Interconnectivity between genome packagaing and other viral functions

基因组包装与其他病毒功能之间的相互联系

• 批准号: 7642618
• 负责人: A L Rao
• 金额: $ 18.73万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7642618
• 关键词: Address; Adopted; Agrobacterium; Alphavirus; Animal Hepatitis; Animals; Binding Sites; Biochemical; Brome mosaic virus; Capsid Proteins; Cells; Cellular biology; Cis-Acting Sequence; Complex; Coupled; Cucumber mosaic virus; Disease; Electron Microscopy; Endoplasmic Reticulum; Engineering; Event; Foundations; Genetic Transcription; Genome; Genome Components; Glean; Housing; Human; Immunogold Technics; Insecta; Knowledge; Laboratories; Life Cycle Stages; Mediating; Membrane; Messenger RNA; Mitochondria; Molecular; Oral cavity; Organism; Plant Viruses; Plants; Play; Poliomyelitis; Principal Investigator; Process; RNA; RNA Viruses; Replicon; Research; Research Proposals; Role; Series; Signal Transduction; Silver; Site; Solid; Specificity; System; Testing; Translating; Translations; United States National Institutes of Health; Venezuelan Equine Encephalitis Virus; Viral; Viral Genome; Viral Physiology; Virion; Virus; Virus Assembly; Virus Diseases; Work; base; design; foot; human disease; improved; in vivo; infancy; interdisciplinary approach; intimate behavior; mitochondrial membrane; novel; programs; promoter; public health relevance; replicase; research study

DESCRIPTION (provided by applicant): Information gleaned from recent studies with single-stranded, positive-sense RNA viruses pathogenic to humans and animals (polio and alphaviruses) and insects (flock house virus; FHV) revealed that the mechanism of genome packaging in these viral systems is functionally coupled to replication. Recently our laboratory adopted a novel in vivo system referred to as Agrobacterium-mediated transient expression (agroinfiltration) to study encapsidation in plants. This system not only allowed efficient expression of viral genome components either autonomously or synchronously in plant cells, but also effectively uncoupled replication from packaging. Application of the agroinfiltration system to brome mosaic virus (BMV, a plant infecting RNA virus) allowed us to hypothesize that packaging in BMV is also functionally coupled to replication. In addition, co-expression of BMV and FHV in plant cells using agroinfiltration revealed that for specific RNA packaging to occur, synchronization of replication and transcription of coat protein (CP) mRNAs from homologous replication machinery is obligatory. This two-year exploratory project is designed to evaluate, at the sub-cellular level, the intimacy of replication to packaging. An agroinfiltration system competent to synchronously infect the same plant cell with BMV and FHV will be used through out these studies. Our working hypothesis is that translation of CP followed by virus assembly occurs very close to the sites of viral replication. Thus in Aim 1, we propose to temporally and sequentially localize and identify the sub-cellular compartment(s) where translation of CP and virus assembly of BMV and FHV occurs. In addition to the molecular and biochemical characterization, delineation of CP translation and virus assembly sites at the sub-cellular level will be investigated by electron microscopy using a novel Silver Enhancement-Controlled Sequential Immunogold technique (SECSI). BMV and FHV differentially replicate on the outer membranes of endoplasmic reticulum (ER) and mitochondria respectively. We found that packaging is non-specific when BMV CP or FHV CP was expressed either transiently or via heterologous replication. Thus, experiments outlined in Aim 2 are focused in addressing, for the packaging specificity occur, whether viral progeny RNA need to be tethered to the same membrane near which it's CP is being actively synthesized. This will be investigated by retargeting the FHV replicase complex to the ER, where the synthesis of FHV CP from genetically engineered BMV RNA will be synchronized. At the completion of the project we should know whether translation of CP and assembly of virions occur at or near the replication sites and whether tethering of viral progeny RNA to the same membrane near which it's CP is being actively synthesized is obligatory to confer packaging specificity. Results obtained from this research proposal would improve our understanding concerning the mechanism of replication-coupled packaging in RNA viruses pathogenic to humans, animals and plants. PUBLIC HEALTH RELEVANCE: RNA viruses cause serious diseases in humans, animals and crop plants of agronomic importance. Since spread of these viruses between respective host organisms is dependent of formation of infectious virions, our proposed research aims will employ a multidisciplinary approach in unraveling the mechanisms that regulate the assembly process.

描述（由申请人提供）：从最近对人类和动物的单链，阳性RNA病毒致病的研究中收集的信息（脊髓灰质炎和α病毒）和昆虫（羊群家用病毒； FHV）揭示了这些病毒系统中基因组包装的机制在这些病毒系统中有效地促进了复制。最近，我们的实验室采用了一种新型的体内系统，称为农杆菌介导的瞬态表达（Agroinflatration）来研究植物中的封装。该系统不仅允许在植物细胞中自主或同步对病毒基因组成分的有效表达，而且还可以有效地从包装中取消复制。将农业浸润系统应用于Brome Mosaic病毒（BMV，一种感染RNA病毒），使我们能够假设BMV中的包装在功能上也与复制耦合。此外，使用AgroInfortration在植物细胞中对BMV和FHV的共表达表明，要发生特定的RNA包装，从同源复制机制中复制和转录的复制和转录同步是必须的。这个为期两年的探索性项目旨在评估复制对包装的亲密关系。在这些研究中，将使用有能力同步感染同一植物细胞的农业泄露系统。我们的工作假设是，CP的翻译随后是病毒组装的转换非常接近病毒复制部位。因此，在AIM 1中，我们建议在临时和依次定位并确定BMV和FHV的CP和病毒组装的翻译。除了分子和生化表征外，通过电子显微镜使用一种新型的银色增强控制的顺序免疫元技术（SECSI）研究了亚细胞水平的CP翻译和病毒组装位点的描述。 BMV和FHV分别对内质网（ER）和线粒体的外膜进行差异化复制。我们发现，当BMV CP或FHV CP瞬时或通过异源复制表示时，包装是非特异性的。因此，AIM 2中概述的实验集中在解决包装特异性的情况下，是否需要将病毒后代RNA束缚在积极合成其CP附近的同一膜上。这将通过将FHV复制酶复合物重新定位到ER进行研究，在该ER中，将同步从基因工程的BMV RNA中合成FHV CP。项目完成时，我们应该知道CP的翻译和病毒粒子的组装是否发生在复制位置或附近，以及将病毒后代RNA的束缚在积极合成的CP附近的同一膜上是否允许赋予包装特异性。从这项研究提案中获得的结果将提高我们对RNA病毒复制偶联包装机制的理解，以使人，动物和植物致病。公共卫生相关性：RNA病毒在人类，动物和作物植物中引起严重的疾病，具有农艺重要性。由于这些病毒在各个宿主生物之间的传播取决于传染病的形成，因此我们提出的研究目标将采用多学科方法来揭示调节装配过程的机制。