Protein determinants of deltaretrovirus RNA packaging

δ逆转录病毒RNA包装的蛋白质决定因素

基本信息

批准号：
7364594
负责人：
Louis M Mansky
金额：
$ 10.66万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7364594
关键词：
Address Adult Amino Acids Animal Model Animals Anti-Retroviral Agents Area Basic Amino Acids Binding Biological Models Birds Bovine Leukemia Virus Bovine Lymphoma Bovine leukemia Cancer Etiology Categories Cattle Cells Chronic Lymphocytic Leukemia Clonality Coculture Techniques Cultured Cells DNA Integration DNA Packaging Data Defect Deltaretrovirus Disruption Educational process of instructing Ensure Family Gagging Gene Expression Genes Genetic Genetic Transcription Genus Capra Goals Goat Homologous Gene Human Human T-lymphotropic virus 1 Human T-lymphotropic virus 2 In Vitro Independent Scientist Award Infection Life Cycle Stages Lymphocyte Lymphoma Malignant Neoplasms Maps Molecular Mutation Nucleocapsid Nucleocapsid Proteins Oncogenes Oncogenic Prevalence Prevention Productivity Proteins RNA RNA Viruses Research Research Personnel Retroviridae Role Science Sheep Signal Transduction Structure Surveys T-Cell Leukemia Tertiary Protein Structure Testing Time Tropical Spastic Paraparesis Vaccines Viral Viral Genome Viral Packaging Viral Reverse Transcription Virion Virus Virus Diseases Virus-like particle Wages beef career cell transformation falls gag Gene Products human disease insight leukemia virus leukemia/lymphoma member mortality mutant neoplastic cell nervous system disorder novel prevent programs stem tax Genes viral DNA viral RNA

项目摘要

DESCRIPTION (provided by applicant): The candidate's long-term career goal is to maintain a strong research program in an area of importance to the biomedical sciences. To date, the candidate's research has made a substantial contribution to the understanding of retrovirus RNA packaging. The salary support provided by an Independent Scientist Award (K02) would relieve the candidate of a significant amount of his teaching load as well as provide relief from administrative duties. This relief would ensure that 75 to 80% of the candidate's time is free for research. The additional research time made available by this Independent Scientist Award will have an immediate and positive effect on the candidate's research productivity. The present proposal extends research being conducted by the candidate to address the protein determinants involved in deltaretrovirus RNA packaging. The members of the deltaretroviruses, which include human T-cell leukemia viruses type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukemia virus (BLV), are medically important because they cause human cancers that cannot be cured and have no vaccines for prevention. The deltaretroviruses replicate to low titers in their natural hosts and are poorly infectious in cell culture. Cocultivation is typically used to infect permissive host cells. Because of these difficulties, information regarding the molecular details of their life cycles, including viral RNA packaging, is limited. The objectives of this proposal are to map the determinants in the Gag polyprotein that are involved in deltaretrovirus RNA packaging. Two observations have been made with deltaretroviruses (specifically BLV) which indicate that the mechanism of RNA packaging is unique among retroviruses. First, the primary packaging signal is located exclusively in the gag gene and not the 5' untranslated leader region. Second, the matrix protein domain of Gag binds specifically to BLV RNA overlapping the primary packaging signal and mutation of basic amino acid residues in the matrix domain can influence RNA packaging efficiencies. In this proposal, genetic approaches will be employed using a newly developed virus-like particle (VLP) model system in order to identify the amino acid residues and/or protein domains in Gag that are involved in RNA packaging. This proposal has 3 specific aims. Specific Aim 1 will determine if the BLV matrix (MA) and nucleocapsid (NC) protein domains of Gag act cooperatively in viral RNA packaging. This aim will test the hypothesis that combination of MA and NC mutations can act in an additive manner to decrease viral RNA packaging efficiencies. Specific Aim 2 will determine the interplay between RNA packaging defects caused by mutations in the BLV MA or NC and disruptions of the stable RNA stem-loop structures that represent the BLV packaging signal. This aim will test the hypothesis that combination of MA or NC mutants with mutations that disrupt RNA stem-loop structures of the RNA packaging signal will aid in associating RNA packaging defects caused by MA or NC protein mutants to RNA stem-loop structures. Specific Aim 3 will determine if both the HTLV MA and NC domains are involved in viral RNA packaging. This aim will test the hypothesis that mutations in the HTLV MA and NC protein domains will correlate with RNA packaging defects. Completion of the aims in this proposal will provide further insight into HTLV/BLV RNA packaging, a group of retroviruses that package viral RNA by novel interactions with the Gag polyprotein precursor. This information should be useful in developing antiretroviral strategies to block deltaretrovirus replication and to prevent retrovirus-induced cancer.

