Bim regulation by HHV-8 vIRF-1

描述(由申请人提供)：病毒的生产性复制依赖于对病毒感染、基因表达和复制所触发的促凋亡刺激的充分控制。病毒已经进化出足以对抗这种抗病毒凋亡反应的机制，从而能够有效地产生病毒。虽然在人类疱疹病毒8型(HHV-8)中已经确定了几种被认为有助于这一功能的抗凋亡蛋白，但这些蛋白不太可能构成HHV-8机制的全部内容，这些机制共同有效地对抗细胞的抗病毒防御。其他非经典的促生存病毒蛋白可能包括信号转导受体和配体，如我们发现的促进细胞存活和病毒生产性复制的vGPCR和v-趋化因子。在我们的研究中，我们已经确定Bim是一个促凋亡的BH3-Only Bcl-2家族成员，它被特异性地诱导在裂解再激活阳性细胞中，发挥着HHV-8复制效率的关键调节作用，并成为v-趋化因子抑制的靶标。此外，我们观察到，在裂解感染的细胞中，Bim几乎只定位在细胞核中，这表明了通过核隔离抑制Bim的另一种全新的机制。Bim的这种核定位仅见于支持HHV-8裂解再激活的细胞，而不是对其他也触发Bim表达和激活的应激诱导因素(如血清饥饿)的反应。对参与Bim核转位的病毒蛋白的研究发现，病毒干扰素调节因子-1(vIRF-1)是一个核心角色。该蛋白与病毒诱导的细胞干扰素、促凋亡的P53和GRIM19形成抑制性相互作用，在体内外与Bim形成络合物，促进Bim在转基因细胞中的核转位和功能抑制，并在HHV-8感染的内皮(Time)细胞裂解再激活时与Bim共定位。本申请的重点是进一步表征BIM和vIRF-1之间的物理和功能相互作用(目标1)以及这种相互作用在HHV-8生产性复制中的作用(目标2)。这项工作将描述vIRF-1的一个新的和重要的功能，并研究调节Bim的新范式，Bim是HHV-8复制效率的关键决定因素。公共卫生相关性人类疱疹病毒8型(HHV-8)与艾滋病相关的内皮肿瘤卡波西氏肉瘤(KS)有关，卡波西氏肉瘤在非洲一些国家非常流行和侵袭性，还与两种罕见的B细胞淋巴瘤有关，也发生在艾滋病患者中。本申请中提出的工作将探索HHV-8使用的一种新的凋亡抑制机制(有效病毒复制所必需的)，涉及病毒干扰素调节因子(vIRF-1)及其与细胞促凋亡蛋白Bim的抑制相互作用，该蛋白在细胞应激(如病毒感染)时被激活。这项研究的数据将为治疗抗病毒策略的发展提供相关信息。

DESCRIPTION (provided by applicant): Virus productive replication is dependent on adequate control of pro-apoptotic stimuli triggered by virus infection, gene expression, and replication. Viruses have evolved mechanisms to counter this anti-viral apoptotic response sufficiently to enable efficient virus production. While several anti-apoptotic proteins believed to contribute to this function have been identified in human herpesvirus 8 (HHV-8), these are unlikely to comprise the full repertoire of HHV-8 mechanisms that together effectively counter the cell's anti-viral defenses. Other, non-classical pro-survival viral proteins may include signal-transducing receptors and ligands, such as vGPCR and v-chemokines that we have found promote both cell survival and virus productive replication. In our studies, we have identified Bim, a pro-apoptotic BH3-only Bcl-2 family member, as being induced specifically in lytic reactivation-positive cells, functioning as a key regulator of HHV-8 replication efficiency, and being a target of suppression by the v-chemokines. Furthermore, we have observed that in lytically-infected cells, Bim is localized almost exclusively in the nucleus, indicating an additional and entirely novel mechanism of Bim inhibition, via nuclear sequestration. Such nuclear localization of Bim was seen only in cells supporting HHV-8 lytic reactivation, not in response to other stress-inducers (e.g., serum starvation) that also trigger Bim expression and activation. Investigations to identify viral proteins involved in Bim nuclear translocation have identified viral interferon regulatory factor-1 (vIRF-1) as a central player. This protein, previously reported to form inhibitory interactions with virus-induced cellular interferons and pro-apoptotic p53 and GRIM19, was found to complex with Bim in vitro and in vivo, to promote nuclear translocation and functional inhibition of Bim in transfected cells, and to co-localize with Bim during lytic reactivation in HHV-8 infected endothelial (TIME) cells. This application is focused on characterizing further the physical and functional interactions between Bim and vIRF-1 (Aim 1) and the role of such interactions in HHV-8 productive replication (Aim 2). The work proposed will characterize a novel and important function of vIRF-1 and investigate a new paradigm in the regulation of Bim, a pivotal determinant of HHV-8 replication efficiency. PUBLIC HEALTH RELEVANCE Human herpesvirus 8 (HHV-8) is linked etiologically with the AIDS-associated endothelial tumor Kaposi's sarcoma (KS), very prevalent and aggressive in some countries in Africa, and also with two rare B cell lymphomas, also arising in AIDS patients. Work proposed in this application will explore a novel mechanism of apoptotic inhibition (necessary for efficient virus replication) used by HHV-8, involving a viral interferon regulatory factor (vIRF-1) and its inhibitory interactions with a cellular pro-apoptotic protein, Bim, that is activated in response to cell stress, such as virus infection. Data from this study will provide information of relevance to the development of therapeutic anti-viral strategies.