Prothymosin-Alpha- A Novel Antiviral Restriction Factor

描述(由申请人提供)：来自原代和转化的CD8细胞的培养上清具有强大的HIV抑制活性。并不是所有的活性都可以用包括2-趋化因子在内的已知活性成分来解释。使用一种设计用于在病毒进入后在初级巨噬细胞中专门筛选抑制艾滋病毒的新因素的策略，我们鉴定并报告了在人类免疫缺陷病毒转化的CD8T细胞系K#150K的细胞培养液中发现的一种蛋白质--前胸腺素α(ProT1)，具有强大的艾滋病毒-1抑制活性(41)。从CD8细胞上清液中去除HIV-1抑制组分的天然ProT1可以消除抑制活性，支持其通过可溶性介质发挥抑制作用。ProT1是一种丰富的酸性多肽，已被报道定位于细胞核内，与细胞增殖和转录激活有关。ProT1抑制HIV-1复制，其活性是靶细胞特异性的，并且抑制主要发生在病毒整合之后(41)。天然和重组ProT1蛋白能有效抑制巨噬细胞和树突状细胞中HIV-1LTR驱动的基因表达。ProT1介导的抑制整合HIV-1的机制(S)尚不清楚。我们的假设是，外源ProT1与细胞表面受体相互作用，触发抑制HIV-1基因表达的信号转导通路。这种效应的细胞特异性是由于细胞特异性转录因子参与了HIV的复制。为了设计一种方法来理解ProT1对HIV-1整合后抑制的机制，需要解决一些初步问题，如本R-21提案中所概述的那样。这些包括活性与细胞摄取/核定位之间的关系，主要信号通路在观察到的抑制中的作用，以及HIV生命周期中被抑制的整合后步骤。对这些基本问题的回答将允许合理设计一种实验方法来确定这种寄主限制途径的作用机制。这种初步的方法将利用已在其他系统中确定的关于ProT1的功能和功能域的内容。了解ProT1抑制前病毒HIV-1的机制并鉴定该分子的抗HIV-1活性结构域(S)将为开发针对整合HIV-1的治疗药物开辟新的途径。当HIV-1感染免疫系统的细胞后，病毒基因组将整合到宿主基因组中。在病毒生命周期的后整合阶段，病毒复制会导致新病毒颗粒的产生，这些病毒颗粒从受感染的细胞中脱落，并继续感染其他细胞。这对受感染的巨噬细胞尤其重要，它可以在长达2-3个月的时间内释放病毒颗粒。艾滋病毒-1生命周期中的整合后事件很难中断，因为很少有药物能够干扰病毒生命周期的这一部分。我们最近发现，人类蛋白原胸腺素α(ProT1)在HIV-1生命周期的后整合阶段非常有效地干扰HIV-1，导致病毒产量减少。研究ProT1在抑制HIV-1中的作用机制将导致能够阻断病毒生命周期这一重要步骤的治疗方法的发展。

DESCRIPTION (provided by applicant): Supernatants derived from primary as well as transformed CD8+ cells have potent HIV inhibitory activity. Not all of that activity can be accounted for by known active components including 2-chemokines. Using a strategy designed to screen specifically for novel factors that inhibit HIV in primary macrophages following viral entry we identified and have reported that prothymosin alpha (ProT1), a protein found in the cell-culture media of the HVS-transformed CD8+ T-cell-line, K#1 50K, has potent HIV-1 inhibitory activity (41). Depletion of native ProT1 from an HIV-1 inhibitory fraction of CD8+ cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProT1 is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. ProT1 suppresses HIV-1 replication, its activity is target-cell specific and inhibition predominantly occurs following viral integration (41). Native and recombinant ProT1 protein potently inhibit HIV-1 LTR-driven gene expression in macrophages and dendritic cells. The mechanism(s) of ProT1 -mediated suppression of integrated HIV-1 is not yet known. Our hypothesis is that exogenous ProT1 interacts with a cell surface receptor triggering signal transduction pathways that inhibit HIV-1 gene expression. The cell specificity of the effect is due to the involvement of cell specific transcription factors in HIV replication. In order to design an approach to understanding the mechanism of the post integrational suppression of HIV-1 by ProT1 some initial questions need to be addressed as outlined in this R-21 proposal. These include the relationship between activity and cellular uptake/nuclear localization, the role of major signaling pathways in the observed inhibition and the post-integration step in the HIV life cycle that is inhibited. Answers to these basic questions will allow the rational design of an experimental approach to determine the mechanism of action of this host restriction pathway. This initial approach will take advantage of what has been determined in other systems regarding function and functional domains of ProT1. Understanding the mechanism of suppression of proviral HIV-1 by ProT1 and identification of the active anti-HIV-1 domain(s) of this molecule will open new avenues for developing therapeutics targeting integrated HIV-1.After the human immunodeficiency virus type-1 (HIV-1) infects cells of the immune system the viral genome becomes integrated into the host genome. Viral replication at the post-integration step of the viral life cycle leads to production of new viral particles that are shed from the infected cell and go on to infect other cells. This is of particular importance with infected macrophages which can shed viral particles for up to 2-3 month. Post-integration events in HIV-1 life cycle are difficult to interrupt because there are very few drugs able to interfere with this part of the viral life cycle. We have recently shown that the human protein prothymosin alpha (ProT1) very effectively interferes with HIV-1 at the post-integration step of its life cycle leading to decrease of viral production. Investigation of the mechanism of action of ProT1 in HIV-1 suppression will lead to the development of therapeutics that can block this important step of the viral life cycle.