The Antiviral Activities of Human Interferons

总之，我们的新的组合抗病毒和抗增殖（AP）的测定是第一个允许检查IFN AP和AV活动，同时对悬浮细胞系。使用MTT（3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物）的该测定与广泛的IFN相容，并且适用于不同的细胞系（例如OVCAR-3）。使用MTT AV/AP测定开发的样品的微阵列分析允许鉴定与IFN AV活性相关的25个基因;这25个基因中的10个先前未报道与IFN的AV活性相关。HSH 2D首次被报道为响应于IFN治疗而上调的基因。IFIT 3是上调最多的基因。IFIT 3的siRNA敲低导致A549对两种不同病毒的敏感性增加。IFIT 3在VERO细胞中的过表达导致感染后病毒滴度的降低。因此，本研究的结果表明IFIT 3是IFN-α AV活性的关键元件。这也是第一项鉴定用IFN-α处理的Daudi细胞系的AV相关基因的研究。 IFN α（I型）和γ（II型）都通过Jak/STAT途径发出信号以引发AV活性，但IFN-γ被认为仅通过STAT 1同源二聚体这样做，而I型IFN激活含有STAT 1和STAT 2的复合物，如ISGF 3（由STAT 1、STAT 2和IRF 9组成）。 IFN-γ处理24小时后的基因表达微阵列分析表明诱导了由ISGF 3（通过ISRE启动子序列）诱导并与1型IFN应答相关的抗病毒基因（例如MxA、PKR和OAS 1）。 在生物测定和qRT-PCR中使用针对这些IFN的中和抗体排除了自分泌I型和III型（λ）IFN信号传导对这些基因的诱导。 尽管缺乏I型或III型IFN，IFN-γ处理诱导新转录因子ISGF 3（由非磷酸化STAT 2，磷酸化STAT 1和IRF 9组成）和ISRE结合的形成，如STAT 2共免疫沉淀和PKR启动子的ChIP分析所示。 STAT 2和IRF 9在A549细胞中的敲低逆转了IFN-介导的ISRE诱导和抗病毒活性。 这表明ISGF 3形成是IFN-γ的细胞应答和生物活性的重要组成部分。 干扰素α（IFN-α）是一种在先天性和适应性免疫中具有多种功能的细胞因子，也是一种有效的HIV抑制剂，其部分通过增强载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC 3）家族成员发挥抗病毒活性。虽然IFN-α治疗与降低病毒负荷相关，但这种细胞因子也介导免疫功能障碍和毒性。通过详细绘制IFN-α受体结合位点，我们产生了IFN-α杂交体和突变体，并确定C-螺旋结构的变化改变了IFN限制逆转录病毒的能力。选择性IFN-α构建体差异性地阻断HIV复制，并且它们的定向抑制幅度与APOBEC 3水平相关。重要的是，某些突变体（例如SDM 1）表现出降低的毒性，如诱导的吲哚胺2，3-双加氧酶（IDO）的降低所反映的。这些数据表明其抗病毒和炎症活性之间的谨慎和共享的细胞内信号传导途径。定义IFN相对于APOBEC和其他抗病毒基因的结构和功能，可以设计新的IFN相关分子，保留有益的抗病毒作用，同时最大限度地减少负面影响。

In summary, 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.