USP7 targeting by HHV-8 vIRFs

HHV-8 vIRF 靶向 USP7

基本信息

批准号：
9883702
负责人：
John Nicholas
金额：
$ 40.94万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9883702
关键词：
Ablation Acquired Immunodeficiency Syndrome Address Affect Antiviral Agents Apoptosis Inhibitor Apoptotic Area B lymphoid malignancy Binding Biological Biology Catalytic Domain Cell Survival Cell physiology Cells Consensus Data Development Disease Endothelium Epithelial Cells Etiology Future Genes Herpesviridae Homologous Gene Human Herpesvirus 8 In Vitro Interferons Kaposi Sarcoma Link Lymphoma cell Lytic Lytic Phase MG132 Maps Mediating Methods Modification Multicentric Angiofollicular Lymphoid Hyperplasia Mutate N-terminal Natural Immunity Pathway interactions Pharmaceutical Preparations Phenotype Polyubiquitin Proteins Public Health Publishing Refractory Regulation Reporting Research Role Seminal Signal Transduction Stress Structure Substrate Interaction System TRAF Domain Therapeutic Therapeutic Agents Time Ubiquitin Ubiquitination Variant Viral Viral Proteins Virus Virus Diseases Virus Latency Virus Replication Work Xeroderma Pigmentosum adduct base cell type cellular targeting disorder prevention genetic resistance latency-associated nuclear antigen latent infection lytic replication mutant novel primary effusion lymphoma targeted treatment ubiquitin-specific protease viral interferon regulatory factor viral interferon regulatory factor-3

项目摘要

Human herpesvirus 8 (HHV-8) is involved etiologically in AIDS-associated diseases Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman’s disease; in all, viral latent and lytic functions are believed to contribute. HHV-8 encodes four interferon regulatory factor homologues (vIRFs) that can interact in inhibitory fashion with cellular IRFs and/or a variety of other cellular proteins involved in innate immunity and antiviral stress signaling. However, very few of these interactions have been detected and assessed functionally in the context of virus infection. It has recently been reported that both vIRF-1 and vIRF-4 interact with the deubiquitinase USP7 [ubiquitin-specific protease 7, also called herpesvirus-associated USP (HAUSP)], and we have identified vIRF-3 (published) and vIRF-2 (new data in this revised application) as additional interaction partners of USP7. While vIRF-4 interacts with the USP7 N-terminal TRAF domain via a core motif, ASTS, matching the consensus USP7-interaction motif, A/PxxS, present in many cellular and viral proteins, vIRF-1 and vIRF-3 have, respectively, a single and duplicated TRAF domain-interacting EGPS core motif and vIRF-2 appears to interact with USP7 in a novel manner. vIRFs 1, 2 and 3 are expressed in latently (in addition to lytically) infected PEL cells, but in other examined cell types, all vIRFs appear to be expressed only during lytic replication. Through depletion-based analyses in PEL cells and gene ablation in the context of HHV-8 bacmid (BAC16) virus-infected iSLK (inducible, epithelial) cells, we have determined that vIRF-1, like vIRF-3, is important for the viability of latently infected PEL cells and that vIRF-3, in contrast to vIRF-1, negatively regulates HHV-8 productive replication. Furthermore, using WT or USP7-refractory (EGPS motif-mutated) vIRF-1/3 “rescue” of vIRF depletion phenotypes in PEL cells and BAC16 mutants expressing USP7-refractory variants of vIRFs 1 and 3, we have determined that vIRF-1 and vIRF-3 interactions with USP7 are involved centrally in the respective latent and lytic activities. USP7 depletion revealed directly the importance of the deubiquitinase for latent PEL cell viability and HHV-8 productive replication. We have also identified ubiquitin modifications of vIRFs 1 and 3, raising the possibility of USP7 modulation of vIRF expression and/or activities via ubiquitin editing. In this application we propose to: (1) examine the functional and biological consequences of vIRF-2 targeting of USP7; (2) characterize structurally and functionally vIRF ubiquitination and USP7 editing thereof; (3) identify vIRF- regulated USP7 targets in the context of HHV-8 infected cells and the functional significance of these proteins in HHV-8 biology. The proposed work will, for the first time, characterize vIRF-2 targeting of USP7 and identify mechanisms underlying biological activities mediated via vIRF-USP7 interactions, of demonstrated importance to both latent and lytic HHV-8 biology. The project will provide mechanistic and biological information (at present sparse) about the significance of viral targeting of USP7 and has the potential to inform future development of novel antiviral and therapeutic agents directed against the mechanisms and pathways identified by this project.

