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

描述（由申请人提供）：尽管急性和慢性病毒感染仍然是主要的卫生保健负担，但治疗选择是有限的。一些最令人担忧的病毒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，通常是致命的。对所有病毒的早期防御是由先天免疫系统和模式识别受体引发的反应介导的，模式识别受体诱导细胞因子和趋化因子，这些细胞因子和趋化因子协调了大部分局部和全身的抗病毒防御。然而，除了这些明确定义的先天免疫信号通路外，鲜为人知的细胞自主因子也有助于保护宿主免受病毒感染。基于新出现的数据和我们的初步研究，我们假设宿主抵抗病毒的一个重要策略是“限制性因子”的上调，这些因子可以阻止病毒进入，限制病毒在宿主细胞中感染或复制的能力，或者增加细胞抵御病毒诱导的细胞病变的能力。然而，尽管这些限制因子可能为治疗干预提供了丰富的新靶点，但这些细胞自主宿主因子的身份在很大程度上是未知的。在此，我们提出利用并进一步优化一种利用转座子诱变的新型无偏筛选方法。该策略依赖于三个步骤：(i) piggyBac转座子诱变以生成诱变细胞文库，（ii）选择对病毒诱导的细胞死亡具有抗性的突变克隆，以及（iii）测序以确定转座子插入位点和有助于保护突变细胞免受选择剂影响的候选基因。在本提案的R21阶段，我们的目标是进一步发展和利用这种方法来鉴定抗病毒限制因子。此外，在R33阶段，我们建议使用天然病毒验证我们的新宿主靶点，并修改转座子系统以用于药物发现。

DESCRIPTION (provided by applicant): 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 nfect 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. PUBLIC HEALTH RELEVANCE: Viral infections remain a major health care burden but the therapeutic options are limited. Some of the most concerning of the viral infections include those caused by the Filoviruses and the Arenaviruses. Here we propose to develop and use a novel unbiased screening approach involving transposon mutagenesis to identify new host-targets that protect from infection with viruses. Our long-term goal is to use this to help identify new therapeutic options