Identification and characterization of the antiviral mode of action of interferon-stimulated genes

i型干扰素（ifn）是细胞在病毒感染时产生的主要先天免疫信使。它们触发宿主基因子集的转录上调，称为干扰素刺激基因（ISGs）。数百个表达的isg组成了一道坚固的防线。干扰素用于治疗，但会引起严重的副作用。更好地理解多层ISG势垒对于更有针对性地操纵ISG效应函数至关重要。目前，数百个isg基本上仍未被描述。先前旨在鉴定抗病毒ISG效应物的研究主要集中在病毒感染的早期阶段。因此，抑制病毒感染后期阶段（组装、输出或颗粒感染性）的抗病毒isg的潜在库尚未确定。我提出了一个研究ISG效应函数的项目。首先，我将描述ISG DDX60的抗病毒功能，它最近被证明可以抑制丙型肝炎病毒（HCV）。基于与已知功能蛋白的序列同源性，我推测DDX60可能抑制病毒蛋白翻译或破坏病毒RNA稳定性。为了确定DDX60抑制HCV的机制，我建议进行一些功能分析，包括功能基序的绘制和病毒生命周期的研究。其次，我计划确定特异性抑制病毒生命周期后期阶段的isg。我将修改目前的ISG功能获得试验，并利用先前发表的ISG筛选中的300+ ISG来鉴定甲型流感病毒、丙型肝炎病毒、西尼罗河病毒、登革热病毒和黄热病病毒的抑制剂。我希望这次筛选的结果能够对抑制这些病毒生命周期后期的isg进行并排比较，从而深入了解病毒输出机制的异同。

Type-I interferons (IFNs) are major innate immune messengers that are produced by cells upon viral infection. They trigger transcriptional upregulation of a subset of host genes called interferon stimulated genes (ISGs). The concert of hundreds of expressed ISGs builds a robust line of defense. IFNs are used therapeutically but cause severe side effects. A better understanding of the multi-layered ISG barrier is crucial to allow manipulation of ISG effector function in a more targeted manner. Currently, several hundred ISGs remain largely uncharacterized. Previous studies that aimed to identify antiviral ISG effectors have focused predominantly on early stages of viral infection. As a result, a potential pool of antiviral ISGs that inhibit late stages of virus infection (assembly, egress or particle infectivity) has yet to be identified. I propose a project to investigate ISG effector functions. First, I will characterize the antiviral function of the ISG DDX60, recently shown to inhibit hepatitis C virus (HCV). Based on sequence homology to proteins with known function, I hypothesize that DDX60 might inhibit viral protein translation or undermine viral RNA stability. I propose to perform a number of functional assays, including mapping of functional motifs and viral life cycle studies in order to define the mechanism by which DDX60 inhibits HCV. Second, I plan to identify ISGs that specifically inhibit late stages in virus lifecycles. I will modify a current ISG gain-of-function assay and utilize the 300+ ISGs from a previously published ISG screen to identify inhibitors of influenza A virus, HCV, West Nile virus, Dengue virus, and yellow fever virus. I expect that the results from this screen will enable side-by-side comparison of ISGs that inhibit late lifecycle stages of each of these viruses, providing insight into similarities and differences in viral egress mechanisms.