cell line expressing the US11 and identified gene with an avirulent virus and examining for phenotypic changes.