Determining the functions of novel genes for influenza A and Ebola viruses

确定甲型流感病毒和埃博拉病毒新基因的功能

• 批准号: 8599188
• 负责人: YOSHIHIRO KAWAOKA
• 金额: $ 50.02万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8599188
• 关键词: Affect; Animal Viruses; Animals; Antiviral Agents; Apoptosis; Biochemical; Biochemistry; Biological Assay; Candidate Disease Gene; Categories; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Communicable Diseases; Communities; Complementary DNA; Confocal Microscopy; Coronavirus; DNA Viruses; Data; Development; Dose; Ebola virus; Ebola-like Viruses; Filovirus; Generations; Genes; Genome; Goals; Growth; Herpesviridae; Human; Human Herpesvirus 8; Hypersensitivity; Immune; Immune Sera; Immune response; Infection; Influenza; Influenza A Virus, H5N1 Subtype; Influenza A virus; Institutes; Interferons; Kinetics; Laboratories; Lethal Dose 50; Life Cycle Stages; Lung diseases; Microscopy; Morbidity - disease rate; Mus; Open Reading Frames; Organ; Pathogenesis; Pathogenicity; Pathway interactions; Proteins; Public Health; Publications; RNA; RNA Viruses; Reagent; Receptor Signaling; Regulation; Research; Research Project Grants; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Signal Transduction; Small RNA; Techniques; Testing; Therapeutic; Translating; Untranslated RNA; Viral; Viral Genes; Viral Hemorrhagic Fevers; Viral Proteins; Virus; Virus Diseases; Virus Replication; chemokine; cytokine; data management; experience; genetic manipulation; insight; killings; mortality; mouse model; mutant; novel; novel vaccines; overexpression; pandemic disease; pathogen; positional cloning; protein expression; response; therapeutic development; viral DNA; viral RNA; virus pathogenesis

Influenza A viruses (lAV) and Ebola viruses (EBOV) are negafive-sense RNA viruses that cause human infecfions associated with respiratory disease or hemorrhagic fever, respectively. Both viruses have small genomes, encoding only 11-12 (lAV) or 8 (EBOV) known viral proteins; however, the idenfification of novel proteins in recent years suggests that these viruses may encode additional uncharacterized genes that are transcribed or transcribed/translated, via non-conventional strategies. We hypothesize that these genes and their products likely contribute to the viral life cycle. Therefore, the goal of research project 2 (RP 2) is to identify and determine the functions of uncharacterized lAV and EBOV genes. In Aini 1, we will experimentally prove that hypothetical genes can be expressed from the genomes of lAV and EBOV at the protein level. For all uncharacterized gene categories; 1) proven hypothefical gene candidates, 2) unknown open reading frame candidates (i.e., viral proteins already known to be expressed but not yet functionally characterized), and 3) noncoding viral RNA candidates, we will assess the biochemical function of these uncharacterized gene candidates, including using apoptosis assays in our laboratory. Additional biochemical assays will be perfonned in RP 1 - Baric to examine the role of uncharacterized genes in interferon and tolllike receptor signaling and in RP 3 - Damania to examine the role of uncharacterized genes in inflammasome and p53 signaling. Collecfively, all three RPs will contribute various assays to determine the biochemical funcfion of the uncharacterized genes in this proposal. By using reverse genefics techniques developed in our laboratory for lAV and EBOV, we will generate mutant viruses that no longer encode the uncharacterized gene product. We will then compare the growth kinetics and host responses to infection of wild-type virus and mutant viruses in cell culture (Aim 2) and in a mouse model (Aim 3). In addition, we will perform confocal microscopy imagining (Aim 2) to determine the subcellular localization of the potentially novel lAV and EBOV gene products, and also of potentially novel SARS coronavirus (RP 1) and human herpesviruses-8 (RP 3) gene products. Such microscopy studies will aid in determining the function of these

甲型流感病毒（IAV）和埃博拉病毒（EBOV）是引起人类免疫缺陷病毒（HIV）的负义RNA病毒。 分别与呼吸道疾病或出血热相关的感染。两种病毒都有小 基因组，仅编码11 - 12（IAV）或8（EBOV）种已知病毒蛋白;然而，新的病毒蛋白的纯化是不可能的。 近年来，蛋白质的研究表明，这些病毒可能编码额外的未知基因， 转录或转录/翻译，通过非传统的策略。我们假设这些基因和 他们的产品可能有助于病毒的生命周期。因此，研究项目2（RP 2）的目标是 鉴定和确定未表征的IAV和EBOV基因的功能。在艾尼1号， 实验证明，假设的基因可以从lAV和EBOV的基因组中表达， 蛋白质水平对于所有未表征的基因类别; 1）已证实的假设候选基因，2）未知 开放阅读帧候选（即，已知已表达但尚未发挥功能的病毒蛋白 特征），和3）非编码病毒RNA候选物，我们将评估这些的生化功能， 未表征的候选基因，包括使用我们实验室的细胞凋亡测定。另外的生物化学 将在RP1-Baric中进行检测，以检查未表征的基因在干扰素和toll样蛋白中的作用。 受体信号传导和RP 3-Damania中，以检查未表征的基因在 炎性小体和p53信号转导。总的来说，所有三个RP都将提供各种检测来确定 在这个建议中的未表征的基因的生化功能。通过使用反向遗传学技术 在我们的实验室开发的lAV和EBOV，我们将产生突变病毒，不再编码 未鉴定的基因产物然后，我们将比较生长动力学和宿主对感染的反应， 野生型病毒和突变型病毒在细胞培养物（Aim 2）和小鼠模型（Aim 3）中。此外，我们会 进行共聚焦显微镜成像（目的2），以确定潜在的亚细胞定位 新的IAV和EBOV基因产物，以及潜在的新的SARS冠状病毒（RP 1）和人 疱疹病毒8型（RP 3）基因产物。这样的显微镜研究将有助于确定这些功能，