USP7 targeting by HHV-8 vIRFs

HHV-8 vIRF 靶向 USP7

• 批准号: 10361554
• 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
• 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/10361554
• 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; Substrate Interaction; System; TRAF Domain; Therapeutic Agents; Time; Ubiquitin; Ubiquitination; Variant; Viral; Viral Proteins; Virus; Virus Diseases; Virus Latency; Virus Replication; Work; Xeroderma Pigmentosum; adduct; antiviral drug development; 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

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型(HHV-8)与艾滋病相关疾病卡波西肉瘤的病原学相关 渗出性淋巴瘤(PEL)和多中心Castleman病；总的来说，病毒的潜伏和溶解功能被认为 做出贡献。HHV-8编码四个干扰素调节因子同系物(VIRFs)，它们可以在抑制中相互作用 与细胞IRF和/或与先天免疫和抗病毒有关的各种其他细胞蛋白一起流行 压力信号。然而，这些交互作用中很少在功能上被检测和评估 病毒感染的背景。最近有报道称，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 我已将vIRF-3(已发布)和vIRF-2(本修订申请中的新数据)确定为附加交互 USP7的合作伙伴。而VIRF-4通过核心基序ASTS与USP7 N端TRAF结构域相互作用， 与许多细胞和病毒蛋白中普遍存在的USP7相互作用基序A/PxxS相匹配，vIRF-1和 VIRF-3分别具有单个和重复的TRAF结构域相互作用的EGPS核心基序和vIRF-2 似乎以一种新颖的方式与USP7相互作用。VIRF1、2和3以潜伏的形式表达(除了 裂解)感染的PEL细胞，但在其他被检查的细胞类型中，所有vIRF似乎仅在裂解过程中表达 复制。通过基于PEL细胞的耗竭分析和在HHV-8杆菌背景下的基因消融 (BAC16)病毒感染的iSLK(可诱导的，上皮)细胞，我们已经确定vIRF-1和vIRF-3一样重要 对于潜伏感染的PEL细胞的活性和vIRF-3，而不是vIRF-1，负向调节HHV-8 高效复制。此外，使用WT或USP7-耐火材料(EGPS基序突变)vIRF-1/3对 PEL细胞和表达USP7耐药变异体vIRF 1和BAC16突变体的vIRF耗竭表型 3，我们已经确定vIRF-1和vIRF-3与USP7的相互作用集中在各自的 潜伏性和裂解性活性。USP7的缺失直接揭示了去泛素酶对潜伏性PEL的重要性 细胞活力和HHV-8的生产性复制。我们还确定了vIRF1和3的泛素修饰， 提高了USP7通过泛素编辑调控vIRF表达和/或活性的可能性。在这 我们建议的应用是：(1)检测vIRF-2靶向USP7的功能和生物学后果； (2)从结构和功能上表征vIRF泛素化及其USP7编辑；(3)鉴定vIRF- HHV-8感染细胞中调控的USP7靶点及其功能意义 在HHV-8生物学中。拟议的工作将首次表征以USP7为靶点的vIRF-2并确定 通过vIRF-USP7相互作用介导的生物学活动的潜在机制，具有重要意义 潜伏的和裂解的HHV-8生物学。该项目将提供机械和生物信息(目前 稀疏)关于病毒靶向USP7的重要性，并有可能为未来的发展提供信息 针对本项目确定的机制和途径的新型抗病毒和治疗药物。