Elucidation of cystatin B associated signaling pathways to reduce HIV reservoirs

描述（由申请人提供）：单核细胞、巨噬细胞和小胶质细胞是在先天免疫中重要的单核吞噬细胞，也是中枢神经系统中HIV的关键储存库。这些储存库对根除HIV-1构成挑战，因为尽管存在抗逆转录病毒疗法，它们仍在组织中产生病毒。半胱氨酸蛋白酶抑制剂B表达与HIV复制和单核细胞源性巨噬细胞（MDM）中STAT-1磷酸化（STAT-1 PY）水平降低呈正相关。然而，这种情况发生的参与者和机制尚不清楚。这项工作有望阐明半胱氨酸蛋白酶抑制剂B通过调节巨噬细胞储库中STAT-1 PY促进HIV-1复制的机制。本申请的目的是确定半胱氨酸蛋白酶抑制剂B、STAT-1 PY、干扰素（IFN）和MDM中HIV持续性之间的关系和信号通路。为了达到这一提议的目的，我们将检验这样的假设，即胱抑素B通过与STAT-1 PY和其他蛋白质（包括与IFN信号通路相关的蛋白质）相互作用促进HIV持续存在。这一假设是根据申请人实验室的初步数据提出的。拟议研究的基本原理是，了解胱抑素B在HIV复制中的作用将允许调节这种蛋白质或其相互作用蛋白质，并减少巨噬细胞储库中的HIV。在强有力的初步数据的指导下，将通过追求两个特定目标来检验该假设：1）定义与巨噬细胞中STAT-1磷酸化和HIV复制相关的半胱氨酸蛋白酶抑制剂B/STAT-1信号传导途径和蛋白质;和2）鉴定在HIV感染和IFN-2活化期间半胱氨酸蛋白酶抑制剂B在JAK/STAT-1途径中的作用。在第一个目标下，我们将使用原位邻近连接测定（PLA）（Olink Biosciences）来确认、定位和定量未感染和HIV感染的MDM中的这种蛋白质相互作用，并确定半胱氨酸蛋白酶抑制剂B是否直接或间接与STAT-1相互作用。我们还将使用基于光谱计数的定量来鉴定与半胱氨酸蛋白酶抑制剂相关的特定蛋白质途径，所述蛋白质途径受到磷酸化修饰。在第二个目标下，我们将使用荧光素酶测定来确定半胱氨酸蛋白酶抑制剂B在LTR介导的HIV复制和IFN诱导的抗病毒应答中的作用，并将我们的发现与MDM中发生的情况相关联。这种方法是创新性的，因为半胱氨酸蛋白酶抑制剂B作为HIV复制的调节蛋白的作用还没有被证明使用蛋白质组学方法和相互作用的合作伙伴还没有被发现，拟议的研究是重要的，因为它将揭示新的HIV持久性的机制，可以针对新的治疗方法，直接消除艾滋病毒在巨噬细胞水库。

DESCRIPTION (provided by applicant): Monocytes, macrophages, and microglia are mononuclear phagocytes important in innate immunity, but also key reservoirs of HIV in the central nervous system. These reservoirs represent a challenge to HIV-1 eradication since they remain producing virus in tissue despite the presence of antiretroviral therapy. Cystatin B expression is positively correlated with HIV replication and decreased levels STAT-1 phosphorylation (STAT-1PY) in monocyte-derived macrophages (MDM). However, the players and the mechanism by which this occurs are unknown. This work is expected to elucidate the mechanism by which cystatin B contributes to HIV-1 replication by regulation of STAT-1PY in macrophage reservoirs. The objective of in this particular application is to define the relationships and signaling pathways between cystatin B, STAT-1PY, interferon (IFN), and HIV persistence in MDM. To attain the objective of this proposal, we will test hypothesis that cystatin B promotes HIV persistence by interacting with the STAT-1PY and additional proteins, including those related to IFN signaling pathway. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale of the proposed research is that understanding the role of cystatin B in HIV replication will permit the modulation of this protein or its interacting proteins and decrease HIV within macrophage reservoirs. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Define the cystatin B/STAT-1 signaling pathways and proteins associated to STAT-1 phosphorylation and HIV replication in macrophages; and 2) Identify the role of Cystatin B in the JAK/STAT-1 pathway during HIV infection and IFN-2 activation. Under the first aim we will use In situ Proximity Ligation Assay (PLA) (Olink Biosciences) to confirm, localize and quantify this protein interaction in uninfected and HIV infected MDM and determine if cystatin B interacts directly or indirectly with STAT-1. We will also identify the specific protein pathways associated to cystatin that are subject to modification by phosphorylation using spectral counting based quantification. Under the second aim, we will determine the effect of cystatin B in LTR mediated HIV replication and IFN induced antiviral response using the luciferase assay and correlate our findings with what happens in the MDM. This approach is innovative because, the role of cystatin B as a regulatory protein for in HIV replication has not been demonstrated using proteomics approaches and the interacting partners have not been found. The proposed research is significant because it will reveal novel mechanisms of HIV persistence that could be targeted for new therapeutic approaches directed to eliminate HIV in macrophage reservoirs.