Reovirus Modulation of the Cardiac Innate Response: Type I Interferon and HSP25

描述（由申请人提供）：我们的总体目标是确定宿主先天抗病毒反应的新成分和病毒颠覆这种反应以影响疾病的新机制。心肌细胞是必需的，不可补充的，因此心脏特别依赖于这一线防御。事实上，我们已经证明心脏干扰素（IFN）反应是独特的，并且在呼肠孤病毒诱导的小鼠心肌炎中，IFN反应在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心脏这个高度脆弱的器官的先天反应，并有了两个新的发现。首先，我们发现呼肠孤病毒通过一种全新的机制抑制IFN信号。具体而言，呼肠孤病毒诱导转录因子IRF9的异常核积累，可能反映了在诱导ifn刺激基因时IRF9功能的伴随干扰。这种病毒株特异性效应是由呼肠孤病毒蛋白中的一个氨基酸决定的。我们假设呼肠孤病毒蛋白？2调节IRF9的结构/功能以抑制IFN信号，那？心肌细胞中IFN信号的抑制对心肌炎至关重要。在Specific Aim 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。结果将为IFN信号参与者的功能域和蛋白质结合伙伴提供重要的新见解。其次，利用蛋白质组学发现方法，我们发现了一种新的不依赖于IFN的保护反应，它可以被病毒破坏。我们发现，在感染的心肌细胞中，热休克蛋白25 （Hsp25）被磷酸化（非心肌呼肠孤病毒）或降低（心肌呼肠孤病毒），并且这是细胞类型特异性的，与ifn无关。Hsp25被许多病毒调节，它可以防止压力，特别是在心脏。然而，在任何细胞类型的任何刺激下，Hsp25的降低从未被报道过。高心肌呼肠孤病毒抑制Hsp25，但没有其他刺激，表明Hsp25具有保护作用，可以被病毒破坏。我们假设磷酸化的Hsp25在感染过程中发挥细胞类型特异性保护作用，而病毒可以破坏这种先天反应。在特异性目标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IFN应答的一个组成部分（IRF9）和IFN独立的保护因子（Hsp25），我们对疾病中病毒对细胞因子的调节有了更全面的了解。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物。最后，心肌细胞培养物中病毒作用与小鼠心肌炎之间显著的强相关性为检验体外产生的假设在体内的有效性提供了一个很好的平台。更广泛的影响是增加了可被病毒破坏和操纵用于治疗干预的保护性宿主因子的目录。

DESCRIPTION (provided by applicant): 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 ?2. We hypothesize that reovirus protein ?2 modulates IRF9 structure / function to inhibit IFN signaling, and that ?2 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. PUBLIC HEALTH RELEVANCE: The host's immediate innate response to viral infection can be a critical determinant of disease outcome. We have identified a new mechanism by which viruses can repress that innate response, and a new component of the innate response that may be important for protection against disease. We will investigate both topics in this proposal.