The Antiviral Activities of Human Interferons

综上所述，我们的新型四甲基偶氮唑蓝AV/AP法是第一个允许在悬浮细胞系上同时检测干扰素AP和AV活性的方法。该方法适用于多种不同的细胞株(如OVCAR-3)。利用MTTAV/AP试验对样品进行微阵列分析，可以鉴定出25个与干扰素AV活性相关的基因；这25个基因中有10个以前没有报道过与干扰素的AV活性有关。HSH2D首次被报道为一种基因在干扰素治疗后上调。IFIT3是最上调的基因。IFIT3的siRNA下调导致A549对两种不同病毒的敏感性增加。在Vero细胞中过表达IFIT3导致感染后病毒滴度下降。因此，这项研究的结果表明IFIT3是干扰素-αAV活性的关键元件。这也是第一次鉴定经干扰素-α处理的Daudi细胞系的AV相关基因。 干扰素α和伽马信号都通过JAK/STAT途径来诱导抗病毒活性，然而干扰素-伽马被认为只通过STAT1同源二聚体实现，而I型IFN激活含有STAT1和STAT2的复合体，如ISGF3(由STAT1、STAT2和IRF9组成)。基因表达芯片分析表明，干扰素-γ处理24小时后，诱导了抗病毒基因(如mxA、PKR和OAS1)，这些基因由ISGF3(通过ISRE启动子序列)诱导并与1型干扰素应答相关。在生物学检测和qRT-PCR中，使用针对这些IFN的中和抗体排除了由I型和III型自分泌干扰素信号诱导这些基因的可能性。STAT2免疫共沉淀和PKR启动子芯片分析显示，尽管没有I型或III型IFN，但干扰素-γ处理诱导了ISGF3的形成和ISRE结合。A549细胞中的STAT2和IRF9基因敲除逆转了干扰素介导的ISRE诱导和抗病毒活性。这表明ISGF3的形成是干扰素-γ细胞反应和生物活性的重要组成部分。 我们已经证明DV衣壳(C)蛋白能够降低Huh7细胞对水疱性口炎病毒(VSV)感染的有效反应的能力。将含有DV C基因的表达载体(Huh7-C)稳定地导入Huh7细胞。用聚(I：C)与脂质体复合处理细胞，诱导RIG-I/MDA5通路，可显著降低Huh7细胞中的病毒滴度，但对Huh7-C细胞中病毒滴度无明显影响。Rig-I/MDA5诱导后，Huh7和Huh7-C对IRF-3、NF-kappa B和c-jun转录因子的激活无明显差异。然而，在Huh7-C细胞中，与对照Huh7细胞相比，干扰素-β转录显著降低。用β干扰素处理Huh7或Huh7-C细胞并没有导致STAT1的磷酸化或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，在感染VSV后，Huh7和Huh7-C细胞之间的病毒滴度没有任何差异。DENV C蛋白可能通过干扰一种尚不确定的机制诱导I型干扰素来逃避宿主的抗病毒反应。

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.