描述（由申请人提供）：候选人的长期职业目标是在生物医学科学的重要领域维持强大的研究计划。迄今为止，该候选人的研究为理解逆转录病毒 RNA 包装做出了重大贡献。独立科学家奖（K02）提供的薪资支持将减轻候选人的大量教学负担，并减轻行政职责。这一减免将确保候选人 75% 到 80% 的时间可以自由用于研究。该独立科学家奖提供的额外研究时间将对候选人的研究生产力产生直接和积极的影响。目前的提案扩展了候选人正在进行的研究，以解决 deltaretrovirus RNA 包装中涉及的蛋白质决定因素。 δ逆转录病毒的成员包括人类 T 细胞白血病病毒 1 型和 2 型（HTLV-1 和 HTLV-2）以及牛白血病病毒 (BLV)，它们在医学上具有重要意义，因为它们会导致无法治愈的人类癌症，并且无法治愈。疫苗用于预防。 δ逆转录病毒在其自然宿主中复制的滴度较低，并且在细胞培养物中感染性很差。共培养通常用于感染许可的宿主细胞。由于这些困难，有关其生命周期的分子细节（包括病毒 RNA 包装）的信息有限。该提案的目的是绘制参与 deltaretrovirus RNA 包装的 Gag 多蛋白中的决定簇。对 delta 逆转录病毒（特别是 BLV）进行了两项观察，这表明 RNA 包装机制在逆转录病毒中是独特的。首先，初级包装信号仅位于 gag 基因中，而不是 5' 非翻译前导区。其次，Gag 的基质蛋白结构域与 BLV RNA 特异性结合，与初级包装信号重叠，基质结构域中碱性氨基酸残基的突变会影响 RNA 包装效率。在该提案中，将采用新开发的病毒样颗粒（VLP）模型系统的遗传方法来识别 Gag 中参与 RNA 包装的氨基酸残基和/或蛋白质结构域。该提案有 3 个具体目标。具体目标 1 将确定 Gag 的 BLV 基质 (MA) 和核衣壳 (NC) 蛋白结构域是否在病毒 RNA 包装中协同作用。这一目标将检验以下假设：MA 和 NC 突变的组合可以以累加的方式起作用，从而降低病毒 RNA 包装效率。具体目标 2 将确定 BLV MA 或 NC 突变引起的 RNA 包装缺陷与代表 BLV 包装信号的稳定 RNA 茎环结构破坏之间的相互作用。该目标将检验以下假设：MA 或 NC 突变体与破坏 RNA 包装信号的 RNA 茎环结构的突变的组合将有助于将 MA 或 NC 蛋白突变体引起的 RNA 包装缺陷与 RNA 茎环结构相关联。具体目标 3 将确定 HTLV MA 和 NC 结构域是否都参与病毒 RNA 包装。这一目标将检验 HTLV MA 和 NC 蛋白结构域突变与 RNA 包装缺陷相关的假设。该提案中目标的完成将为 HTLV/BLV RNA 包装提供进一步的见解，HTLV/BLV RNA 包装是一组逆转录病毒，通过与 Gag 多蛋白前体的新型相互作用来包装病毒 RNA。这些信息应该有助于制定抗逆转录病毒策略，以阻止δ逆转录病毒复制并预防逆转录病毒诱发的癌症。