Bim regulation by HHV-8 vIRF-1

描述（由申请方提供）：病毒生产性复制依赖于对由病毒感染、基因表达和复制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，以实现有效的病毒生产。虽然已经在人类疱疹病毒8（HHV-8）中鉴定了几种被认为有助于这种功能的抗凋亡蛋白，但这些蛋白不太可能构成HHV-8机制的全部组成部分，这些机制一起有效地对抗细胞的抗病毒防御。其他非经典的促存活病毒蛋白可能包括信号转导受体和配体，例如我们发现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，我们已经鉴定了Bim，一种促凋亡的BH 3-only Bcl-2家族成员，在裂解性再活化阳性细胞中特异性诱导，作为HHV-8复制效率的关键调节因子发挥作用，并且是V-趋化因子抑制的靶点。此外，我们已经观察到，在裂解感染的细胞中，Bim几乎完全定位在细胞核中，这表明通过核隔离的Bim抑制的另外的和完全新的机制。Bim的这种核定位仅见于支持HHV-8裂解再活化的细胞中，而不是响应于其他应激诱导剂（例如，血清饥饿），其也触发Bim表达和激活。对Bim核转位相关病毒蛋白的鉴定研究已将病毒干扰素调节因子-1（vIRF-1）鉴定为核心参与者。这种蛋白质，以前报道形成抑制性相互作用与病毒诱导的细胞干扰素和促凋亡p53和GRIM 19，被发现复合Bim在体外和体内，以促进核转位和功能抑制Bim在转染细胞，并与Bim共定位在HHV-8感染的内皮细胞（TIME）的裂解再活化过程中。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（Aim 1）以及这种相互作用在HHV-8生产性复制中的作用（Aim 2）。这项工作将描述vIRF-1的一个新的和重要的功能，并研究一个新的模式，在调节Bim，一个关键的决定因素HHV-8复制效率。人类疱疹病毒8型（HHV-8）在病因学上与艾滋病相关的内皮肿瘤卡波西肉瘤（KS）有关，卡波西肉瘤在非洲的一些国家非常普遍和具有侵袭性，并且还与两种罕见的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使用的凋亡抑制（有效病毒复制所必需的）的新机制，涉及病毒干扰素调节因子（vIRF-1）及其与细胞促凋亡蛋白Bim的抑制性相互作用，Bim响应于细胞应激（如病毒感染）而被激活。本研究的数据将提供与开发治疗性抗病毒策略相关的信息。

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.