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的致死率为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。缺乏一个有效的治疗感染汉坦病毒部分归因于一个贫穷的了解这些病毒进入宿主细胞。因此，本研究申请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为广泛目标。这个为期两年的应用程序旨在延续一个

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.