The Antiviral Activities of Human Interferons

综上所述，建立了一种MTT法，可以同时检测IFN的抗增殖和抗病毒活性。基于上述测定的结果，我们能够在非生长抑制但抗病毒保护和非抗病毒保护的条件下对Daudi细胞进行处理。这种治疗后的样本分析使我们能够更精确地鉴定与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浓度与不导致AV保护的浓度之间是普遍和显著不同的。从抗病毒中和实验的结果表明，协会的鉴定基因和蛋白质与IFN AV活性。因此，这项工作不仅代表了一个例子，如何精确的结果，从生物测定可以帮助设计的实验，导致功能基因和蛋白质的鉴定，以阐明IFN AV的效果，但这也是我们所知的第一个研究涉及AV相关基因的分析B细胞系。 关于ISGF 3复合物和IFN-γ信号传导，蛋白质印迹分析显示，当用IFN-α处理时，磷酸化的Stat 1（Y 701，作为IFN活化的替代物）在2小时达到峰值，保持在低水平长达48小时。 用IFN-γ处理的细胞显示出相同的趋势，直到15小时，此时通过持续到24小时的额外信号传导峰检测到pStat 1的增加。 IFN-γ处理24小时后的基因表达微阵列分析表明，通常与1型IFN应答相关的抗病毒蛋白质水平增加，如Mx 1、PKR和OAS 1。 在生物测定中使用中和抗体和通过qRT-PCR排除了自分泌I型和III型IFN信号传导对这些基因的诱导。 尽管不存在任何其它IFN，但在IFN-γ处理24小时后通过免疫共沉淀分离ISGF 3转录因子复合物。 这是可能的，这种转录因子复合物的诱导在转录抗病毒基因和随后的保护由IFN-γ介导的病毒中发挥作用。

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.