Activities of HHV-8 vIRF-1 in Virus Biology

描述(申请人提供)：人类疱疹病毒8型(HHV-8)指定四种病毒干扰素调节因子同系物(VIRFs)，除抑制细胞IRFs外，还能抑制细胞IRFs 到细胞防御通路的其他组成部分，促进细胞周期停滞和细胞凋亡，以应对病毒感染。VIRF-1靶向抑制的细胞蛋白包括P53、ATM、GRIM19、Smad转录因子，以及IRF介导的反应所需的p300/CBP转录共激活因子。这个实验室发现了一类全新的相互作用， 在vIRF-1和应激反应、促凋亡的BH3-Only蛋白(BOP)之间，Bim和Bid被vIRF-1结合和其他新发现的BOP靶点抑制。此外，我们还发现vIRF-1部分定位于线粒体，表明vIRF-1可以直接在作用部位失活BOPs。VIRF-1诱导的BIM核移位代表了一种继发性的、独特的失活模式。VIRF-1：BOP相互作用是通过vIRF-1(残基170-187)的BID-BH3B相关BOP结合结构域(BBD)和靶向BOP的BH3结构域实现的。Bim和Bid都是由HHV-8裂解复制诱导的，Bim至少是一种强大的病毒生产抑制因子，这表明Bim和其他BOP需要被HHV-8有效地控制才能发生病毒复制。事实上，vIRF-1 BBD介导的相互作用有助于促进HHV-8的生产性复制和vIRF-1介导的细胞凋亡抑制。然而，目前还不清楚每个vIRF-1：BOP相互作用的确切贡献以及其他vIRF-1：蛋白质相互作用在病毒生物学中的意义。此外，我们新发现的vIRF-1的线粒体定位还有待于功能评估。这项应用提出：鉴定BOP/BH3靶向和vIRF-1抑制的分子/特异性决定因素(目标1)；分子解剖和分离vIRF-1的其他蛋白结合和功能结构域(目标2)；确定每个vIRF-1的贡献：在HHV-8感染的背景下vIRF-1的蛋白质相互作用和线粒体定位的活性(目标3)。该项目包括对HHV-8蛋白的全面而重点的分析，该蛋白在病毒复制中具有积极作用，并为研究多个(vIRF-1靶向)细胞防御途径在病毒生物学中的相对重要性提供了一个独特的有价值的工具。这项研究产生的信息可能为新的抗病毒疗法的开发提供基础。

DESCRIPTION (provided by applicant): Human herpesvirus 8 (HHV-8) specifies four viral interferon regulatory factor homologues (vIRFs) that function to inhibit cellular IRFs in addition to other components of cellular defense pathways that promote cell cycle arrest and apoptosis in response to virus infection. Cellular proteins targeted for inhibition by vIRF-1 include p53, ATM, GRIM19, Smad transcription factors, and p300/CBP transcriptional co-activators required for IRF-?mediated responses. This laboratory has identified an entirely novel class of interaction, between vIRF-1 and stress-responsive, pro-apoptotic BH3-only proteins (BOPs) Bim and Bid, demonstrated to be inhibited by vIRF-1 binding, and other newly recognized BOP targets. Furthermore, we have identified partial localization of vIRF-1 to mitochondria, suggesting that vIRF-1 can inactivate BOPs directly at their site of action. vIRF-1- induced nuclear translocation of Bim represents a secondary, unique mode of inactivation. vIRF-1:BOP interaction is via the Bid-BH3B-related BOP-binding domain (BBD) of vIRF-1 (residues 170-187) and the BH3 domains of targeted BOPs. Both Bim and Bid are induced by HHV-8 lytic replication and Bim, at least, is a powerful inhibitor of virus production, suggesting that Bim and probably other BOPs need to be controlled effectively by HHV-8 for virus replication to occur. Indeed, vIRF-1 BBD-mediated interactions contribute specifically and significantly to promotion of HHV-8 productive replication and vIRF-1-mediated apoptotic inhibition. However, the precise contributions of each vIRF-1:BOP interaction and the significance of other vIRF-1:protein interactions in virus biology are at present unknown. Furthermore, our newly-identified mitochondrial localization of vIRF-1 has yet to be assessed functionally. This application proposes: identification of the molecular/specificity determinants of BOP/BH3 targeting and inhibition by vIRF-1 (Aim 1); molecular dissection and dissociation of other protein-binding and functional domains of vIRF-1 (Aim 2); establishing the contributions of each vIRF-1:protein interaction and mitochondrial-localized activity of vIRF-1 in the context of HHV-8 infection (Aim 3). The project comprises a comprehensive, yet focused analysis of an HHV-8 protein with a demonstrated positive role in virus replication and which provides a uniquely valuable tool to study the relative importance of multiple (vIRF-1-targeted) cellular defense pathways in virus biology. Information generated from this study could provide the basis for development of novel anti-viral therapies.