, an MTT assay was developed for suspension cells in which we can test IFN antiproliferative as well as antiviral activities at the same time. Based on results from the above mentioned assay, we were able to perform treatment on Daudi cells under conditions of non-growth inhibition but antiviral protection, and non-antiviral protection. The analysis of samples after this treatment allowed us to more precisely identify genes associated with IFN-alpha antiviral activity. Based on microarray results there were 26 genes that are commonly and significantly different between an IFN concentration leading to AV protection versus a concentration not causing AV protection. Results from antiviral neutralizing experiments suggested the association of the identified genes and proteins with IFN AV activity. Thus, this work not only represents an example of how precise results from biological assays can help in the design of experiments leading to identification of functional genes and proteins to elucidate the IFN AV effect, but this is also to our knowledge the first study involving an analysis of AV associated genes on a B cell line. With respect to the ISGF3 complex and IFN-gamma signaling, western blot analysis revealed that phosphorylated Stat1 (Y701, as a surrogate for IFN activation) peaked at 2h when treated with IFN-alpha, remaining at low levels for up to 48h. Cells treated with IFN-gamma showed the same trend until 15h, when an increase in pStat1 was detected by an additional signaling peak that continued through 24h. Gene expression microarray analysis following IFN-gamma treatment for 24h indicated increased levels of antiviral proteins normally associated with a type-1 IFN response, such as Mx1, PKR, and OAS1. Induction of these genes by autocrine type-I and type-III IFN signaling was ruled out using both neutralizing antibodies in biological assays and by qRT-PCR. Despite the absence of any other IFNs, the ISGF3 transcription factor complex was isolated by co-immunoprecipitation after IFN-gamma treatment for 24h. It is possible that induction of this transcription factor complex plays a role in transcribing antiviral genes and the subsequent protection from viruses mediated by IFN-gamma.