人类疱疹病毒 8 (HHV-8) 在病因学上涉及艾滋病相关疾病卡波西肉瘤、原发性渗出性淋巴瘤 (PEL) 和多中心卡斯尔曼病；人们相信病毒具有潜伏和溶解功能； HHV-8 编码四种可以在抑制中相互作用的干扰素调节因子同源物 (vIRF)。与细胞 IRF 和/或参与先天免疫和抗病毒的各种其他细胞蛋白的时尚然而，这些相互作用很少被检测到并在功能上进行评估。最近有报道称，vIRF-1 和 vIRF-4 均与病毒感染相互作用。去泛素酶 USP7 [泛素特异性蛋白酶 7，也称为疱疹病毒相关 USP (HAUSP)]，我们已将 vIRF-3（已发布）和 vIRF-2（本修订后的应用程序中的新数据）确定为附加交互而 vIRF-4 通过核心基序 ASTS 与 USP7 N 末端 TRAF 结构域相互作用，匹配常见的 USP7 相互作用基序 A/PxxS，存在于许多细胞和病毒蛋白 vIRF-1 和 vIRF-3 分别具有单个和重复的 TRAF 域交互 EGPS 核心基序，而 vIRF-2 vIRF 1、2 和 3 似乎以一种新颖的方式与 USP7 相互作用（除了裂解）感染的 PEL 细胞，但在其他检查的细胞类型中，所有 vIRF 似乎仅在裂解过程中表达通过 PEL 细胞中基于耗竭的分析和 HHV-8 bacmid 背景下的基因消融。 (BAC16) 病毒感染的 iSLK（诱导型上皮细胞）细胞，我们确定 vIRF-1 与 vIRF-3 一样，很重要对于潜伏感染的 PEL 细胞的活力，与 vIRF-1 相比，vIRF-3 负向调节 HHV-8 此外，使用 WT 或 USP7 难治性（EGPS 基序突变）vIRF-1/3“拯救” PEL 细胞和表达 vIRF 1 和 USP7 难治性变体的 BAC16 突变体中 vIRF 耗尽表型如图 3 所示，我们确定 vIRF-1 和 vIRF-3 与 USP7 的相互作用集中参与各自的潜在和裂解活性直接揭示了去泛素酶对潜在 PEL 的重要性。我们还鉴定了 vIRF 1 和 3 的泛素修饰，提高了 USP7 通过泛素编辑调节 vIRF 表达和/或活性的可能性。我们建议的应用是：（1）检查 vIRF-2 靶向 USP7 的功能和生物学后果； (2)表征vIRF泛素化及其USP7编辑的结构和功能；(3)鉴定vIRF-； HHV-8 感染细胞中调节的 USP7 靶点以及这些蛋白质的功能意义在 HHV-8 生物学中，拟议的工作将首次描述 USP7 的 vIRF-2 靶向特征并确定。通过 vIRF-USP7 相互作用介导的生物活性机制，具有重要意义该项目将提供潜伏和裂解 HHV-8 生物学信息（目前）。稀疏）了解病毒靶向 USP7 的重要性，并有可能为未来的发展提供信息针对该项目确定的机制和途径的新型抗病毒和治疗药物。