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

描述(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，但治疗选择有限。一些最令人担忧的病毒感染包括由丝状病毒和阿雷纳病毒引起的病毒感染。这些RNA病毒会导致严重的出血热，通常是致命的。对所有病毒的早期防御是通过先天免疫系统和模式识别受体触发的反应来调节的，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系统抗病毒防御。然而，除了这些定义明确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。根据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上调限制因子，这些限制因子阻止病毒进入宿主细胞，限制病毒在宿主细胞中的感染或复制能力，或增加细胞抵御病毒诱导的细胞病变的能力。然而，尽管这些限制因素可能为治疗干预提供大量新的靶点，但这些保护病毒的细胞自主宿主因素的身份在很大程度上尚不清楚。在这里，我们建议利用并进一步优化一种使用转座子突变的新的无偏筛选方法。这一策略依赖于三个步骤：(I)猪Bac转座子突变以产生诱变细胞库，(Ii)选择抗病毒诱导的细胞死亡的突变克隆和(Iii)测序以确定有助于保护突变细胞免受选择剂攻击的转座子插入位点和候选基因。在这项提案的R21阶段，我们的目标是进一步开发和利用这种方法来识别抗病毒限制因子。此外，在R33阶段，我们建议使用本地病毒验证我们的新宿主靶标，以及修改转座子系统以用于药物发现。 公共卫生相关性：病毒感染仍然是主要的卫生保健负担，但治疗选择有限。一些最令人担忧的病毒感染包括由丝状病毒和阿雷纳病毒引起的病毒感染。在这里，我们建议开发和使用一种涉及转座子突变的新的无偏筛选方法来识别新的宿主靶标，以保护其免受病毒感染。我们的长期目标是利用这一点来帮助确定新的治疗方案

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