HSV-1 US11 MEDIATED EVASION OF HOST SHUTOFF

HSV-1 US11 介导的主机关闭规避

• 批准号: 2885691
• 负责人: Kevin A Cassady
• 金额: $ 8.66万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2885691
• 关键词: cell line; chromatography; gel electrophoresis; herpes simplex virus 1; host organism interaction; immunoprecipitation; molecular pathology; protein biosynthesis; protein kinase; protein structure function; virus genetics; virus infection mechanism; virus protein; yeast two hybrid system

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.

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并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，并开展剖析病毒感染和人类疾病的分子和遗传基础的研究。 指导本次培训的导师是单纯疱疹病毒学专家。 候选人的职业目标是成为一名儿科医生科学家，提供对病毒发病机制的见解，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：特别是病毒如何逃避宿主内在和免疫防御系统。该提案研究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，即干扰素诱导蛋白激酶（PKR）。 研究发现，第二个位点突变 (US11) 侧翼的 HSV 基因的动力学表达变化有助于这些病毒恢复致病性。 这些研究并没有解决一个显着的悖论：US11蛋白的早期合成使得病毒能够逃避PKR，但病毒携带的预制蛋白却无效。该提议的工作假设是，虽然 PKR 抑制的这些功能差异可能反映了预制 US11 和合成 US11 之间固有的生化差异，但这更有可能反映了合成 US11 募集辅助感染细胞蛋白的相对能力。生化技术（色谱、2-D 电泳、体内磷酸化）将用于分离和分析病毒体相关和合成的 US11。 随后将使用 PKR 体外激酶测定来测试功能差异。 使用生物化学（免疫沉淀、蛋白质亲和力）和遗传方法（酵母两种杂交系统）的亲和力研究将评估 US11 或 PKR 是否招募参与修改 PKR 途径的感染细胞蛋白。体外 PKR 激酶测定将测试已识别蛋白质的功能意义。 最后，将通过创建表达 US11 的细胞系并用无毒病毒鉴定基因并检查表型变化来评估生物功能。