Daclatasvir

Stock solutions of daclatasvir [10 mM in dimethyl sulfoxide (DMSO); Selleck Chemicals], sofosbuvir (10 mM in DMSO; Selleck Chemicals), favipiravir (20 mM in water; Atomax Chemicals Co. Ltd.), and ribavirin (100 mM in PBS; Sigma) were prepared, sterilized by filtration, and stored at−70°C, as detailed previously (Sheldon et al., 2014 (link); Gallego et al., 2016 (link), 2018 (link)). Drugs were diluted in DMEM before their use to reach the desired concentrations for the experiments. In all experiments, 4 x 10⁵ Huh-7.5 reporter cells were infected with either HCV p0 or HCV p200 at a multiplicity of infection (MOI) of 0.03 TCID₅₀/cell. After 5 h of virus adsorption to cells, the inoculum was removed, cells were washed, and fresh medium without or with the antiviral compounds was added. In different protocols, the time of addition of antiviral compounds relative to the initiation of infection and the duration of the infection varied, as indicated for each experiment. For serial viral passages, 0.5 ml of the cell culture supernatant from the previous infection was used to infect 4 x 10⁵ Huh-7.5 reporter cells. The infection continued in the absence or presence of the drugs for 72 to 96 h, as indicated in the relevant experiment.

The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, shown to be required for the processive polymerase activity of nsp12, were cloned and purified as described^{11 (link),20 (link)}. The 3′-exonuclease, referred to as nsp14, and its protein cofactor, nsp10, were cloned and expressed based on the SARS-CoV-2 genome sequence. Remdesivir triphosphate (RDV-TP) and NHC triphosphate were purchased from MedChemExpress (Monmouth Junction, NJ), Sofosbuvir triphosphate (SOF-TP) was purchased from Sierra Bioresearch (Tucson, AZ), Tenofovir diphosphate (Tfv-DP) was purchased from Alfa Chemistry (Ronkonkoma, NY), and Favipiravir triphosphate and AT-9010 were purchased from NuBlocks LLC (Oceanside, CA). Pibrentasvir was purchased from Cayman Chemical Company (Ann Arbor, MI) and Ombitasvir, Daclatasvir, Ritonavir and Lopinavir were purchased from Selleckchem (Houston, TX). UTP, GTP and ATP were purchased from Fisher Scientific. The RNA oligonucleotides (template-loop-primers) were purchased from Dharmacon (Horizon Discovery, Lafayette, CO).