The Antiviral Activities of Human Interferons

人干扰素的抗病毒活性

基本信息

批准号：
8336259
负责人：
Kathryn Zoon
金额：
$ 41.69万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336259
关键词：
A549 Aftercare Antiviral Agents Antiviral Response Binding Binding Sites Biological Biological Assay Biological Process Bromides Cell Line Cells Co-Immunoprecipitations Complex Data Elements Exhibits Family member Gene Expression Gene Expression Microarray Analysis Gene Proteins Genes Genetic Transcription Goals Growth HIV Human Human Activities Hybrids Immune System Diseases Infection Inflammatory Interferon Type I Interferon Type II Interferon-alpha Interferons Link Maps Measures Mediating Microarray Analysis Natural Immunity Pathway interactions Play Protein Family Proteins RNA Interference Relative (related person)Reporting Reverse Transcriptase Polymerase Chain Reaction Role STAT1 gene STAT2 gene Sampling Signal Pathway Signal Transduction Small Interfering RNA Structure Suspension substance Suspensions Time Toxic effect Transfection Tryptophan 2,3 Dioxygenase Up-Regulation Viral Viral Load result Virus Virus Replication Western Blotting adaptive immunity apolipoprotein B mRNA editing enzyme autocrine cytokine design gene induction inhibitor/antagonist interest interferon alpha receptor interferon-stimulated gene factor 3 knock-down mutant neutralizing antibody novel overexpression polypeptide promoter protein function receptor binding research study response transcription factor

项目摘要

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.

总之，我们的新型联合抗病毒和抗增殖 (AP) 测定是第一个允许在悬浮细胞系上同时检查 IFN AP 和 AV 活性的方法。该测定使用 MTT（3-(4,5-二甲基噻唑-2-Yl)-2,5-二苯基四唑溴化物）与多种 IFN 兼容，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 个基因中有 10 个此前未曾报道与 IFN 的 AV 活性有关。 HSH2D 首次被报道为响应 IFN 治疗而上调的基因。 IFIT3 是上调最多的基因。 IFIT3 的 siRNA 敲低导致 A549 对两种不同病毒的敏感性增加。 VERO细胞中IFIT3的过度表达导致感染后病毒滴度下降。因此，本研究的结果表明 IFIT3 是 IFN-α AV 活性的关键要素。这也是第一项鉴定经 IFN-α 处理的 Daudi 细胞系的 AV 相关基因的研究。 IFN α（I 型）和 IFN γ（II 型）均通过 Jak/STAT 通路发出信号以引发 AV 活性，但 IFN-gamma 被认为仅通过 STAT1 同二聚体来实现这一点，而 I 型 IFN 则激活 STAT1- 和 STAT1- 和 STAT1- 。含有 STAT2 的复合物，例如 ISGF3（由 STAT1、STAT2 和 IRF9 组成）。 IFN-γ处理24小时后的基因表达微阵列分析表明抗病毒基因（例如MxA、PKR和OAS1）的诱导，这些基因由ISGF3（通过ISRE启动子序列）诱导并与1型IFN反应相关。在生物测定和 qRT-PCR 中使用这些 IFN 的中和抗体排除了自分泌 I 型和 III 型 (lambda) IFN 信号传导对这些基因的诱导。尽管不存在 I 型或 III 型 IFN，IFN-γ 治疗仍诱导新型转录因子 ISGF3（由未磷酸化 STAT2、磷酸化 STAT1 和 IRF9 组成）的形成和 ISRE 结合，如 STAT2 免疫共沉淀和 ChIP 分析所示。 PKR 发起人。 A549 细胞中 STAT2 和 IRF9 敲低可逆转 IFN-γ 介导的 ISRE 诱导和抗病毒活性。这表明 ISGF3 的形成是 IFN-γ 的细胞反应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，通过增强载脂蛋白 B mRNA 编辑酶催化多肽样 3 (APOBEC3) 家族发挥抗病毒活性成员。尽管 IFN-α 疗法与减少病毒负荷有关，但这种细胞因子也会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，我们生成了 IFN-α 杂合体和突变体，并确定 C 螺旋的结构变化改变了 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，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）表现出毒性降低，这反映在诱导吲哚胺 2,3-双加氧酶 (IDO) 的减少上。这些数据表明其抗病毒和炎症活性之间存在谨慎且共享的细胞内信号传导途径。定义与 APOBEC 和其他抗病毒基因相关的 IFN 结构和功能，可能有助于设计新型 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。