PKC-omerga and HIV-1 transcriptional regulator Tat co-exist at the LTR promoter in CD4+ T cells

Maria Lopez-Huertas, Jasmine Li, Anjum ZAFAR, Sara Rodriguez-Mora, Carlota Garcia-Dominguez, Elena Mateos, Jose Alcami, Sudha RAO, Mayte Coiras

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Abstract

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.

Original languageEnglish
Article number69
Pages (from-to)1-14
Number of pages14
JournalFrontiers in Immunology
Volume7
Issue numberFEB
DOIs
Publication statusPublished - 2016

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Lopez-Huertas, M., Li, J., ZAFAR, A., Rodriguez-Mora, S., Garcia-Dominguez, C., Mateos, E., ... Coiras, M. (2016). PKC-omerga and HIV-1 transcriptional regulator Tat co-exist at the LTR promoter in CD4+ T cells. Frontiers in Immunology, 7(FEB), 1-14. [69]. https://doi.org/10.3389/fimmu.2016.00069