Mechanisms of Antiviral Action of Human Interferon-alpha

1.我们测量了干扰素和干扰素刺激的基因产生，以响应不同病毒的细胞感染，以了解在诱导某些干扰素亚型时激活的不同信号调节器。我们发现，一组亚型仅在干扰素受体刺激后诱导，而另一组亚型仅在模式识别受体刺激后诱导。我们还发现，根据病毒浓度的不同，诱导出不同的干扰素刺激基因集。 2.我们的研究表明，未知蛋白FlJ11286(Flj)是由登革热病毒感染以干扰素依赖的方式诱导的，并在抗病毒应答中发挥作用。Flj全长291aa，含有保守的半胱氨酸残基，在脊椎动物分类群中具有同源物。在干扰素的刺激下，Flj表达上调，而通过siRNA介导的干扰素调节因子9的下调，干扰素信号的中断导致干扰素治疗后Flj的诱导减少。Flj具有抗DENV和脑心肌炎病毒的活性。我们还证明了Flj是一种核酸结合蛋白，具有与登革热病毒mRNA结合的能力，并与多种登革热和宿主蛋白有关。这些数据证明flj是一个新的干扰素刺激基因，具有抗DENV活性。 3.通过检测IFIT3对水泡性口炎病毒(VSV)、脑心肌炎病毒(EMCV)和登革病毒(DV)病毒滴度的影响，探讨IFIT3在抗病毒反应中的作用。我们的数据显示，IFIT3的存在会导致病毒滴度的下降。虽然确切的机制尚不清楚，但IFIT3对几种不同的病毒显示出明显的抗病毒效果。 4.利巴韦林增强干扰素-α2的抗病毒活性：干扰素和利巴韦林联合治疗病毒感染是成功的，但这些药物如何共同作用的作用机制尚不清楚。为了验证这一点，我们利用水泡性口炎病毒(VSV)感染A549人肺上皮细胞作为实验模型。与未处理的细胞相比，利巴韦林单独或与干扰素联合处理的细胞在24小时VSV滴度下降了4-log10。我们发现，与单独使用干扰素相比，pSTAT1和pSTAT2的激活增加且持续时间延长，总的STAT1、STAT2和IRF9蛋白的表达增加。此外，与单独使用干扰素相比，单独使用利巴韦林或与干扰素联合使用时，干扰素刺激基因(ISGs)mxa、IFIT3、PKR和ISG15的表达增强。PSTAT1和pSTAT2的激活以及ISG表达的增加在未感染的细胞中不存在，这表明RBV诱导的活性增强需要主动的病毒感染。干扰素信号的这种延长和增强的活性以及基因表达的诱导可能在干扰素和利巴韦林共同治疗病毒感染的方式中发挥作用。

1.We measured interferon and interferon stimulated gene production in response to cellular infection of different viruses in order to gain an understanding of the different signaling modulators that are activated when certain interferon subtypes are induced. We found that a group of subtypes are exclusively induced following stimulation of the IFN receptor, while another group of subtypes are exclusively induced following pattern recognition receptor stimulation. We also found that distinct sets of interferon stimulated genes are induced depending on virus concentration. 2. We have shown that the uncharacterized protein FLJ11286 (FLJ) is induced by dengue virus infection in an IFN dependent manner and plays a role in antiviral response. FLJ is 291 aa in length, contains conserved cysteine residues and has homologues across the vertebrate taxon. FLJ is upregulated in response to stimulation with IFN, and that disruption of IFN signaling, through siRNA mediated knockdown of IFN regulatory factor 9, results in decreased induction of FLJ after IFN treatment. FLJ displays antiviral activity against DENV as well as against Encephalomyocarditis virus. We also have demonstrated that FLJ is a nucleic acid binding protein with the ability to bind to dengue viral mRNA and associates with a variety of dengue and host proteins. These data serve to identify FLJ as a novel IFN stimulated gene with antiviral activity against DENV. 3.We also examined the role of IFIT3 in the antiviral response by studying the effect of IFIT3 on virus titers of VSV (vesicular stomatitis virus), EMCV (encephalomyocarditis virus) and DV (Dengue virus). Our data show that presence of IFIT3 leads to a decrease in virus titers. Although the precise mechanism is still unknown, IFIT3 demonstrated a clear antiviral effect against several different viruses. 4. Enhancement of the antiviral activity of IFN-alpha2 by Ribavirin: Combination treatments of interferon and ribavirin are successful in treating viral infections, however the mechanism of action for how these agents work together remains unclear. To examine this, we utilized vesicular stomatitis virus (VSV) infection on A549 human lung epithelial cells as an experimental model. Treating cells with ribavirin alone or in combination with interferon resulted in a 4-log10 decrease in VSV titer at 24h compared to untreated cells. We found increased and prolonged activation of pSTAT1 and pSTAT2, and in increases in total STAT1, STAT2, and IRF9 protein expression compared to interferon alone. In addition, expression of the Interferon Stimulated Genes (ISGs) MxA, IFIT3, PKR, and ISG15 showed enhanced expression in the presence of ribavirin alone or in combination with interferon when compared to interferon alone. The increases in pSTAT1 and pSTAT2 activation as well as ISG expression were not present in uninfected cells, suggesting that the enhanced activity induced by RBV required an active viral infection. This prolonged and enhanced activity of interferon signaling and induction of gene expression may play a role in how interferon and ribavirin work together to treat viral infections.