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对不同病毒的细胞感染的反应，以了解当某些干扰素亚型被诱导时激活的不同信号调节剂。我们发现，一组亚型是专门诱导后刺激的IFN受体，而另一组亚型是专门诱导后模式识别受体刺激。我们还发现，不同的干扰素刺激基因的诱导依赖于病毒浓度。 2.我们已经表明，未表征的蛋白FLJ 11286（FLJ）是由登革病毒感染以IFN依赖的方式诱导的，并在抗病毒反应中发挥作用。 FLJ长291个氨基酸，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。FLJ对IFN刺激的应答上调，并且通过siRNA介导的IFN调节因子9的敲低，IFN信号传导的破坏导致IFN处理后FLJ的诱导减少。 FLJ显示抗DENV以及抗脑心肌炎病毒的抗病毒活性。 我们还证明了FLJ是一种核酸结合蛋白，具有结合登革病毒mRNA的能力，并与多种登革和宿主蛋白相关联。 这些数据用于鉴定FLJ为具有抗DENV的抗病毒活性的新型IFN刺激基因。 3.通过观察IFIT 3对VSV（水泡性口炎病毒）、EMCV（脑心肌炎病毒）和DV（登革病毒）病毒滴度的影响，探讨IFIT 3在抗病毒反应中的作用。我们的数据显示IFIT 3的存在导致病毒滴度的降低。虽然确切的机制仍然未知，但IFIT 3对几种不同的病毒表现出明显的抗病毒作用。 4.利巴韦林增强IFN-α 2的抗病毒活性：干扰素和利巴韦林的联合治疗在治疗病毒感染方面是成功的，但是这些药物如何一起工作的作用机制仍不清楚。为了检验这一点，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，用利巴韦林单独或与干扰素组合处理细胞导致24小时VSV滴度降低4-log 10。 我们发现与单独使用干扰素相比，pSTAT 1和pSTAT 2的激活增加并延长，并且总STAT 1，STAT 2和IRF 9蛋白表达增加。此外，干扰素刺激基因（ISG）MxA、IFIT 3、PKR和ISG 15的表达在单独利巴韦林或与干扰素组合存在下与单独干扰素相比显示增强的表达。pSTAT 1和pSTAT 2激活以及ISG表达的增加在未感染的细胞中不存在，这表明由RBV诱导的增强的活性需要主动的病毒感染。 这种干扰素信号传导的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如何一起工作以治疗病毒感染中起作用。

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.