Elucidation of cystatin B associated signaling pathways to reduce HIV reservoirs

描述(申请人提供)：单核细胞、巨噬细胞和小胶质细胞是天然免疫中重要的单核吞噬细胞，但也是中枢神经系统中艾滋病毒的关键储存库。这些蓄水池对根除艾滋病毒-1构成了挑战，因为尽管存在抗逆转录病毒治疗，但它们仍然在组织中产生病毒。Cystatin B的表达与单核细胞来源的巨噬细胞(MDM)中HIV复制和STAT-1磷酸化水平降低(STAT-1PY)呈正相关。然而，这种情况发生的参与者和机制尚不清楚。这项工作有望阐明胱抑素B通过调节巨噬细胞储存库中的STAT-1PY而促进HIV-1复制的机制。在这个特殊的应用中，目的是确定在MDM中Cystatin B、STAT-1PY、干扰素(IFN)和HIV持久性之间的关系和信号通路。为了达到这个建议的目的，我们将检验一种假设，即胱抑素B通过与STAT-1PY和其他蛋白质相互作用来促进HIV的持久性，包括那些与干扰素信号通路相关的蛋白质。这一假设是根据申请者实验室提供的初步数据提出的。这项拟议研究的基本原理是，了解胱抑素B在艾滋病毒复制中的作用将允许调节该蛋白或其相互作用的蛋白，并减少巨噬细胞储存库中的艾滋病毒。在强大的初步数据指导下，这一假说将通过追求两个特定目标来检验：1)确定与巨噬细胞中STAT-1磷酸化和HIV复制相关的Cystatin B/STAT-1信号通路和蛋白；2)确定Cystatin B在HIV感染和干扰素-2激活过程中JAK/STAT-1通路中的作用。在第一个目标下，我们将使用原位邻近连接分析(LA)(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cystatin 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方法来确定与半胱氨酸氨基转移酶相关的特定蛋白通路，这些通路可以通过磷酸化进行修饰。在第二个目标下，我们将使用荧光素酶分析来确定cystatin B在LTR介导的HIV复制和干扰素诱导的抗病毒反应中的作用，并将我们的发现与MDM中发生的情况相关联。这种方法是创新的，因为，胱抑素B作为一种调节蛋白质在艾滋病毒复制中的作用尚未用蛋白质组学方法证明，也没有找到相互作用的伙伴。这项拟议的研究具有重要意义，因为它将揭示艾滋病毒持续存在的新机制，可能成为针对消除巨噬细胞储存库中的艾滋病毒的新治疗方法的目标。

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.