人类疱疹病毒8(HHV-8),与卡波西肉瘤有关,原发性渗出性淋巴瘤(PEL),多中心Castleman病,编码四个干扰素调节因子同源物,vIRF1-4,与宿主细胞防御病毒感染的各种介质相互作用并抑制它们。所有vIRF靶向的细胞蛋白是泛素特异性蛋白酶7(USP7);虽然已针对vIRF1-3鉴定了复制调节和潜伏感染的USP7靶向PEL细胞促活力表型,但vIRF-4与USP7相互作用的重要性仍未确定。在这里我们展示,通过遗传消融感染细胞中的vIRF-4-USP7相互作用,vIRF-4与USP7的结合对于vIRF-4的最佳表达和正常的HHV-8复制是必需的。来自转染和感染细胞的实验结果通过K48连接和USP7结合相关的vIRF-4泛素化抑制鉴定了vIRF-4的泛素化,在受感染的细胞中,vIRF-4表达增加。作为vIRF-4的功能的IFN-I诱导和相关信号传导的分析及其与USP7的相互作用鉴定了各自在先天免疫抑制中的作用。最后,发现通过K63-聚泛素化的先天免疫信号传导介质TRAF3的激活在受感染细胞中以USP7结合相关方式被vIRF-4抑制,但不是在转染的细胞中,可能是通过vIRF-4的结合调节表达。一起,我们的数据确定了vIRF泛素化的第一个例子和USP7的vIRF底物,通过与USP7相互作用增强vIRF-4的表达,以及vIRF-4的TRAF3抑制活性.调查结果地址,第一次,vIRF-4与USP7相互作用的生物学意义,并揭示了vIRF-4通过TRAF3调节介导的先天免疫逃避和前复制活性的机制。
目的:细胞干扰素调节因子(IRFs)的HHV-8同系物,参与宿主细胞防御病毒感染,以抑制方式与IRF和其他抗病毒先天免疫介质相互作用。这些互动对于成功的小学来说具有证明或假设的重要性,生产性(裂解性),和HHV-8潜伏(持续)感染。虽然已知HHV-8vIRF-4与USP7去泛素酶物理相互作用,各种细胞蛋白质的关键调节因子,相互作用的功能和生物学意义尚未得到解决。本研究确定了这种相互作用对HHV-8生产性复制很重要,的确,对于vIRF-4的表达,并通过抑制TRAF3的活性揭示了vIRF-4的新功能,TRAF3是通过激活细胞IRF和诱导I型干扰素的宿主细胞抗病毒活性的关键介质。这些发现确定了开发新型抗HHV-8药物的潜在目标。例如能够破坏vIRF-4-USP7相互作用或vIRF-4稳定USP7活性的那些。
Human herpesvirus 8 (HHV-8), associated with Kaposi sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease, encodes four interferon regulatory factor homologs, vIRFs 1-4, that interact with and inhibit various mediators of host-cell defense against virus infection. A cellular protein targeted by all the vIRFs is ubiquitin-specific protease 7 (USP7); while replication-modulatory and latently infected PEL-cell pro-viability phenotypes of USP7 targeting have been identified for vIRFs 1-3, the significance of the interaction of vIRF-4 with USP7 has remained undetermined. Here we show, through genetic ablation of the vIRF-4-USP7 interaction in infected cells, that vIRF-4 association with USP7 is necessary for optimal expression of vIRF-4 and normal HHV-8 replication. Findings from experiments on transfected and infected cells identified ubiquitination of vIRF-4 via K48-linkage and USP7-binding-associated suppression of vIRF-4 ubiquitination and, in infected cells, increased vIRF-4 expression. Analysis of IFN-I induction and associated signaling as a function of vIRF-4 and its interaction with USP7 identified a role of each in innate-immune suppression. Finally, activation via K63-polyubiquitination of the innate-immune signaling mediator TRAF3 was found to be suppressed by vIRF-4 in a USP7-binding-associated manner in infected cells, but not in transfected cells, likely via binding-regulated expression of vIRF-4. Together, our data identify the first examples of vIRF ubiquitination and a vIRF substrate of USP7, enhanced expression of vIRF-4 via its interaction with USP7, and TRAF3-inhibitory activity of vIRF-4. The findings address, for the first time, the biological significance of the interaction of vIRF-4 with USP7 and reveal a mechanism of vIRF-4-mediated innate-immune evasion and pro-replication activity via TRAF3 regulation.
OBJECTIVE: HHV-8 homologs of cellular interferon regulatory factors (IRFs), involved in host-cell defense against virus infection, interact in an inhibitory fashion with IRFs and other mediators of antiviral innate immunity. These interactions are of demonstrated or hypothesized importance for successful primary, productive (lytic), and latent (persistent) infection by HHV-8. While HHV-8 vIRF-4 is known to interact physically with USP7 deubiquitinase, a key regulator of various cellular proteins, the functional and biological significance of the interaction has not been addressed. The present study identifies the interaction as important for HHV-8 productive replication and, indeed, for vIRF-4 expression and reveals a new function of vIRF-4 via inhibition of the activity of TRAF3, a pivotal mediator of host-cell antiviral activity through activation of cellular IRFs and induction of type-I interferons. These findings identify potential targets for the development of novel anti-HHV-8 agents, such as those able to disrupt vIRF-4-USP7 interaction or vIRF-4-stabilizing USP7 activity.