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

摘要。我们的总体目标是确定宿主先天性抗病毒药物的新成分， 病毒破坏这种反应而导致疾病的新机制。 心肌细胞是不可再生的，因此心脏异常地依赖于这一第一线的细胞。 防御事实上，我们已经证明，心脏I型干扰素（IFN）反应是独特的， 在呼肠孤病毒诱导的小鼠心肌炎中，不同病毒株的IFN应答不同 并且对于保护至关重要。我们继续使用这种强大的病毒工具包来探测 在一个非常脆弱的器官，心脏的先天反应，并取得了两个新的发现。 首先，我们发现呼肠孤病毒通过一种以前没有发现的机制抑制IFN信号传导 任何病毒。具体地说，呼肠孤病毒蛋白质2抑制IFN信号传导，并且抑制是病毒 菌株特异性，并与转录因子IRF 9的异常核积累有关， 这可能反映了伴随干扰其正常参与IFN-γ的诱导。 刺激基因我们假设呼肠孤病毒蛋白质2调节IRF 9的结构/功能 抑制IFN信号传导，心肌细胞中IFN信号传导的抑制是 心肌炎在具体目标1中，我们将：i）确定2是否改变IRF 9结构或细胞 结合伴侣; ii）鉴定IRF 9效应所需的IRF 2和IRF 9结构域，和iii）确定 呼肠孤病毒诱导的心肌炎是否需要IRF 9的调节。第二，使用 通过蛋白质组学发现方法，我们发现了一种新的IFN-依赖性保护反应， 而这些都可以被病毒破坏。我们发现呼肠孤病毒诱导磷酸化（非- 心肌呼肠孤病毒）或热休克蛋白-25（HSP 25）降解（心肌呼肠孤病毒）。 心肌细胞，这是细胞类型特异性和干扰素无关。成员七人 病毒家族诱导HSP 25表达或磷酸化，但不降解，但只有一种 研究已经解决了HSP 25的抗病毒作用。许多心脏损伤诱导HSP 25 磷酸化而不是降解，过表达的HSP 25可以保护机体免受应激。 导致心脏损伤。我们假设HSP 25发挥细胞类型特异性抗病毒作用， 磷酸化的HSP 25是抗病毒的，病毒已经进化出了逃避病毒的机制， 这种与生俱来的反应在具体目标2中，我们将确定：i）呼肠孤病毒的细胞类型特异性 ii）呼肠孤病毒改变HSP 25的机制，和iii）HSP 25的改变机制， 其中HSP 25抑制呼肠孤病毒感染。我们的研究的更广泛的影响是增加 一系列可以被病毒破坏的保护性宿主因子，可能提供新的 治疗靶点，特别是对于仍然是难治性疾病的病毒性心肌炎。

ABSTRACT. Our overall goal is to identify novel components of the host innate antiviral response and novel mechanisms by which viruses subvert this response to cause disease. Cardiac myocytes are non-replenishable; thus the heart is unusually dependent on this first-line defense. Indeed, we have shown that the cardiac Type I 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 a mechanism not previously shown for any virus. Specifically, reovirus protein ¿2 represses IFN signaling, and repression is virus strain-specific and is associated with unusual nuclear accumulation of transcription factor IRF9, likely reflecting concomitant interference with its normal participation in induction of IFN- stimulated genes. 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 required for myocarditis. In Specific Aim 1, we will: i) determine whether ¿2 alters IRF9 structure or cellular binding partners; ii) identify ¿2 and IRF9 domains required for IRF9 effects, and iii) determine whether ¿2 modulation of IRF9 is required for reovirus-induced myocarditis. Second, using a proteomic discovery approach, we identified a new IFN-independent protective response which can be subverted by virus. We found that reoviruses induce phosphorylation (non- myocarditic reovirus) or degradation (myocarditic reovirus) of Heat Shock Protein-25 (HSP25) in cardiac myocytes, and that this is cell type-specific and IFN-independent. Members of seven virus families induce HSP25 expression or phosphorylation but not degradation, yet only one study has addressed HSP25 antiviral effects. Many cardiac insults induce HSP25 phosphorylation but not degradation, and over-expressed HSP25 protects against stress- induced cardiac damage. We hypothesize that HSP25 plays a cell type-specific antiviral role, that phosphorylated HSP25 is antiviral, and that viruses have evolved mechanisms to evade this innate response. In Specific Aim 2, we will determine: i) cell type-specificity for reovirus alteration of HSP25, ii) mechanisms by which reovirus alters HSP25, and iii) mechanisms by which HSP25 inhibits reovirus infection. The broader impact of our studies is to increase the catalog of protective host factors that can be sabotaged by viruses, potentially providing new therapeutic targets, particularly for viral myocarditis which remains an intractable disease.