USP7 targeting by HHV-8 vIRFs

HHV-8 vIRF 靶向 USP7

基本信息

批准号：
10581544
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 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10581544
关键词：
Ablation Acquired Immunodeficiency Syndrome Affect Apoptosis Inhibitor Apoptotic Area B lymphoid malignancy Binding Biological Biology Catalytic Domain Cell Survival Cell physiology Cells Consensus Data Development Disease Endothelium Epithelium 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 Productivity Proteins Public Health Publishing Refractory Regulation Reporting Research Role Seminal Signal Transduction Stress Substrate Interaction System TRAF Domain Therapeutic Therapeutic Agents Time Transfection Ubiquitin Ubiquitination Variant Viral Viral Proteins Virus Virus Diseases Virus Latency Virus Replication Work Xeroderma Pigmentosum adduct antiviral drug development cell type cellular targeting disorder prevention genetic resistance latency-associated nuclear antigen latent infection lytic replication mutant novel primary effusion lymphoma targeted treatment ubiquitin isopeptidase 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.

人疱疹病毒8（HHV-8）参与病因与艾滋病相关疾病Kaposi的肉瘤，主要出现淋巴瘤（PEL）和多中心骑员氏病；总之，人们相信病毒潜在和裂解功能做出贡献。 HHV-8编码可以在抑制性相互作用的四个干扰素调节因子同源物（VIRF）带有细胞IRF和/或各种其他细胞蛋白的时尚与先天免疫和抗病毒有关应力信号传导。但是，在功能上检测到的这些相互作用中很少病毒感染的背景。最近据报道，VIRF-1和VIRF-4都与去泛素酶USP7 [泛素特异性蛋白酶7，也称为疱疹病毒相关USP（HAUSP）]，我们已经将VIRF-3（已发布）和VIRF-2（本修订应用程序中的新数据）确定为附加交互 USP7的合作伙伴。虽然VIRF-4通过核心基序ASTS与USP7 N末端TRAF域相互作用，但与许多细胞和病毒蛋白中存在的A/PXX相匹配，A/PXXS，VIRF-1和 VIRF-3分别具有单个和重复的TRAF结构域相互作用的EGP核心基序和Virf-2 似乎以新颖的方式与USP7互动。 VIRFS 1、2和3以潜在的方式表达（除了在纵向上）感染的pel细胞，但在其他检查的细胞类型中，所有VIRF似乎仅在裂解期间表达复制。通过在HHV-8 BACMID的背景下，通过基于PEL细胞的耗竭分析和基因消融（BAC16）感染病毒的ISLK（诱导，上皮）细胞，我们确定像VIRF-3这样的VIRF-1很重要与VIRF-1相比，为了延迟感染的PEL细胞和VIRF-3的生存能力，对HHV-8进行负调节生产性复制。此外，使用WT或USP7难治性（EGPSTOBIF-MUTIF-MUTIF）VIRF-1/3“救援” PEL细胞和BAC16突变体中的VIRF耗竭表型，表达VIRF的USP7难治性变体1和 3，我们确定VIRF-1和VIRF-3与USP7的相互作用集中参与了相对潜在的和裂解的活动。 USP7耗竭直接揭示了去泛素酶对潜在PEL的重要性细胞活力和HHV-8产品复制。我们还确定了VIRFS 1和3的泛素修饰，通过泛素编辑提高了USP7调节VIRF表达和/或活动的可能性。在这个我们建议的应用：（1）检查USP7靶向VIRF-2的功能和生物学后果；（2）在结构和功能上的泛素化及其编辑中表征；（3）确定virf- 在HHV-8感染细胞的背景下，调节USP7靶标和这些蛋白质的功能意义在HHV-8生物学中。拟议的工作将首次表征USP7的VIRF-2目标并确定通过VIRF-USP7相互作用介导的生物学活性的机制，其进口对潜在和裂解HHV-8生物学。该项目将提供机械和生物学信息（目前）稀疏）关于USP7病毒靶向的重要性，并有可能告知未来的发展新型抗病毒和热剂针对该项目确定的机制和途径。