Time-of-Drug-Addition Assay for HCV Infection

The time-of-drug-addition assay was performed using an earlier described method22 (link), by adding DHMD (50 μM) to Huh-7.5 cell monolayers (1 × 10⁴ cells/well of a 96-well plate) either 24 h before, concurrently with, or subsequent to HCVcc inoculation (multiplicity of infection [MOI] = 0.01). Wash steps were included to ensure that the drug was only present during the specific treatment period (Fig. 3a). Following 72 h of incubation, the supernatant was collected and luciferase reporter signals reflecting viral infectivity was determined using the Gaussia luciferase assay kit (New England Biolabs; Pickering, ON, Canada) and a luminometer (Promega; Madison, WI, USA) as previously published20 (link). Data are expressed as log₁₀ of relative light units (log₁₀ RLU). For comparison, IFN-α (1,000 international unit [IU]/ml; Sigma) and DMSO (0.5%) served as positive and negative control treatments, respectively.