Genetic determinants of efficacy of antiviral drugs revealed by genome-wide CRISPR screens.

Nucleoside and nucleobase analog antiviral drugs are pivotal in antiviral therapy, but comprehensive methods to understand their cellular response mechanisms and genetic regulators are still lacking. Here, we show that Eμ-Myc; Arf-/- mouse lymphoma cells, which are highly apoptosis-prone, enabled genome-wide CRISPR-Cas9 screening on such drugs to identify genes that modulate their efficacy. Using retroviral sgRNA libraries and MAGeCK analysis, we uncovered key regulators of drug transport, activation, and inactivation for these drugs. For ribavirin, adenosine kinase (ADK) and adenylsuccinate synthase (ADSS) were critical for nucleotide metabolism and bioactivation. Remdesivir uptake and activation depended on the transporter SLC29A3 and phosphoamidase HINT1, whereas favipiravir resistance was linked to NT5C2-mediated dephosphorylation. Viral replication assays in Huh7 cells validated that knockout of SLC29A3, HINT1, or NT5C2 significantly altered antiviral efficacy. This study delineates the genetic network governing nucleotide analog response, providing mechanistic insights and potential biomarkers for personalized antiviral therapy.