The Antiviral Activities of Human Interferons

人干扰素的抗病毒活性

基本信息

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
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/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

项目摘要

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.

总而言之，为悬浮细胞开发了MTT测定法，我们可以同时测试IFN抗增殖和抗病毒活性。根据上述测定的结果，我们能够在不增长抑制但抗病毒保护和非抗病毒保护条件下对Daudi细胞进行治疗。对这种处理后的样品的分析使我们可以更精确地识别与IFN-Alpha抗病毒活性相关的基因。基于微阵列结果，有26个基因在导致AV保护的IFN浓度之间通常且显着差异，而不是引起AV保护的浓度。抗病毒中和实验的结果表明，已鉴定的基因和蛋白质与IFN AV活性的关联。因此，这项工作不仅代表了生物测定结果如何精确导致的一个例子，可以帮助设计实验的设计，从而鉴定功能基因和蛋白质以阐明IFN AV效应，但据我们所知，这也是第一个研究，涉及对B细胞系上AV相关基因的分析。关于ISGF3复合物和IFN-GAMMA信号传导，Western印迹分析表明，用IFN-Alpha处理磷酸化的STAT1（Y701，作为IFN激活的替代物）在2H处达到峰值，保持低水平的水平最高为48h。用IFN-GAMMA处理的细胞直到15H都显示出相同的趋势，当时PSTAT1的增加通过持续到24小时的额外信号传导峰的增加。 IFN-GAMMA处理后24小时后，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水平增加，例如MX1，PKR和OAS1。使用生物学测定中的中和抗体和QRT-PCR排除了Autocrine-I型和III型IFN信号传导诱导这些基因。尽管没有任何其他IFN，但在IFN-GAMMA处理后，通过共免疫沉淀分离ISGF3转录因子复合物24小时。该转录因子复合物的诱导可能在转录抗病毒基因和随后对IFN-GAMMA介导的病毒的保护作用。