Rk 33

PD98059 (Selleck, Houston, Texas, USA), which is a ligand of aryl hydrocarbon receptor (AHR) and functions as an antagonist, was used as a specific inhibitor of MEK 25 . RK33 (Selleck, Houston, Texas, USA), which inhibits DDX3 helicase activity 26 (link), was used as a specific inhibitor of DDX3.

Jurkat cells and J-Lat 11.1 cells (Jurkat cells containing integrated full-length HIV-1 genome mutated in env gene and GFP replacing nef)^{39 (link)} were maintained in RPMI-1640 medium (Sigma-Aldrich) supplemented with 7% FBS and 100 μg/ml penicillin-streptomycin at 37 °C in a humidified, 5% CO₂ atmosphere. J-Lat latent proviruses were reactivated by adding 10 µM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) to the culture media for 18 h. Primary CD4+ T cells were isolated from buffy coats from healthy donors by Ficoll gradient followed by separation via negative selection with RosetteSep Human CD4+ T-cell Enrichment Kit (StemCells Technologies) according to the manufacturer’s instructions. After isolation, CD4+ T cells were maintained in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS and 100 μg/ml penicillin-streptomycin at 37 °C in a humidified, 5% CO₂ atmosphere. Cells were activated with 100 ng/ml PMA and 1 µg/ml Ionomycin (Sigma-Aldrich). HEK293T cells were cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% FBS and 100 μg/ml penicillin-streptomycin at 37 °C in a humidified, 5% CO₂ atmosphere. RK-33 (SelleckChem) was used at a concentration of 2 µM unless indicated otherwise. FH-1321 was provided by First Health Pharmaceuticals and used at concentrations of 1 µM. YM155 (SelleckChem) was used at concentrations of 200 nM.

Anti-RPL13, anti-DDX3, and anti-G3BP1 monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-green fluorescent protein (GFP), anti-FLAG, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Anti-hemagglutinin (HA) monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal pig antiserum directed against FMDV was prepared and stored in our laboratory. Lipofectamine LTX Reagent for the transfection of recombinant plasmids and Lipofectamine RNAiMax Reagent for the transfection of siRNA were purchased from Invitrogen (CA, USA). The QuikChange Site-Directed Mutagenesis Kit was purchased from Agilent Technologies (CA, USA). RNA was extracted with TRIzol Reagent, purchased from Invitrogen (CA, United States). The specific inhibitors of helicase DDX3, RK-33 and ketorolac salt, were purchased from Selleck Chemicals (Houston, TX, USA).