Reovirus modulation of the cardiac innate response: Type I interferon and HSP25

呼肠孤病毒对心脏先天反应的调节：I 型干扰素和 HSP25

• 批准号: 8644636
• 负责人: BARBARA SHERRY
• 金额: $ 1.9万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8644636
• 关键词: Address; Amino Acids; Antiviral Agents; Antiviral Response; Binding; Cardiac; Cardiac Myocytes; Cataloging; Catalogs; Cells; Complex; Disease; Disease Outcome; Evolution; Family; Fibroblasts; Genes; Genetic Transcription; Goals; Heart; Heat shock proteins; In Vitro; Infection; Integration Host Factors; Interferon Type I; Interferons; Mus; Myocarditis; Nuclear; Obstruction; Organ; Participant; Phenotype; Phosphorylation; Play; Protein Binding Domain; Proteins; Proteomics; Reovirus; Reporting; Repression; Role; STAT1 gene; STAT2 gene; Signal Pathway; Signal Repression; Signal Transduction; Stimulus; Stress; Structure; Sudden Death; Sum; Testing; Therapeutic Intervention; Viral; Virus; Virus Diseases; cell injury; cell type; cytokine; human disease; in vivo; insight; member; mouse model; new therapeutic target; novel; novel therapeutics; protective effect; receptor; response; therapeutic target; tool; transcription factor; virus tropism; young adult

Our overall goal is to identify novel components of the host innate antiviral response and novel mechanisms for viral subversion of this response to impact disease. Cardiac myocytes are essential and non-replenishable, thus the heart is exceptionally dependent on this first-line defense. Indeed, we have shown that the cardiac interferon (IFN) response is unique, and that in reovirus-induced murine myocarditis the IFN response differs between virus strains and is critical for protection. We continue to use this powerful tool-kit of viruses to probe the innate response in a highly vulnerable organ, the heart, and have made two new discoveries. First, we found that reovirus inhibits IFN signaling by an entirely novel mechanism. Specifically, reovirus induces unusual nuclear accumulation of transcription factor IRF9, likely reflecting concomitant interference with IRF9 function in induction of IFN-stimulated genes. This virus strain specific-effect is determined by a single amino acid in reovirus protein u2. We hypothesize that reovirus protein u2 modulates IRF9 structure / function to inhibit IFN signaling, and that u2 repression of IFN signaling in cardiac cells is critical for myocarditis. In Specific Aim 1, we will determine the mechanism for this modulation and its impact on myocarditis and other disease. Results will provide significant new insights into functional domains and protein binding partners of participants in IFN signaling. Second, using a proteomic discovery approach, we identified a new IFN- independent protective response which can be subverted by virus. We found that Heat Shock Protein-25 (Hsp25) is phosphorylated (non-myocarditic reoviruses) or decreased (myocarditic reovirus) in infected cardiac myocytes, and that this is cell type-specific and IFN-independent. Hsp25 is modulated by many viruses and it is protective against stress, particularly in the heart. However, an Hsp25 decrease has never been reported for any stimulus in any cell type. Inhibition of Hsp25 by a highly myocarditic reovirus, but no other stimulus, suggests that Hsp25 is protective and can be subverted by viruses. We hypothesize that phosphorylated Hsp25 plays a cell type-specific protective role during infection, and that viruses can subvert this innate response. In Specific Aim 2, we will determine the protective effects of Hsp25, the mechanisms by which viruses modulate it, and the impact on virus tropism and disease. Results will define a completely new protective response against viral infection. In sum, by studying both a component of the IFN response (IRF9) and an IFN-independent protective factor (Hsp25), we gain a more complete picture of virus modulation of cell factors in disease. By studying cells critically dependent on innate antiviral responses, we uncover effectors that may be present in many cell types but not readily detected. Finally, the remarkably strong correlation between viral effects in cardiac myocyte cultures and murine myocarditis provides an outstanding platform to test hypotheses generated in vitro for their validity in vivo. The broader impact is to increase the catalog of protective host factors that can be sabotaged by viruses and manipulated for therapeutic intervention.

我们的总体目标是确定宿主先天抗病毒反应的新成分，以及病毒颠覆这种反应的新机制，以应对冲击性疾病。心肌细胞是必需的和不可补充的，因此心脏特别依赖于这一第一线防御。事实上，我们已经证明了心脏干扰素(干扰素)的反应是独特的，在呼肠孤病毒诱导的小鼠心肌炎中，干扰素的反应在不同的病毒株之间是不同的，并且对保护至关重要。我们继续使用这个强大的病毒工具包来探测高度脆弱的器官--心脏--的先天反应，并有了两项新发现。首先，我们发现呼肠孤病毒通过一种全新的机制抑制干扰素信号转导。具体地说，呼肠孤病毒诱导转录因子IRF9异常的核积累，可能反映了在诱导干扰素刺激的基因过程中伴随着对IRF9功能的干扰。这种病毒株的特异性效应是由呼肠孤病毒U2蛋白中的单一氨基酸决定的。我们假设呼肠孤病毒蛋白U2调节IRF9的结构/功能以抑制干扰素信号，并且U2抑制心肌细胞中的干扰素信号是心肌炎的关键。在具体目标1中，我们将确定这种调节的机制及其对心肌炎和其他疾病的影响。研究结果将对干扰素信号转导参与者的功能结构域和蛋白结合伙伴提供重要的新见解。其次，利用蛋白质组发现的方法，我们发现了一种新的干扰素非依赖的保护性反应，可以被病毒破坏。我们发现，在感染的心肌细胞中，热休克蛋白-25(HSP25)被磷酸化(非心肌炎性呼肠孤病毒)或减少(心肌炎性呼肠孤病毒)，并且这是细胞类型特异性的，并且不依赖于干扰素。HSP25受许多病毒的调节，它对压力有保护作用，特别是在心脏。然而，在任何类型的细胞中，从来没有报道过任何刺激作用下HSP25的降低。用一种高度心肌性呼肠孤病毒抑制HSP25，但没有其他刺激，表明HSP25具有保护性，可以被病毒颠覆。我们假设，磷酸化的HSP25在感染过程中发挥着细胞类型特异性的保护作用，并且病毒可以颠覆这种固有的反应。在特定的目标2中，我们将确定HSP25的保护作用，病毒调节它的机制，以及对病毒趋向性和疾病的影响。结果将定义一种全新的针对病毒感染的保护性反应。总之，通过研究干扰素应答的组成部分(IRF9)和干扰素非依赖性保护因子(HSP25)，我们获得了病毒在疾病中对细胞因子的调控的更完整的图景。通过研究严重依赖于先天抗病毒反应的细胞，我们发现了可能存在于许多细胞类型中但不易检测到的效应器。最后，心肌细胞培养中的病毒效应与小鼠心肌炎之间的显著相关性为检验体外产生的假说在体内的有效性提供了一个出色的平台。更广泛的影响是增加了保护性宿主因素的目录，这些因素可以被病毒破坏并被操纵进行治疗干预。