Transposon Mutagenesis for Host-Target and Drug Discovery in Infectious Disease

虽然急性和慢性病毒感染仍然是一个主要的卫生保健 负担，治疗选择有限。一些最令人担忧的病毒式传播 感染包括由丝状病毒和沙粒病毒引起的感染。这些RNA 病毒引起严重的出血热，常常是致命的。早期防御所有 病毒是由先天免疫系统介导的， 诱导细胞因子和趋化因子的模式识别受体， 局部和全身的抗病毒防御系统。但是，除了这些好的-- 明确的先天免疫信号通路，不太清楚的细胞自主 这些因子也有助于保护宿主免受病毒感染和在病毒感染期间保护宿主。基于 新兴的数据和我们的初步研究，我们假设，一个重要的战略， 宿主对病毒的抗性是“限制因子”的上调， 进入，限制病毒在宿主细胞中感染或复制的能力，或增加病毒在宿主细胞中感染或复制的能力。 抵抗病毒引起的细胞病变。尽管这些限制因素 可能为治疗干预提供大量新靶点， 这种保护免受病毒侵害的细胞自主宿主因子在很大程度上是未知的。 在这里，我们建议利用和进一步优化一种新的无偏筛选方法 使用转座子诱变。该策略依赖于三个步骤：（i）piggyBac转座子 诱变以产生诱变细胞文库，（ii）选择突变克隆 对病毒诱导的细胞死亡具有抗性，和（iii）测序以鉴定转座子插入 有助于保护突变细胞免受选择的位点和候选基因 剂在本提案的R21阶段，我们的目标是进一步开发和利用这一点， 用于鉴定抗病毒限制因子的方法。此外，在R33阶段 我们建议使用原生病毒验证我们的新主机目标，并修改 转座子系统用于药物发现。

Although both acute and chronic viral infections remain a major health care burden, the therapeutic options are limited. Some of the most concerning of the viral infections include those caused by the Filoviruses and the Arenaviruses. These RNA viruses cause severe hemorrhagic fevers that are often lethal. Early defense against all viruses is mediated by the innate immune system and the responses triggered by pattern recognition receptors that induce the cytokines and chemokines that orchestrate much of the local and systemic anti-viral defenses. However, in addition to these well- defined innate immune signaling pathways, less well-understood cell autonomous factors also contribute to protect the host from and during viral infection. Based on emerging data and our preliminary studies, we hypothesize that an important strategy of host resistance against viruses is the upregulation of `restriction factors' that prevent viral entry, restrict the ability of viruses to infect or replicate in host cells or increase the ability of cells to withstand viral-induced cytopathy. However, although these restriction factors are likely to provide a wealth of new targets for therapeutic intervention, the identities of such cell-autonomous host factors that protect against viruses are largely unknown. Here we propose to utilize and further optimize a novel unbiased screening approach using transposon mutagenesis. This strategy relies three steps: (i) piggyBac transposon mutagenesis to generate a library of mutagenized cells, (ii) selection of mutant clones resistant to viral-induced cell death and (iii) sequencing to identify transposon insertions sites and candidate genes that contribute to protect the mutant cells from the selection agent. In the R21 phase of this proposal we aim to further develop and utilize this approach for identification of antiviral restriction factors. Additionally, in the R33 phase we propose to both validate our new host targets using native virus as well as modify the transposon system for drug discovery.