A haploid cell-based screen for the discovery of Andes Virus entry factors

用于发现安第斯病毒进入因子的基于单倍体细胞的筛选

• 批准号: 8668716
• 负责人: Kenneth Edward Briley
• 金额: $ 0.51万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668716
• 关键词: Address; Americas; Andes Virus; Antiviral Agents; Area; Binding; Candidate Disease Gene; Case Fatality Rates; Cell Line; Cells; Cessation of life; Classification; Complement; Consensus; Data; Defect; Development; Disease; Ebola virus; Endocytosis; Event; Family; Genes; Genetic; Genus Phlebovirus; Glycoproteins; Goals; Hantavirus; Hantavirus Infections; Hantavirus Pulmonary Syndrome; Haploid Cells; Haploidy; Hemorrhagic Fever with Renal Syndrome; Human; Human Cell Line; Infection; Insecta; Integration Host Factors; Invaded; Libraries; Licensing; Location; Mammalian Cell; Maps; Medical; Methodology; Molecular; Molecular Biology Techniques; Morbidity - disease rate; Nature; North America; Ontology; Orthobunyavirus; Pathway interactions; Plague; Population; Predisposition; Prevention; RNA Interference; Recombinants; Refractory; Research; Research Personnel; Resistance; Rodent; Sin Nombre virus; South America; Surface; Technology; Testing; Therapeutic; Transcription Coactivator; Translating; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Proteins; Virus; Virus Diseases; Work; base; clinical application; combat; deep sequencing; design; effective therapy; falls; insight; knockout gene; member; mortality; nuclease; pathogen; protein complex; public health relevance; receptor; screening; therapeutic target; tool; virus envelope

DESCRIPTION (provided by applicant): Hantaviruses are found throughout most of the Americas and are the causative agents of Hantavirus Pulmonary Syndrome (HPS). HPS has a case fatality rate of 40 percent and there is currently no cure for this disease or licensed vaccines for the prevention of hantavirus infections. The lack of an effective treatment for hantavirus infection is partially attributable to a poor understanding of how these viruses gain entry to their host cell. Thus, the aim of this research application is to dissect the molecular entry pathway of a representative Hantavirus, Andes virus (ANDV), with the broad goal of identifying potential therapeutic targets. This two year application is designed to carry forward a current human haploid screening project that has already identified several clonal cell lines differentially refractory to a replication competent recombinant vesicular stomatitis virus bearing only the ANDV envelope (rVSV:ANDV). These cell lines were cloned from an insertionally-mutagenized population of human haploid cells following a lethal selection with rVSV:ANDV and tested positive for wild type rVSV susceptibility. This indicated that the defects in infectivity wre determined by the ANDV protein envelope and likely at the point of entry. Aim 1 of this application is to identify and verify new human factors required for the entry of Andes Virus. This aim compiles the use of standard molecular biology techniques and cutting edge 454 deep sequencing technologies to map the location of genes required for Andes Virus entry. By ranking the number of independent insertions into known genes within these two populations, it has been possible to statistically identify genes important for viral entry. The importance of these genes will need to be validated, and the final portion of Aim 1 describes a strategy for creating expression knockdowns and knockouts of these genes to retest infectivity with wild type BSL-3 Andes Virus. Aim 2 will establish the point of defect along the Andes Virus entry pathway (binding, endocytosis, fusion), and test if other bunyaviruses utilize any of the identified host factors during their entry. Together, these aims serve to complete a body of research that will yield basic scientific data that may translate to clinical applications for the treatment of HPS an prevention of ANDV infection.

描述（由申请人提供）：在整个美洲的大多数地区都发现了汉坦病毒，并且是汉塔病毒肺综合征（HPS）的致病药物。 HPS的病例死亡率为40％，目前尚无治愈该疾病或获得持牌疫苗可预防汉坦病毒感染。缺乏有效的汉坦病毒感染治疗方法部分归因于对这些病毒如何进入其宿主细胞的不良理解。因此，该研究应用的目的是剖析代表性汉塔病毒（Andes病毒（ANDV））的分子进入途径，其广泛的目标是识别潜在的治疗靶标。这两年的申请旨在提出 当前的人类单倍筛查项目已经确定了几个克隆细胞系，将差异性固定性差异为复制，可复制的重组囊炎病毒病毒病毒轴承 仅ANDV信封（RVSV：ANDV）。这些细胞系从用RVSV进行致命的选择后，从插入的人类单倍体细胞的插入式植物群克隆：ANDV，对野生型RVSV敏感性呈阳性。这表明感染性缺陷wre由ANDV蛋白包膜确定，并且可能在进入点。本应用的目标1是识别和验证安第斯山脉病毒所需的新人为因素。这 AIM编译了标准分子生物学技术和尖端454深度测序技术的使用，以绘制安第斯山脉病毒进入所需的基因位置。通过对这两个人群中的独立插入到已知基因的数量进行排名，可以从统计上识别对病毒进入重要的基因。这些基因的重要性将需要得到验证，AIM 1的最后部分描述了一种创建表达敲低和敲除这些基因的策略，以用野生型BSL-3 Andes病毒重新造成感染。 AIM 2将在安第斯山脉病毒进入途径（结合，内吞作用，融合）上建立缺陷点，并测试其他Bunyaviruses在进入期间是否使用任何已鉴定的宿主因子。这些目的共同完成了一项研究，该研究将产生基本的科学数据，这些数据可能转化为治疗HPS预防ANDV感染的临床应用。