, 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.
