USP7 targeting by HHV-8 vIRFs

HHV-8 vIRF 靶向 USP7

• 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
• 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

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）在艾滋病相关疾病的病因学上参与其中， 渗出性淋巴瘤（PEL）和多中心Castleman病;总之，病毒潜伏和溶解功能被认为是 作出贡献. HHV-8编码四个干扰素调节因子同源物（viRF），其可以在抑制HHV-8的表达中相互作用。 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质结合 压力信号然而，这些相互作用中很少有被检测到，并在功能上进行了评估， 病毒感染的背景。最近有报道，vIRF-1和vIRF-4两者都与 去泛素化酶USP 7 [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（已发表）和vIRF-2（本修订申请中的新数据）确定为额外的相互作用 USP 7的合作伙伴虽然vIRF-4通过核心基序ASTS与USP 7 N-末端TRAF结构域相互作用， 与存在于许多细胞和病毒蛋白中的共有USP 7相互作用基序A/PxxS相匹配，vIRF-1和 vIRF-3和vIRF-2分别具有单个和重复的TRAF结构域相互作用EGPS核心基序和 似乎以一种新的方式与USP 7相互作用。vIRF 1、2和3在细胞中潜伏表达（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， 复制的通过在PEL细胞中进行基于耗竭的分析和在HHV-8杆粒背景下进行基因消融， （BAC 16）病毒感染的iSLK（可诱导的，上皮）细胞，我们已经确定vIRF-1，像vIRF-3，是重要的， vIRF-3与vIRF-1相反，负调控HHV-8 生产性复制。此外，使用WT或USP 7难治性（EGPS基序突变的）vIRF-1/3“拯救”了 PEL细胞和表达vIRF 1的USP 7难治性变体的BAC 16突变体中的vIRF耗竭表型， 3，我们已经确定vIRF-1和vIRF-3与USP 7的相互作用主要涉及各自的免疫应答。 潜伏和裂解活性。USP 7缺失直接揭示了去泛素化酶对潜伏性PEL的重要性。 细胞活力和HHV-8生产性复制。我们还鉴定了vIRF 1和3的泛素修饰， 提高了USP 7通过泛素编辑调节vIRF表达和/或活性的可能性。在这 应用方面，我们提出：（1）研究vIRF-2靶向USP 7的功能和生物学后果; (2)在结构和功能上表征vIRF泛素化及其USP 7编辑;（3）鉴定vIRF- 在HHV-8感染细胞的背景下调节USP 7靶点以及这些蛋白质的功能意义 HHV-8生物学拟议的工作将首次表征USP 7的vIRF-2靶向，并确定 通过vIRF-USP 7相互作用介导的生物学活性的潜在机制，已证明其重要性 潜伏性和裂解性HHV-8生物学。该项目将提供机械和生物信息（目前 稀疏）关于USP 7病毒靶向的意义，并有可能告知未来的发展， 针对该项目确定的机制和途径的新型抗病毒剂和治疗剂。