The Antiviral Activities of Human Interferons

人干扰素的抗病毒活性

• 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
• 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

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.

总而言之，我们的新型抗病毒和抗脂肪性（AP）测定是第一个允许在悬浮细胞系上同时检查IFN AP和AV活动的测定法。该测定法使用MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）与广泛的IFN兼容，并且适用于不同的细胞系（例如，ovcar-3）。使用MTT AV/AP测定法进行了对样品的微阵列分析允许鉴定与IFN AV活性相关的25个基因。以前没有报道这25个基因中的十个与IFN的AV活性有关。首次报道了HSH2D，因为响应于IFN治疗，基因被上调。 IFIT3是最上调的基因。 IFIT3的siRNA敲除导致A549对两种不同病毒的敏感性提高。 Vero细胞中IFIT3的过表达导致感染后病毒滴度减少。因此，这项研究的结果表明IFIT3是IFN-Alpha AV活性的关键要素。这也是第一项研究，鉴定了用IFN-Alpha处理的Daudi细胞系的AV相关基因。 IFN alpha（I型）和伽马（II型）通过JAK/Stat途径发出信号，以引起AV活动，但是，人们认为仅通过STAT1同型二聚体来进行IFN-Gamma，而I型IFNS则激活了STAT1-和Stat2含量符合STAT1和ISGF3（例如STAT1，STAT1，STAT2，IRF9），以及IRF9。 IFN-GAMMA处理后24小时进行的基因表达微阵列分析表明，由ISGF3（通过ISRE启动子序列）诱导并与1型IFN响应相关联的抗病毒基因（例如MXA，PKR和OAS1）。 通过使用对生物学分析和QRT-PCR中这些IFN的中和抗体排除了Autocrine-I型和III型（Lambda）IFN信号传导诱导这些基因的诱导。 尽管没有I型或III类IFN，但IFN-GAMMA治疗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，如STAT2 Co-Muneprecipition and Chip促进剂分析所示。 A549细胞中的STAT2和IRF9敲低反转IFN-γ介导的ISRE诱导和抗病毒活性。 这表明ISGF3的形成是IFN-GAMMA细胞反应和生物学活性的重要组成部分。 干扰素α（IFN-Alpha）是一种具有多种功能在先天和适应性免疫和HIV的有效抑制剂中的细胞因子，部分地通过增强载脂蛋白B mRNA-MRNA-编辑酶催化酶催化性多肽样3（Apobobec3（apobec3））。尽管IFN-α疗法与病毒负担减轻有关，但该细胞因子还介导免疫功能障碍和毒性。通过详细的IFN-Alpha受体结合位点的映射，我们产生了IFN-Alpha杂种和突变体，并确定C-螺旋中的结构变化会改变IFN限制逆转录病毒的能力。选择性IFN-alpha构建了差异性阻断HIV复制及其抑制的定向幅度与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，这反映了诱导的吲哚胺2,3-二氧酶（IDO）。这些数据表明其抗病毒和炎症活性之间的谨慎并共享细胞内信号通路。定义相对于APOBEC和其他抗病毒基因的IFN结构和功能可以实现新型IFN相关分子的设计，以保留有益的抗病毒作用，同时最大程度地减少负面影响。