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.

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8
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2015
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