, our novel combined antiviral and antiproliferative (AP) assay is the first to allow examination of IFN AP and AV activities simultaneously on a suspension cell line. This assay, using MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide) 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 (Type I) and gamma (Type II) signal through the Jak/STAT pathway in order to elicit AV 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 (lambda) 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 formation of the noveltranscription factor ISGF3 (composed of unphosphorylated STAT2, phosphorylated STAT1 and IRF9) 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. Interferon alpha (IFN-alpha), a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of HIV, exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family members. Although IFN-alpha therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFN-alpha receptor binding sites, we generated IFN-alpha hybrids and mutants and determined that structural changes in the C-helix alter the ability of IFN to limit retroviral. Selective IFN-alpha constructs differentially block HIV replication and their directional magnitude of inhibition correlates with APOBEC3 levels. Importantly, certain mutants (e.g. SDM1) exhibited reduced toxicity as reflected by a reduction of induced indoleamine 2,3-dioxygenase (IDO). These data suggest discreet and shared intracellular signaling pathways between its antiviral and inflammatory activities. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects.
