BH3-only protein targeting by HHV-8

描述(申请人提供)：人类疱疹病毒8型(HHV-8)与内皮性卡波西氏肉瘤(KS)和B细胞恶性肿瘤、原发渗出性淋巴瘤和多中心Castleman病有关，所有这些都主要发生在艾滋病毒合并感染的背景下。潜伏的和裂解的基因产物都被认为与这些病理有关，而有效的复制和增加的病毒载量与KS有关。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从而治疗或预防HHV-8相关疾病提供机会。HHV-8和其他病毒复制的关键是限制应激诱导的凋亡信号的病毒机制，这是一种针对病毒感染的天生防御机制。HHV-8编码几种蛋白质，这些蛋白质可以抑制这种促凋亡途径。这些蛋白包括病毒干扰素调节因子-1(vIRF-1)、病毒G蛋白偶联受体(VGPCR)和病毒趋化因子vCCL-1和vCCL-2，所有这些都被证明既能抑制细胞凋亡，又是病毒有效复制所必需的。该实验室已经确定了vIRF-1与复制诱导的促凋亡BH3-Only蛋白(BOPS)BIM和BID的抑制作用，并揭示了控制这些蛋白对病毒有效复制的关键重要性。通过转导shRNAs抑制Bim或Bid的表达导致病毒复制显著增加，并破坏vIRF-1：BOP相互作用抑制病毒生产 并促进裂解感染细胞的凋亡。除了Bim和Bid外，vIRF-1还通过其功能BH3结构域靶向其他BOP(Bik，Bmf，HRK，Noxa)，表明这些BOP也具有生物学意义，需要在裂解复制过程中抑制它们的活性。此外，病毒趋化因子受体(VGPCR)和CCR8激动剂vCCL-1和vCCL-2激活了导致抑制Bim表达的信号通路。因此，趋化因子受体信号可能部分通过BOP调节来促进HHV-8的生产性复制。这项应用提出：(1)阐明vGPCR和vCCL抑制HHV-8复制的主要抑制剂Bim的机制；(2)识别vIRF-1的多肽和药理抑制剂：BOP相互作用，以特异性抑制BOP调节的这一机制；(3) 确定裂解周期诱导的BOP的抗病毒活性，以及通过BOP控制，vCCLS、vGPCR和vIRF-1对HHV-8生产性复制的单独和联合贡献。该项目包括重点分析BOP的功能和在HHV-8裂解复制过程中vCCLS、vGPCR和vIRF-1的靶向，以及评估可能被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.