The Antiviral Activities of Human Interferons

人干扰素的抗病毒活性

• 批准号: 7964667
• 负责人: Kathryn Zoon
• 金额: $ 41.55万
• 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/7964667
• 关键词: Aftercare; Antiviral Agents; Antiviral Response; B-Lymphocytes; Binding Sites; Biological Assay; Biological Process; Burkitt Lymphoma; Cell Line; Cells; Co-Immunoprecipitations; Complex; Dengue; Dengue Virus; Disease; Flavivirus; Gene Expression; Gene Expression Microarray Analysis; Gene Proteins; Genes; Growth; Human; Human Activities; Hybrids; Infection; Interferon Activation; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-2c; Interferon Type I; Interferon Type II; Interferon-alpha; Interferons; Knowledge; Laboratories; Mediating; Mus; Play; Property; Protein Family; Proteins; RNA Interference; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Transduction; Suspension substance; Suspensions; System; Testing; Time; Virus; Virus Replication; Western Blotting; Work; autocrine; base; design; gene induction; in vivo; interest; interferon-stimulated gene factor 3; neutralizing antibody; protein function; research study; response; transcription factor; trend

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.

总之，我们开发了一种针对悬浮细胞的 MTT 测定法，我们可以同时测试 IFN 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。治疗后的样本分析使我们能够更准确地识别与 IFN-α 抗病毒活性相关的基因。根据微阵列结果，导致 AV 保护的 IFN 浓度与不引起 AV 保护的浓度之间有 26 个基因存在常见且显着的差异。抗病毒中和实验的结果表明，已识别的基因和蛋白质与 IFN AV 活性相关。因此，这项工作不仅代表了生物测定的精确结果如何帮助设计实验，从而识别功能基因和蛋白质以阐明 IFN AV 效应，而且据我们所知，这也是第一项涉及B 细胞系上 AV 相关基因的分析。 关于 ISGF3 复合物和 IFN-γ 信号传导，蛋白质印迹分析显示，磷酸化 Stat1（Y701，作为 IFN 激活的替代物）在用 IFN-α 处理时在 2 小时达到峰值，并在长达 48 小时内保持低水平。 用 IFN-γ 处理的细胞在 15 小时之前表现出相同的趋势，此时通过持续到 24 小时的额外信号峰值检测到 pStat1 的增加。 IFN-γ 治疗 24 小时后的基因表达微阵列分析表明，通常与 1 型 IFN 反应相关的抗病毒蛋白（例如 Mx1、PKR 和 OAS1）水平升高。 使用生物测定中的中和抗体和 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。 尽管不存在任何其他 IFN，但在 IFN-γ 处理 24 小时后，通过免疫共沉淀分离了 ISGF3 转录因子复合物。 这种转录因子复合物的诱导可能在抗病毒基因转录和随后的 IFN-γ 介导的病毒防护中发挥作用。