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

描述（由申请人提供）：汉坦病毒遍布美洲大部分地区，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，也没有预防汉坦病毒感染的许可疫苗。汉坦病毒感染缺乏有效治疗的部分原因是对这些病毒如何进入宿主细胞的理解不足。因此，本研究应用的目的是剖析代表性汉坦病毒，安第斯山脉病毒（ANDV）的分子进入途径，其广泛目标是鉴定潜在的治疗靶点。这项为期两年的申请旨在推进一项 目前的人类单倍体筛选项目已经鉴定了几种克隆细胞系，这些细胞系对携带有复制能力的重组水泡性口炎病毒的 只有ANDV包膜（rVSV：ANDV）。这些细胞系是在用rVSV：ANDV进行致死选择后，从插入诱变的人单倍体细胞群体克隆的，并对野生型rVSV易感性检测为阳性。这表明感染性的缺陷是由ANDV蛋白包膜决定的，可能是在进入点。本申请的目的1是识别和验证安第斯山脉病毒进入所需的新人为因素。这 aim利用标准的分子生物学技术和尖端的454深度测序技术，绘制了安第斯山脉病毒进入所需基因的位置。通过对这两个群体中已知基因中独立插入的数量进行排序，有可能在统计学上确定对病毒进入重要的基因。这些基因的重要性需要验证，目标1的最后部分描述了一种策略，用于产生这些基因的表达敲低和敲除，以重新测试野生型BSL-3安第斯山脉病毒的感染性。目标2将沿着安第斯山脉病毒进入途径（结合、内吞、融合）建立沿着缺陷点，并检测其他布尼亚病毒在进入过程中是否利用任何已鉴定的宿主因子。总之，这些目标有助于完成一系列研究，这些研究将产生基础科学数据，这些数据可能转化为治疗HPS和预防ANDV感染的临床应用。

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.