The Antiviral Activities of Human Interferons

总之，我们为悬浮细胞开发了一种MTT试验，可以同时测试IFN的抗增殖和抗病毒活性。根据上述实验结果，我们能够在无生长抑制但有抗病毒保护和无抗病毒保护的条件下对Daudi细胞进行处理。这种处理后的样品分析使我们能够更精确地识别与ifn - α抗病毒活性相关的基因。基于微阵列结果，有26个基因在导致AV保护的IFN浓度与不引起AV保护的IFN浓度之间普遍且显著不同。抗病毒中和实验结果表明，所鉴定的基因和蛋白与IFN AV活性有关。因此，这项工作不仅代表了生物测定的精确结果如何有助于设计实验，从而鉴定功能基因和蛋白质，以阐明IFN AV效应，而且据我们所知，这也是第一个涉及B细胞系AV相关基因分析的研究。

In summary, 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.