Novel antiviral activities of tumor necrosis factor-alpha

描述（由申请人提供）：肿瘤坏死因子-a（TNF 1）具有有效的直接抗病毒活性，但由于其全身毒性作用，尚未用作抗病毒治疗。我们已经观察到用低水平的TNF 1处理的原代人肝细胞和肝癌细胞在体外对丙型肝炎病毒（HCV）的感染具有抗性。这种效应依赖于TNF 1与其受体的相互作用，并且在缺乏可检测的IFN 1或IFN 2基因表达的情况下观察到。辛德毕斯病毒是一种甲病毒，感染Huh-7肝癌细胞，对TNF 1预处理不敏感。然而，TNF 1与IFN 2协同作用以阻断辛德毕斯病毒感染。我们建议使用最先进的技术来鉴定TNF 1单独和与IFN 2组合的抗病毒作用的关键介质。我们提出三个具体目标。在第一个目标中，我们将使用药理学抑制剂以及TNF 1信号转导的显性负调节剂，通过TNF 1与其受体结合激活的主要途径来测试信号转导的要求。在第二个目标中，我们将使用新的病毒学和细胞生物学工具来定义TNF 1治疗后抑制的HCV感染阶段。在第三个目标中，我们将使用微阵列和下一代测序方法来确定TNF 1对转录组的影响，确定可能介导TNF 1处理细胞中抗病毒状态发展的候选基因，并通过敲低和过表达研究来测试这些基因的重要性。这项研究的长期目标是确定介导TNF 1抗病毒活性的特定细胞途径，并确定是否有可能将这些活性与TNF 1的全身毒性作用分离开来。

DESCRIPTION (provided by applicant): Tumor necrosis factor-a (TNF1) has potent, direct antiviral activity but has not been exploited as an antiviral therapy because of its systemic toxic effects. We have observed that primary human liver cells and hepatoma cells treated with low levels of TNF1 are resistant to infection with the hepatitis C virus (HCV) in vitro. This effect is dependent on TNF1 interaction with its receptor and is seen in the absence of detectable IFN1 or IFN2 gene expression. Sindbis virus, an alphavirus, infects Huh-7 hepatoma cells and is insensitive to TNF1 pretreatment. However, TNF1 synergizes with IFN2 to block Sindbis virus infection. We propose to use state- of-the-art techniques to identify key mediators of TNF1's antiviral effects alone and in combination with IFN2. We propose three Specific Aims. In the first Aim, we will test the requirements for signal transduction through the major pathways activated by TNF1 binding to its receptor, using pharmacologic inhibitors as well as dominant negative regulators of TNF1 signal transduction. In the second Aim, we will use novel virological and cell biological tools to define the stages in HCV infection that are inhibited following TNF1 treatment. In the third Aim, we will use microarray and next-generation sequencing approaches to define the effects of TNF1 on the transcriptome, identify candidate genes that may mediate the development of an antiviral state in TNF1- treated cells, and test the importance of these genes by knockdown and overexpression studies. The long-term goals of this research are to identify specific cellular pathways that mediate TNF1's antiviral activity, and to determine whether it is possible to isolate these activities from TNF1's systemic toxic effects.