BH3-only protein targeting by HHV-8

描述（由申请方提供）：人疱疹病毒8型（HHV-8）与内皮卡波西肉瘤（KS）和B细胞恶性肿瘤、原发性渗出性淋巴瘤和多中心Castleman病相关，所有这些疾病主要发生在HIV合并感染的情况下。潜伏和裂解基因产物被认为有助于这些病理学，并且生产性复制和增加的病毒载量与KS相关。了解成功裂解复制所需的过程将为靶向基本病毒功能以阻断病毒复制提供机会，从而治疗或预防HHV-8相关疾病。HHV-8和其他病毒复制的关键是限制应激诱导的细胞凋亡信号传导的病毒机制，该信号传导作为针对病毒感染的先天防御机制。HHV-8编码几种抑制这种促凋亡途径的蛋白质。这些蛋白质包括病毒干扰素调节因子-I（vIRF-1）、病毒G蛋白偶联受体（vGPCR）和病毒趋化因子vCCL-1和vCCL-2，所有这些蛋白质都已被证明既抑制细胞凋亡又是有效病毒复制所需的。该实验室已经鉴定了vIRF-1与复制诱导的促凋亡BH 3-only蛋白（BOP）Bim和Bid的抑制剂相互作用，并揭示了控制这些蛋白对有效病毒复制发生的至关重要性。通过转导的shRNA抑制Bim或Bid表达导致病毒复制非常显著的增加，并且破坏vIRF-1：BOP相互作用抑制病毒产生 并促进裂解感染细胞的凋亡。除了Bim和Bid之外，vIRF-1通过其功能性BH 3结构域靶向其他BOP（Bik、Bmf、Hrk、Noxa），这也表明这些BOP的生物学重要性以及在裂解复制期间抑制其活性的需要。此外，病毒趋化因子受体（vGPCR）和CCR 8激动剂vCCL-1和vCCL-2激活导致Bim表达抑制的信号传导途径。因此，趋化因子受体信号可能部分通过BOP调节来增强HHV-8的生产性复制。本申请建议：（1）阐明Bim（一种已鉴定的HHV-8复制的主要抑制剂）的vGPCR和vCCL抑制机制;（2）鉴定vIRF-1：BOP相互作用的肽和药理学抑制剂，以特异性抑制BOP调节机制;（3） 确定裂解周期诱导的BOP的抗病毒活性以及vCCL、vGPCR和vIRF-1通过BOP对照对HHV- 8生产性复制的单独和组合贡献。该项目包括在HHV-8裂解复制期间重点分析BOP功能和vCCL，vGPCR和vIRF-1的靶向，并评估可能开发用于抗病毒目的的抑制试剂和方法。

DESCRIPTION (provided by applicant): Human herpesvirus 8 (HHV-8) is associated with endothelial Kaposi's sarcoma (KS) and B cell malignancies primary effusion lymphoma and multicentric Castleman's disease, all of which occur predominantly in the context of HIV coinfection. Both latent and lytic gene products are believed to contribute to these pathologies, and productive replication and increased viral loads are associated with KS. Understanding the processes required for successful lytic replication will provide opportunities for targeting essential viral functions to block virus replication and thereby treat or prevent HHV-8 associated disease. Key to replication by HHV-8 and other viruses are viral mechanisms that limit stress-induced apoptotic signaling that serves as an innate defense mechanism against virus infection. HHV-8 encodes several proteins that inhibit such pro-apoptotic pathways. These proteins include viral interferon regulatory factor-1 (vIRF-1), viral G protein coupled receptor (vGPCR), and viral chemokines vCCL-1 and vCCL-2, all of which have been demonstrated to both inhibit apoptosis and be required for efficient virus replication. This laboratory has identified inhibitor interactions of vIRF-1 with replication-induced pro-apoptotic BH3-only proteins (BOPs) Bim and Bid and revealed the critical importance of controlling these proteins for efficient virus replicaton to occur. Suppression of Bim or Bid expression via transduced shRNAs leads to very significant increases in virus replication, and disruption of vIRF-1:BOP interactions inhibits virus production and promotes apoptosis in lytically infected cells. In addition to Bim and Bid, vIRF-1 targets other BOPs (Bik, Bmf, Hrk, Noxa), via their functional BH3 domains, indicating the biological importance of these BOPs also and the need to inhibit their activities during lytic replication. Furthermore, the viral chemokine receptor (vGPCR) and CCR8 agonists vCCL-1 and vCCL-2 activate signaling pathways that lead to suppression of Bim expression. Thus, chemokine receptor signaling is likely to enhance HHV-8 productive replication in part via BOP regulation. This application proposes to: (1) elucidate the mechanisms of vGPCR and vCCL suppression of Bim, an identified major inhibitor of HHV-8 replication; (2) identify peptide and pharmacological inhibitors of vIRF-1:BOP interactions to specifically inhibit this mechanism of BOP regulation; (3) determine the antiviral activities of lytic cycle-induced BOPs and the individual and combined contributions, via BOP control, of vCCLs, vGPCR, and vIRF-1 to HHV- 8 productive replication. The project comprises a focused analysis of BOP function and targeting by vCCLs, vGPCR and vIRF-1 during HHV-8 lytic replication and assessment of inhibitory reagents and methods that could potentially be developed for antiviral purposes.