The Antiviral Activities of Human Interferons

综上所述，我们的新型抗病毒和抗增殖(AP)联合试验首次允许在悬浮细胞系上同时检测干扰素AP和病毒活性。该方法使用四甲基偶氮唑盐(3-(4，5-Dimethylthiazol-2-Yl)-2，5-Diphenyltetrazolium溴化物)，与多种干扰素相容，适用于不同的细胞系(如卵巢癌细胞株3)。利用MTTAV/AP试验对样品进行微阵列分析，可以鉴定出25个与干扰素AV活性相关的基因；这25个基因中有10个以前没有报道过与干扰素的AV活性有关。HSH2D首次被报道为一种基因在干扰素治疗后上调。IFIT3是最上调的基因。IFIT3的siRNA下调导致A549对两种不同病毒的敏感性增加。在Vero细胞中过表达IFIT3导致感染后病毒滴度下降。因此，这项研究的结果表明IFIT3是干扰素-αAV活性的关键元件。这也是第一次鉴定经干扰素-α处理的Daudi细胞系的AV相关基因。 干扰素α(I型)和伽马(II型)都通过JAK/STAT途径发出信号来诱导AV活性，然而干扰素-伽马被认为只通过STAT1同源二聚体来实现，而I型IFN激活含有STAT1和STAT2的复合体，如ISGF3(由STAT1、STAT2和IRF9组成)。基因表达芯片分析表明，干扰素-γ处理24小时后，诱导了抗病毒基因(如mxA、PKR和OAS1)，这些基因由ISGF3(通过ISRE启动子序列)诱导并与1型干扰素应答相关。在生物学检测和qRT-PCR中，使用针对这些IFN的中和抗体排除了由I型和III型(Lambda)自分泌干扰素信号诱导这些基因的可能性。STAT2免疫共沉淀和PKR启动子芯片分析表明，尽管没有I型或III型IFN，但干扰素-γ处理诱导了新的转录因子ISGF3的形成(由未磷酸化的STAT2、磷酸化的STAT1和IRF9组成)和ISRE结合。A549细胞中的STAT2和IRF9基因敲除逆转了干扰素介导的ISRE诱导和抗病毒活性。这表明ISGF3的形成是干扰素-γ细胞反应和生物活性的重要组成部分。 干扰素α是一种在先天免疫和获得性免疫中具有多种功能的细胞因子，也是一种有效的HIV抑制因子，其抗病毒活性部分是通过促进载脂蛋白B基因编码酶催化多肽样3(APOBEC3)家族成员发挥的。虽然干扰素-α治疗与减少病毒负担有关，但这种细胞因子也介导了免疫功能障碍和毒性。通过对干扰素-α受体结合位点的详细定位，我们产生了干扰素-α杂交体和突变体，并确定C-螺旋的结构变化改变了干扰素限制逆转录病毒的能力。选择性的干扰素-α结构以不同的方式阻断HIV的复制，其定向抑制程度与APOBEC3水平相关。重要的是，某些突变体(例如SDM1)表现出降低的毒性，这反映在诱导的吲哚胺2，3-双加氧酶(IDO)的减少。这些数据表明，其抗病毒和炎症活性之间存在谨慎和共享的细胞内信号通路。确定与APOBEC和其他抗病毒基因相关的干扰素的结构和功能可能有助于设计新的干扰素相关分子，保留有益的抗病毒作用，同时将负面影响降至最低。

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.