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、基因表达和复制引发的促凋亡刺激的充分控制。病毒已经进化出了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。虽然已经在人类疱疹病毒8 （HHV-8）中发现了几种抗凋亡蛋白，这些蛋白被认为有助于这一功能，但它们不太可能包括有效对抗细胞抗病毒防御的HHV-8机制的全部功能。其他非经典促存活病毒蛋白可能包括信号转导受体和配体，如vGPCR和v-趋化因子，我们发现它们既能促进细胞存活，又能促进病毒的多产复制。在我们的研究中，我们已经确定了Bim，一个促凋亡的BH3-only Bcl-2家族成员，在裂解再激活阳性细胞中被特异性诱导，作为HHV-8复制效率的关键调节因子，并且是v-趋化因子抑制的靶标。此外，我们还观察到，在溶解性感染的细胞中，Bim几乎完全定位于细胞核中，这表明通过核隔离有一种额外的、全新的Bim抑制机制。这种Bim的核定位只在支持HHV-8裂解再激活的细胞中发现，而在其他应激诱导因子（如血清饥饿）也能触发Bim的表达和激活时没有发现。对参与Bim核易位的病毒蛋白的研究已经确定病毒干扰素调节因子-1 （vIRF-1）是一个核心角色。该蛋白先前报道与病毒诱导的细胞干扰素和促凋亡的p53和GRIM19形成抑制相互作用，在体外和体内发现与Bim复合物，促进转染细胞中Bim的核易位和功能抑制，并在HHV-8感染内皮细胞（TIME）的裂解再激活过程中与Bim共定位。该应用程序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的一个新颖而重要的功能，并研究hbv -8复制效率的关键决定因素Bim调控的新范式。人类疱疹病毒8型（HHV-8）在病原学上与艾滋病相关的内皮肿瘤卡波西肉瘤（KS）有关，卡波西肉瘤在非洲一些国家非常普遍和具有侵袭性，还与两种罕见的B细胞淋巴瘤有关，也出现在艾滋病患者中。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的抑制相互作用，Bim在细胞应激（如病毒感染）下被激活。这项研究的数据将为治疗性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.