The Antiviral Activities of Human Interferons

人干扰素的抗病毒活性

• 批准号: 8157036
• 负责人: Kathryn Zoon
• 金额: $ 51.36万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157036
• 关键词: Antiviral Agents; Human Activities; Interferons; Antiviral Agents; Human Activities; Interferons

In summary, our novel MTT AV/AP assay is the first to allow examination of IFN AP and AV activities simultaneously on a suspension cell line. This assay is compatible with a broad range of IFNs and is applicable to different cell lines (e.g OVCAR-3). The microarray analysis of samples developed using the MTT AV/AP assay allowed identification of 25 genes associated with IFN AV activity; ten out of these 25 genes have not been previously reported as linked to AV activity of IFN. HSH2D is reported for the first time as a gene being upregulated in response to IFN treatment. IFIT3 was the most upregulated gene. The siRNA knock-down of IFIT3 results in increased sensitivity of A549 to two different viruses. Overexpression of IFIT3 in VERO cells led to a decrease in viral titer after infection. Thus, results of this study suggested IFIT3 as key element of IFN-alpha AV activity. This is also the first study identifying AV-associated genes of the Daudi cell line treated with IFN-alpha. Both IFN alpha and gamma signal through the Jak/STAT pathway in order to elicit antiviral activity, yet IFN-gamma is thought to do so only through STAT1 homodimers while type-I IFNs activate both STAT1- and STAT2-containing complexes such as ISGF3 (composed of STAT1, STAT2, and IRF9). Gene expression microarray analysis following IFN-gamma treatment for 24h indicated an induction of antiviral genes (e.g. MxA, PKR, and OAS1) that are induced by ISGF3 (via ISRE promoter sequences) and associated with a type-1 IFN response. Induction of these genes by autocrine type-I and type-III IFN signaling was ruled out using both neutralizing antibodies to these IFNs in biological assays and qRT-PCR. Despite the absence of type-I or type-III IFNs, IFN-gamma treatment induced ISGF3 formation and ISRE binding, as shown by STAT2 co-immunoprecipitation and ChIP analysis of the PKR promoter. STAT2 and IRF9 knockdown in A549 cells reversed IFN-γ-mediated ISRE induction and antiviral activity. This suggests that ISGF3 formation is a significant component of the cellular response and biological activity of IFN-gamma. We have shown that the DV capsid (C) protein is able to reduce Huh7 cells ability to mount an effective response to infection with Vesicular stomatitis virus (VSV). Huh7 cells were stably transfected with an expression vector containing the DV C gene (Huh7-C). Induction of RIG-I/MDA5 pathways by treatment of cells with poly (I:C) complexed with lipofectamine resulted in significant reduction of viral titers in Huh7 cells, but not in Huh7-C cells. After induction of RIG-I/MDA5, there were no observable differences in activation of IRF-3, NF-kappa B or c-Jun transcription factors between Huh7 and Huh7-C. However, there was a significant reduction of interferon-beta transcription in Huh7-C cells compared to control Huh7 cells. Treatment of Huh7 or Huh7-C cells with interferon-beta did not result in differences in phosphorlyation of STAT1 or in transcription of type I interferon stimulated genes MX1 or OAS1. Pretreatment of cells with interferon-beta did not result in any difference in viral titers between Huh7 and Huh7-C cells after infection with VSV. The DENV C protein is likely involved in evasion of host antiviral response by disrupting the induction of type I interferon by an as yet undetermined mechanism.

总而言之，我们的新型MTT AV/AP分析是第一个允许在悬浮细胞系上同时检查IFN AP和AV活动的方法。该测定与广泛的IFN兼容，适用于不同的细胞系（例如ovcar-3）。使用MTT AV/AP测定法进行了对样品的微阵列分析允许鉴定与IFN AV活性相关的25个基因。以前没有报道这25个基因中的十个与IFN的AV活性有关。首次报道了HSH2D，因为响应于IFN治疗，基因被上调。 IFIT3是最上调的基因。 IFIT3的siRNA敲除导致A549对两种不同病毒的敏感性提高。 Vero细胞中IFIT3的过表达导致感染后病毒滴度减少。因此，这项研究的结果表明IFIT3是IFN-Alpha AV活性的关键要素。这也是第一项研究，鉴定了用IFN-Alpha处理的Daudi细胞系的AV相关基因。 通过JAK/Stat途径发出IFN Alpha和Gamma信号，以激发抗病毒活性，但是，IFN-Gamma仅通过STAT1同型二聚体来进行IFN-GAMMA，而I型IFNS则激活了STAT1-和STAT2的复合物，例如ISGF3（例如STAT1，STAT1，Stat2和IRF9）。 IFN-GAMMA处理后24小时进行的基因表达微阵列分析表明，由ISGF3（通过ISRE启动子序列）诱导并与1型IFN响应相关联的抗病毒基因（例如MXA，PKR和OAS1）。 使用对生物测定和QRT-PCR中这些IFN的两种中和抗体排除了通过自分泌I型和III III型IFN信号传导诱导这些基因的。 尽管没有I型或III型IFN，但IFN-GAMMA处理仍诱导ISGF3形成和ISRE结合，如STAT2共免疫沉淀和PKR启动子的芯片分析所示。 A549细胞中的STAT2和IRF9敲低反转IFN-γ介导的ISRE诱导和抗病毒活性。 这表明ISGF3的形成是IFN-GAMMA细胞反应和生物学活性的重要组成部分。 我们已经表明，DV CAPSID（C）蛋白能够降低HuH7细胞对用囊泡口腔炎病毒（VSV）进行有效反应的有效反应的能力。 用含有DV C基因（HUH7-C）的表达载体稳定转染HuH7细胞。 通过用与Lipofectamine复合的聚（I：C）处理细胞的细胞来诱导RIG-I/MDA5途径，从而导致HUH7细胞中的病毒滴度显着降低，但在HUH7-C细胞中却没有显着降低。 诱导RIG-I/MDA5后，HUH7和HUH7-C之间的IRF-3，NF-KAPPA B或C-JUN转录因子的激活没有明显的差异。 然而，与对照HuH7细胞相比，HUH7-C细胞中的干扰素β转录显着降低。 用干扰素β对HUH7或HUH7-C细胞的处理不会导致STAT1的磷光磷酸化或I型干扰素刺激基因MX1或OAS1的转录差异。 用干扰素β对细胞进行预处理并没有导致Huh7和Huh7-C细胞在感染VSV后的病毒滴度差异。 DENV C蛋白可能通过通过尚未确定的机制破坏I型干扰素的诱导来逃避宿主抗病毒反应。