Screening DExD/H Box Helicases in KSHV Reactivation

Vero rKSHV.219 cells were seeded in individual wells of a 48-well plate at 200 μL of 9.5 × 10⁴ cells/mL in DMEM/10% FBS without antibiotics and grown overnight. A Lipofectamine RNAiMAX transfection (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was performed with a pool of 5 ON-TARGETplus siRNA duplexes that target a particular DExD/H box helicase or scrambled control (Dharmacon, Horizon Discovery, Lafayette, CO, USA) for a final siRNA concentration of 50 nM as per the manufacturer’s instructions. The list of the 22 DExD/H box helicase targets used in the knockdown screen [40 (link)] is presented in Table S1. The sequences of siRNAs against specific helicases and controls are in Table S2. After 28 h, the knock-down of control RNAi GFP was confirmed and all siRNA containing media was replaced with 10% FBS media containing 20 ng/mL TPA, and 2 mM NaB to induce lytic reactivation. Fluorescence images were acquired on a Leica DMI 4000 microscope, cell counts were recorded, and DNA was extracted at 48 h post-induction. Three random fields of vision were selected to quantify the adherent Vero cells prior to the 29 h post-induction time point. After the 36 h time point, lytic Vero cells were no longer adherent and could not be quantified.