Interferon Action and Protein Phosphorylation

干扰素作用和蛋白质磷酸化

基本信息

批准号：
7781640
负责人：
Charles E Samuel
金额：
$ 35.6万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7781640
关键词：
5&apos Flanking Region Adenoviruses Affect Apoptosis Apoptotic Binding Binding Sites Biochemical Biological Biological Assay Biological Process Cells Cellular Tropism Characteristics Chimeric Proteins Clinic Clone Cells Complement Complex Conserved Sequence Cultured Cells DNA DNA Binding DNase-I Footprinting Dependence Double-Stranded RNA E3L protein Elements Engineering Fishes Gel Gene Expression Gene Silencing Genes Genetic Transcription Growth Health Hela Cells Human Human Cell Line Human Characteristics IRF3 gene Immune response Interferons Investigation Maps Measles virus Mediating Messenger RNA Molecular Mus Mutagenesis Normal Cell Nucleotides Oncolytic Phenotype Phosphorylation Phosphotransferases Positioning Attribute Production Property Protein Binding Protein Engineering Proteins RNA RNA Recognition Motif RNA-Binding Proteins Regulation Relative (related person)Research Response Elements Role Signal Transduction Site Stimulus Structure Therapeutic Tissues Trans-Activators Vaccines Vaccinia virus Viral Virus Virus Diseases Virus Replication adapter protein animal tissue base cDNA Expression cell growth eIF-2 Kinase insight mutant novel promoter protein activation protein function public health relevance research study response stem

5&apos Flanking Region Adenoviruses Affect Apoptosis Apoptotic Binding Binding Sites Biochemical Biological Biological Assay Biological Process Cells Cellular Tropism Characteristics Chimeric Proteins Clinic Clone Cells Complement Complex Conserved Sequence Cultured Cells DNA DNA Binding DNase-I Footprinting Dependence Double-Stranded RNA E3L protein Elements Engineering Fishes Gel Gene Expression Gene Silencing Genes Genetic Transcription Growth Health Hela Cells Human Human Cell Line Human Characteristics IRF3 gene Immune response Interferons Investigation Maps Measles virus Mediating Messenger RNA Molecular Mus Mutagenesis Normal Cell Nucleotides Oncolytic Phenotype Phosphorylation Phosphotransferases Positioning Attribute Production Property Protein Binding Protein Engineering Proteins RNA RNA Recognition Motif RNA-Binding Proteins Regulation Relative (related person)Research Response Elements Role Signal Transduction Site Stimulus Structure Therapeutic Tissues Trans-Activators Vaccines Vaccinia virus Viral Virus Virus Diseases Virus Replication adapter protein animal tissue base cDNA Expression cell growth eIF-2 Kinase insight mutant novel promoter protein activation protein function public health relevance research study response stem

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项目摘要

DESCRIPTION (provided by applicant): The OVERALL OBJECTIVE of the proposed investigation is to elucidate the roles of the interferon-inducible RNA-regulated protein kinase (PKR) in the actions that interferons (IFN) mediate on viral and host functions. The three SPECIFIC AIMS of our proposed continuation studies are as follows: (1) To further delineate the roles of the PKR protein in the host responses to virus infection. To characterize the function of the PKR protein in human cells in culture, through the use of established HeLa, U, and Huh cell clones that are stably deficient in PKR because of targeted gene silencing compared to appropriate knockdown-control cell clones. These human cell clones will be examined for the mechanisms by which PKR affects virus replication, with emphasis on vaccinia virus (wild-type and E3L mutant), and measles virus (wild- type, V and C mutants) and adenovirus (VAI mutant), viruses with human cell tropism, and for the cellular apoptotic and IFN inducing phenotypes in response to different stimuli of the knockdown compared to control cell clones. (2) To elucidate the biochemical mechanism by which the PKR protein confers the PKR-dependent biological phenotypes characterized under aim 1. To attempt to determine the biochemical functions of the PKR protein (RNA binding activity; catalytic activity; PKR domain) necessary to complement the biological phenotypes (including apoptosis, virus growth and IFN production) and biochemical changes characteristic of the human PKR knockdown cells, using wild-type and mutant forms of cDNA expression constructs for the human PKR protein engineered to circumvent knockdown, and the mouse PKR and fish PKZ proteins. (3) To further delineate the structure of the 5'-flanking region of the Pkr gene and identity of trans-acting factors required for interferon-inducible as well as basal transcriptional activity. To map the major Pkr transcription sites, using RNA from uninfected and infected and untreated and IFN-treated cells and tissues, and to elucidate the basis of the tissue-specific differences in Pkr mRNA size multiplicity. To delineate the importance of Sp1 and Sp3 protein binding to the novel 15-bp DNA element and upstream sites in IFN inducible as well as basal transcriptional activity. PUBLIC HEALTH RELEVANCE: The health relatedness and relevance of the proposed research stems from the likelihood that the studies may contribute to a better understanding of regulatory mechanisms operative in normal cells as well as virus- infected cells, including cells infected with oncolytic or vaccine strain viruses for therapeutic or preventative strategies. Elucidation of the actions of interferon at the molecular level is of immediate importance in view of the use of interferon in the clinic.

描述（由申请人提供）：拟议研究的总体目标是阐明干扰素诱导的RNA调节蛋白激酶（PKR）在干扰（IFN）介导病毒和宿主功能的作用中的作用。我们提出的延续研究的三个特定目的如下：（1）进一步描述PKR蛋白在宿主对病毒感染的反应中的作用。通过使用已建立的HELA，U和HUH细胞克隆来表征PKR蛋白在培养物中的功能，这些克隆在PKR中稳定缺乏，因为与适当的敲低控制细胞克隆相比，靶向基因沉默。将检查这些人类细胞克隆的pKR影响病毒复制的机制，重点是疫苗病毒（野生型和E3L突变体），以及麻疹病毒（野生型，v和c突变体）和腺病毒（VAI突变体），与人类细胞抗体的反应相同的病毒，以及Infortip intuce of Human apopti intuce of the Munder Apopti，IFNING APOPITIT量与对照细胞克隆相比，敲低。（2）阐明PKR蛋白通过目标1的特征于PKR蛋白赋予PKR依赖性的生物学表型的生化机制，以确定PKR蛋白（RNA结合活性；催化活性； PKR域； PKR域）的生物化学功能，以补充生物学的生长（包括Apoptes of Apoptis），以补充PKR蛋白（RNA结合活性； PKR结构型）所必需的生物化学功能。人类PKR敲低细胞，使用野生型和突变形式的cDNA表达构建体的人类PKR蛋白设计为绕过敲低的人，以及小鼠PKR和FISH PKZ蛋白。（3）进一步描绘了PKR基因的5'-频式区域的结构以及干扰素诱导和基础转录活性所需的反式作用因子的身份。绘制主要的PKR转录位点，使用未感染和感染和未经处理和IFN处理的细胞和组织的RNA，并阐明PKR mRNA大小多重性的组织特异性差异的基础。为了描述SP1和SP3蛋白与新型15 bp DNA元素的重要性以及在IFN诱导和基础转录活性中的上游位点的重要性。公共卫生相关性：拟议研究的健康相关性和相关性源于研究可能有助于更好地理解正常细胞中在正常细胞中起作用的调节机制以及感染病毒感染的细胞，包括感染癌或疫苗菌株的细胞，用于治疗或预防策略。鉴于在诊所中使用干扰素，阐明干扰素在分子水平上的作用至关重要。