HHV-8 vIRF interactions in the context of infection

 描述（由申请人提供）：人疱疹病毒8型（HHV-8）指定了4种病毒干扰素调节因子同源物（vIRF），代表了一组新的病毒编码蛋白，迄今为止仅在HHV-8和密切相关的恒河猴Rhadinovirus中发现。HHV-8 vIRF的功能是抑制细胞IRF活性和细胞防御途径的其他组分，这些组分促进细胞周期停滞和细胞凋亡以响应病毒感染。已证明vIRF-1和vIRF-3中的两种vIRF显著促进病毒生产性复制（在内皮细胞中）或潜伏感染细胞活力[在原发性灌注淋巴瘤（PEL）细胞中];每种vIRF在潜伏（PEL）和裂解复制期间均表达。病毒生命周期的两个阶段对HHV-8恶性发病机制都很重要：卡波西肉瘤、PEL和多中心Castleman病。vIRF-1的已知靶向细胞蛋白包括IRF、IRF介导的应答所需的p300/CBP转录共激活因子、IFN/视黄酸诱导的GRIM 19、p53、p53激活ATM激酶和Bcl-2家族的某些仅BH 3（促凋亡）成员; vIRF-3靶向IRF、p300/CBP和p53。然而，这些多效性蛋白质的抑制性相互作用的全部谱还远不清楚，并且在潜伏和裂解复制的背景下尚未充分调查相互作用。虽然我们已经确定vIRF-1与BH 3-only蛋白的相互作用对于内皮细胞中成功的生产性复制是至关重要的，但其他vIRF-1相互作用在这些和其他细胞中的裂解性复制中的作用以及vIRF-1及其相互作用对PEL潜伏期的贡献仍然未知。类似地，vIRF-3及其相互作用在生产性复制中的作用以及特定vIRF-3相互作用在潜伏期中的作用仍有待确定。为了解决这些问题，我们建议：（1）从潜伏感染的PEL和裂解感染的PEL和内皮细胞中进行vIRF 1和3的亲和沉淀，然后对共沉淀的细胞蛋白进行质谱分析;（2）使用抑制、过表达和结合竞争分析来鉴定vIRF 1和3（它们的细胞靶标）的贡献，以及特定的vIRF-蛋白质相互作用对潜伏感染的细胞活力和生产性复制的影响 并开发抑制剂。该项目包括一个广泛的，但概念和实验重点分析独特的病毒先天免疫逃避蛋白，其功能和机制的贡献，病毒生物学目前还不清楚。由于这些蛋白质对潜伏期和生产性复制都非常重要，因此本研究产生的信息可以为开发新的抗病毒和临床疗法提供基础。

 DESCRIPTION (provided by applicant): Human herpesvirus 8 (HHV-8) specifies four viral interferon regulatory factor homologues (vIRFs) that represent a novel set of virus-encoded proteins, so-far identified only in HHV-8 and closely related rhesus rhadinovirus. The HHV-8 vIRFs function to inhibit cellular IRF activities and other components of cellular defense pathways that promote cell cycle arrest and apoptosis in response to virus infection. Two of the vIRFs, vIRF-1 and vIRF-3, have been demonstrated to contribute significantly to either virus productive replication (in endothelial cells) or to latently infected cell viability [in primary efusion lymphoma (PEL) cells]; each vIRF is expressed during both latent (PEL) and lytic replication. Both phases of the virus life cycle are important for HHV-8 malignant pathogenesis: Kaposi's sarcoma, PEL, and multicentric Castleman's disease. Known targeted cellular proteins of vIRF-1 include IRFs, p300/CBP transcriptional co-activators required for IRF-mediated responses, IFN/retinoic acid-induced GRIM19, p53, p53-activating ATM kinase, and certain BH3-only (pro-apoptotic) members of the Bcl-2 family; vIRF-3 targets IRFs, p300/CBP, and p53. However, the full repertoire of inhibitory interactions of these pleiotropic proteins is far from clear and the interactions have not been adequately surveyed in the context of latent and lytic replication. Whilst we have determined that vIRF-1 interactions with BH3-only proteins are critical for successful productive replication in endothelial cells, the roles of other vIRF-1 interactions in lytic replication in these and other cells and the contributions of vIRF-1 and its interactions to PEL latency remain unknown. Similarly, the roles of vIRF-3 and its interactions in productive replication and of specific vIRF-3 interactions in latency remain to be determined. To address these issues, we propose to: (1) undertake affinity precipitations of vIRFs 1 and 3 from latently infected PEL and lytically infected PEL and endothelial cells followed by mass spectrometry analysis of co-precipitated cellular proteins; (2) use suppression, overexpression, and binding-competition analyses to identify the contributions of vIRFs 1 and 3, their cellular targets, and particular vIRF-protein interactions to latently infected cell viability and productive replication and to develop inhibitory agents. The project comprises a broadly-visioned but conceptually and experimentally focused analysis of unique viral innate immune evasion proteins whose functional and mechanistic contributions to virus biology are presently unclear. As these proteins are centrally important to both latency and productive replication, information generated from this study could provide the basis for development of novel antiviral and clinical therapies.