Data in support of NFκB and JNK pathways involvement in TLR3-mediated HIV-1 transactivation, expression of IL-6 and transcription factors associated with HIV-1 replication

In the present article, using human monocyte-derived macrophages and cell lines containing integrated copies of the HIV-1 promoter, we show the effects of TLR3 ligands on the pro-inflammatory cytokine IL-6. We further show the effects of TLR3 ligands on HIV-1 transactivation and transcription factors involved in HIV-1 replication. This article complements the data reported by the authors, “Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways, and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication” (Bhargavan et al., 2015) [1], and the interpretation of these data can be found in the research article published by the authors in Cellular Signaling in 2015 (Bhargavan et al., 2015) [1].

Others interested in studying the functional effects of receptor-ligand interactions could learn from our approach.
This approach would be of interest when determining the critical role of time on gene transcription and expression.

Data
To determine whether the TLR3 ligand polyinosinic-polycytidylic acid (PIC) can interact with HIV-1 to affect gene transcriptional regulation in human monocyte-derived macrophages (MDM), time-dependent and concentration-dependent real-time PCR analyses were performed on uninfected or HIV-1 infected human MDM treated with PIC. Genes analyzed included the pro-inflammatory cytokine IL-6 and transcription factors known to regulate the HIV-1 promoter activity (STAT-1, REL-B, JUN, CEBPA, and CEBPG). To investigate the role and involvement of these transcription factors in TRL3-mediated HIV-1 transactivation, pharmacological inhibitors targeting their signaling pathways were used, with and without TLR3 ligands, in luciferase and chloramphenicol acetyltransferase (CAT) ELISA assays on TZM-bl and U38 cells, both of which contained integrated copies of the HIV-1 promoter.

Luciferase and chloramphenicol acetyltransferase (CAT) assays
TZM-bl and U38 cells were treated for 48 h with PIC (25 or 50 mg/ml), with or without the inhibitor of NFκB transcriptional activation (481406, 20 nM), the JNK inhibitor ( 420119, 10 mM), the inhibitor of c-Jun/JNK complex (420130, 5 mM), and the MEKK7/MKK7 inhibitor (5ZO, 5 mM). Each experimental condition was performed in triplicate and following treatment, cells were harvested, washed with phosphate-buffered saline, and lysed as described [1]. Cell lysates were then used to quantify the luciferase activity (Fig. 3A) and the CAT activity (Fig. 3B) in each sample using the Luciferase Assay System (Promega, Madison, WI) and the CAT ELISA kit (Roche Diagnostics Indianapolis, IN), as described in the main manuscript [1].
In separate experiments, cells were treated for 48 h with PIC (25 mg/ml), with or without the AP-1 inhibitor (SR11302, 2 mM and 10 mM), the JNK inhibitor V (420129, 10 mM and 20 mM), and the IRAK-1/ 4 inhibitor (5 mM and 10 mM). Each experimental condition was performed in triplicate and following treatment, the effects of PIC and inhibitors on HIV-1 transactivation was quantified in TZM-bl cells (Fig. 4A) or U38 cells (Fig. 4B). The main manuscript [1] includes the manufacturer's names, catalog numbers, and mechanisms of action of each inhibitor.