López-Huertas María Rosa, Li Jasmine, Zafar Anjum, Rodríguez-Mora Sara, García-Domínguez Carlota, Mateos Elena, Alcamí José, Rao Sudha, Coiras Mayte
AIDS Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III , Madrid , Spain.
Department of Microbiology and Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne , Melbourne, VIC , Australia.
Front Immunol. 2016 Feb 29;7:69. doi: 10.3389/fimmu.2016.00069. eCollection 2016.
PKCθ is essential for the activation of CD4(+) T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4(+) T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T(538) residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4(+) T cells and could be used as a therapeutic target.
蛋白激酶Cθ（PKCθ）对于CD4(+) T细胞的激活至关重要。在TCR/CD28刺激下，PKCθ发生磷酸化并迁移至免疫突触，诱导细胞转录因子如NF-κB的激活以及对HIV-1复制至关重要的激酶如ERK的激活。我们之前证明PKCθ对于HIV-1复制也是必需的，但具体机制尚不清楚。高效的HIV-1转录和延伸绝对依赖于NF-κB与病毒调节蛋白Tat之间的协同作用。Tat通过结合病毒mRNA帽位点近端的一个RNA茎环结构（称为TAR）发挥其功能。此外，由于其对细胞代谢途径的影响，Tat会使受感染的CD4(+) T细胞发生深刻变化，如NF-κB和ERK的激活。我们推测Tat介导的NF-κB和ERK激活的异常上调是通过PKCθ信号传导发生的。事实上，具有稳定且可被强力霉素抑制表达Tat的Jurkat TetOff细胞（Jurkat-Tat）表达高水平的PKCθ mRNA。在这些细胞中，位于质膜的PKCθ在未分裂细胞中、在无刺激的情况下，其苏氨酸（T）538残基被磷酸化。用强力霉素处理可抑制Jurkat-Tat细胞中PKCθ的磷酸化，表明Tat表达与PKCθ的激活直接相关。NF-κB和Ras/Raf/MEK/ERK信号通路在Jurkat-Tat细胞中均被显著激活，这与HIV-1长末端重复序列（LTR）启动子的高反式激活相关。即使在存在Tat的情况下，针对PKCθ的RNA干扰也会抑制NF-κB和ERK活性以及LTR介导的反式激活。除了Tat在细胞质中介导的PKCθ激活外，我们通过连续染色质免疫沉淀法证明Tat和PKCθ共存于结合在HIV-1 LTR启动子上的同一复合物中，特别是在包含TAR环的区域。总之，PKCθ - Tat相互作用似乎对于CD4(+) T细胞中的HIV-1复制至关重要，并且可作为一个治疗靶点。
