Urokinase plasminogen activator inhibits HIV virion release from macrophage-differentiated chronically infected cells via activation of RhoA and PKCε.
Urokinase plasminogen activator inhibits HIV virion release from macrophage-differentiated chronically infected cells via activation of RhoA and PKCε.
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DOI：
10.1371/journal.pone.0023674
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Alfano M
中科院分区：
文献类型：
--
作者：
Graziano F;Elia C;Laudanna C;Poli G;Alfano M
HIV在单核吞噬细胞内的复制是一个由病毒和细胞蛋白调控的多步骤过程，具有病毒粒子出芽和在细胞质内囊泡内积累的特点。尿激酶型纤溶酶原激活剂（uPA）与其细胞表面受体（uPAR）的相互作用已被证明有利于病毒粒子在原代单核细胞源性巨噬细胞的亚细胞室中积累，而慢性感染的单核细胞源性U1细胞经肉豆酸酯phorbol acetate （PMA）刺激分化为巨噬细胞样细胞。通过采用后一种模型系统，我们在这里研究了uPA/uPAR相互作用触发哪些细胞内信号通路，导致病毒粒子在细胞质内囊泡中积聚的重定向。uPA诱导pma分化的U1细胞中RhoA、PKCδ和PKCε的激活。在相同的条件下，RhoA、PKCδ和PKCε调节了upa诱导的细胞粘附和极化，而只有RhoA和PKCε也负责细胞内囊泡中病毒粒子的重定向。G和F肌动蛋白的分布表明，uPA在贴壁细胞和极化细胞中都重组了细胞骨架。G和F肌动蛋白异构体的作用是通过使用细胞松弛素D来揭示的，细胞松弛素D是一种细胞渗透性真菌毒素，可以阻止F肌动蛋白聚合。细胞松弛素D对受体无关的细胞骨架进行重塑，导致细胞粘附、极化和细胞内HIV病毒粒子的积累，其作用与uPA类似。这些发现说明了uPA/uPAR系统在胞质内囊泡的产生和/或维持中的潜在贡献，这些囊泡积极地积聚病毒粒子，从而维持巨噬细胞起源的HIV储存库的存在。此外，我们的观察结果还提供了证据，表明控制细胞骨架重塑和PKCε激活的途径与设计新的抗病毒策略有关，这些策略旨在干扰感染的人巨噬细胞细胞质内囊泡和质膜之间病毒粒子出芽的分配。
HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA) with its cell surface receptor (uPAR) has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA). By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles. uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA. These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed at interfering with the partitioning of virion budding between intra-cytoplasmic vesicles and plasma membrane in infected human macrophages.
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3.3
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