BH3-only protein targeting by HHV-8

描述（由申请人提供）：人类疱疹病毒 8 (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，所有这些主要发生在 HIV 共感染的情况下。据信潜伏基因产物和裂解基因产物都会导致这些病理学，并且高效复制和病毒载量增加与 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止病毒复制提供机会，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，该信号传导是针对病毒感染的先天防御机制。 HHV-8 编码多种抑制此类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-1 (vIRF-1)、病毒 G 蛋白偶联受体 (vGPCR) 以及病毒趋化因子 vCCL-1 和 vCCL-2，所有这些蛋白质均已被证明能够抑制细胞凋亡，并且是有效病毒复制所必需的。该实验室已经确定了 vIRF-1 与复制诱导的促凋亡 BH3 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于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 表达会导致病毒复制显着增加，而破坏 vIRF-1:BOP 相互作用会抑制病毒产生 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 BH3 结构域还靶向其他 BOP（Bik、Bmf、Hrk、Noxa），这表明这些 BOP 的生物学重要性以及在裂解复制过程中抑制其活性的需要。此外，病毒趋化因子受体 (vGPCR) 和 CCR8 激动剂 vCCL-1 和 vCCL-2 激活信号通路，从而抑制 Bim 表达。因此，趋化因子受体信号传导可能部分通过 BOP 调节增强 HHV-8 的高效复制。本申请旨在：(1) 阐明 vGPCR 和 vCCL 对 Bim 的抑制机制，Bim 是一种已确定的 HHV-8 复制的主要抑制剂； (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 调节的这种机制； (3) 确定裂解循环诱导的 BOP 的抗病毒活性，以及通过 BOP 控制，vCCL、vGPCR 和 vIRF-1 对 HHV-8 生产性复制的单独和组合贡献。该项目包括重点分析 HHV-8 裂解复制过程中 BOP 功能和 vCCL、vGPCR 和 vIRF-1 的靶向，以及评估可能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.