Regulation of Interferon Signaling by INI1/hSNF5 During HIV-1 Replication

描述（由申请人提供）：本提案的长期目标是描述HIV-1复制过程中宿主-病毒的动态相互作用，并利用这些信息开发新的有效策略来对抗艾滋病。INI1/hSNF5是一种HIV-1整合酶（IN）结合宿主因子，是哺乳动物染色质重塑SWI/SNF复合体参与转录调控的一个组分。INI1/hSNF5直接和强结合HIV-1 IN和INI1/hSNF5片段，跨越最小IN结合域，主要负性抑制HIV-1复制。这些观察结果表明，INI1/hSNF5相互作用是HIV-1复制所必需的。最近的研究表明，INI1/hSNF5是一种肿瘤抑制因子，在横纹肌样肿瘤中双等位突变。我们对全球基因表达谱的研究表明，INI1/hSNF5在INI1-/-横纹肌样细胞中高度刺激IFN信号诱导基因（ISGs）和PML核体蛋白。这些结果为INI1/hSNF5在HIV-1复制中的作用提供了新的见解。我们假设INI1是细胞抗病毒防御（先天免疫）的一个组成部分，并且是在HIV-1复制过程中诱导IFN信号传导所必需的，并且HIV-1通过IN直接结合并在其复制过程中捕获INI1/hSNF5来破坏INI1/hSNF5的作用。为了验证这一假设，在特定目的I中，我们将确定INI1/hSNF5是否需要通过IFN， poly(I):(C)和HIV-1复制在T细胞中诱导IFN信号。我们将监测抗病毒基因（如IFIT1、IFITM1、OAS2、MX2和PKR）、PML核体成分（如PML、Sp100、Sp110）和信号转导（如STAT1）的诱导。我们将把INI1和ISG表达下调与HIV-1复制的影响联系起来。这些结果将确定INI1/hSNF5是否是HIV-1复制过程中细胞抗病毒防御的一个组成部分。在特定目的II中，我们将确定HIV-1是否通过与In结合捕获ini1而破坏ini1诱导的抗病毒防御。我们将：(i)使用含有INI1相互作用缺陷的IN突变体（H12Y）和INI1的跨显性突变体（S6）的突变HIV-1病毒粒子；确定in突变体H12Y是否不能捕获INI1并允许IFN信号传导；（ii）确定S6是否隔离IN并允许细胞INI1装载IFN信号，并将S6与IN相互作用缺陷的S6突变体的效果进行比较（E3）；（iii）为了确定Gag-Pol环境下的IN是否能够隔离INI1，我们将研究Gag-Pol表达、ISG表达和HIV-1复制后期内源性INI1再分配之间的时间关系。如果Gag-Pol表达能够将细胞INI1隔离在细胞质中，那么这将导致HIV-1复制期间ISG表达的破坏。这些结果将揭示HIV-1破坏ini1诱导的细胞抗病毒防御的机制。我们希望上述两个具体目标将共同建立一个动态宿主- hiv -1相互作用的新范式，并可能为开发针对艾滋病的新型抗病毒药物和疫苗策略提供见解。艾滋病仍然是一个主要的健康问题，因为由于耐药病毒的出现，目前的药物无法消灭艾滋病毒-1。因此，迫切需要确定新的靶点和开发新的治疗策略来对抗艾滋病。在发病过程中，宿主与病毒之间存在动态相互作用。本应用程序的目的是测试一个新的假设，即INI1/hSNF5是一种结合HIV-1蛋白（整合酶）的细胞蛋白，是细胞抗病毒防御的一个组成部分，并利用这一见解在未来开发针对艾滋病的新型抗病毒策略。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to delineate the dynamic host-virus interactions during HIV-1 replication and use this information to develop novel and effective strategies to combat AIDS. INI1/hSNF5, is a HIV-1 integrase (IN) binding host factor, is a component of the mammalian chromatin-remodeling SWI/SNF complex involved in transcriptional regulation. INI1/hSNF5 directly and strongly binds to HIV-1 IN and a fragment of INI1/hSNF5, spanning the minimal IN-binding domain, dominant negatively inhibits HIV-1 replication. These observations suggested that INI1/hSNF5 interaction is required for HIV-1 replication. Recent studies demonstrate that INI1/hSNF5 is a tumor suppressor, biallelically mutated in rhabdoid tumors. Our attempt to study global gene expression profile revealed that INI1/hSNF5 stimulates a high degree of IFN signal induced genes (ISGs) and PML nuclear body proteins in INI1-/- rhabdoid cells. These results shed new insight on the role of INI1/hSNF5 in HIV-1 replication. We hypothesize that INI1 is a component of the cellular anti-viral defense (innate immunity) and is required for induction of IFN signaling during HIV-1 replication and that HIV-1 subverts the effect of INI1/hSNF5 by directly binding to it via IN and capturing it during its replication. To test this hypothesis, in the specific aim I we will determine if INI1/hSNF5 is required for inducing IFN signaling in T cells by IFNs, poly(I):(C) and during HIV-1 replication. We will monitor the induction of antiviral genes (e.g. IFIT1, IFITM1, OAS2, MX2 and PKR), PML nuclear body components (e.g. PML, Sp100, Sp110), and signal transducers (e.g. STAT1). We will correlate the effect of down-modulation of INI1 and ISG expression to HIV-1 replication. These results will establish if INI1/hSNF5 is a component of the cellular antiviral defense during HIV-1 replication. In the specific aim II we will determine if HIV-1 subverts the INI1-induced antiviral defense by capturing it via binding by IN. We will: (i) use a mutant HIV-1 virion harboring INI1-interaction-defective IN mutants (H12Y) and a transdominant mutant of INI1 (S6); determine if IN-mutants H12Y will be unable to capture INI1 and allow the IFN signaling; (ii) determine if S6 sequesters IN and allows cellular INI1 to mount IFN signaling and compare the effect S6 to an IN-interaction defective mutant of S6 (E3); (iii) To determine if IN within the context of Gag-Pol is able to sequester INI1, we will investigate the temporal relationship between expression of Gag-Pol, ISG expression and redistribution of endogenous INI1 during late events of HIV-1 replication. If Gag-Pol expression is able to sequester cellular INI1 in the cytoplasm, then this will lead to subversion of the ISG expression during HIV-1 replication. These results will shed light on the mechanism by which HIV-1 may subvert INI1-induced cellular antiviral defense. We hope that together the above two specific aims will establish a new paradigm in dynamic host-HIV-1 interaction and may provide insight for developing novel antiviral and vaccine strategies against AIDS. AIDS is still a major health problem because of the inability of current drugs to eliminate HIV-1 due to the emergence of the resistant viruses. Therefore, there is a dire need for identification of new targets and development of novel therapeutic strategies to combat AIDS. There is a dynamic interaction between host and the virus during pathogenesis. The aim of this application is to test a novel hypotheses that INI1/hSNF5, a cellular protein that binds to one of the HIV-1 proteins (integrase), is a component of the cellular antiviral defense and utilize this insight for developing novel antiviral strategies against AIDS in the future.