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。尽管存在抗逆转录病毒治疗，这些储存库仍然在组织中产生病毒，因此对根除HIV-1构成了挑战。单核细胞源性巨噬细胞（MDM）中胱抑素B的表达与HIV复制和STAT-1磷酸化（STAT-1PY）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。这项工作有望阐明胱抑素B通过调控巨噬细胞储存库中的STAT-1PY参与HIV-1复制的机制。在这个特定的应用程序中，目的是定义MDM中胱抑素B、STAT-1PY、干扰素（IFN）和HIV持久性之间的关系和信号通路。为了实现这一提议的目标，我们将验证胱抑素B通过与STAT-1PY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持久性的假设。这一假设是根据申请人实验室产生的初步数据制定的。该研究的基本原理是了解胱抑素B在HIV复制中的作用，将允许调节该蛋白或其相互作用蛋白，并减少巨噬细胞储存库中的HIV。在强有力的初步数据的指导下，这一假设将通过两个特定目标来验证：1)确定巨噬细胞中胱抑素B/STAT-1信号通路和与STAT-1磷酸化和HIV复制相关的蛋白质；2)确定Cystatin B在HIV感染和IFN-2激活过程中在JAK/STAT-1通路中的作用。在第一个目标下，我们将使用原位接近结扎法（PLA） （Olink Biosciences）来确认、定位和量化这种蛋白在未感染和HIV感染MDM中的相互作用，并确定胱抑素B是否直接或间接与STAT-1相互作用。我们还将确定与胱抑素相关的特定蛋白质途径，这些途径可通过基于光谱计数的定量磷酸化修饰。在第二个目标下，我们将使用荧光素酶测定确定胱抑素B在LTR介导的HIV复制和IFN诱导的抗病毒反应中的作用，并将我们的发现与MDM中发生的情况联系起来。这种方法是创新的，因为胱抑素B作为一种调节蛋白在HIV复制中的作用尚未被用蛋白质组学方法证明，而且还没有发现相互作用的伙伴。这项研究具有重要意义，因为它将揭示HIV持续存在的新机制，这可能是针对消除巨噬细胞储存库中的HIV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.