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

描述(申请人提供)：汉坦病毒在美洲大部分地区都有发现，是汉坦病毒肺综合征(HPS)的病原体。HPS的病死率为40%，目前还没有治愈这种疾病的方法，也没有获得许可的疫苗来预防汉坦病毒感染。汉坦病毒感染缺乏有效的治疗方法，部分原因是对这些病毒如何进入宿主细胞缺乏了解。因此，这项研究应用的目的是剖析具有代表性的汉坦病毒-安第斯病毒(ANDV)的分子进入途径，以确定潜在的治疗靶点。这项为期两年的申请旨在推进 目前的人类单倍体筛选计划已经确定了几个对复制能力强的重组水泡性口炎病毒具有不同抵抗力的克隆细胞系 仅ANDV信封(rVSV：ANDV)。这些细胞系是从插入突变的人类单倍体细胞群中克隆出来的，经过rVSV：ANDV的致死选择，并经野生型rVSV敏感性检测呈阳性。这表明感染性缺陷是由ANDV蛋白包膜决定的，很可能是在入口点。这项申请的目的1是确定和核实安第斯病毒进入所需的新的人为因素。这 AIM汇编了标准分子生物学技术和尖端454深度测序技术的使用，以绘制安第斯病毒进入所需基因的位置。通过对这两个群体中已知基因的独立插入数量进行排序，就有可能在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这些基因的表达敲除和敲除的策略，以重新测试野生型BSL-3和安第斯病毒的传染性。目的2将确定沿安第斯病毒进入途径(结合、内吞、融合)的缺陷点，并测试其他布尼亚病毒在进入过程中是否利用任何已确定的宿主因子。总而言之，这些目标有助于完成一系列研究，这些研究将产生基本的科学数据，这些数据可能会转化为HPS的临床应用，预防ANDV感染。

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.