A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways

The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs. Host factors play a critical role in FMDV infection, however, the mechanisms are poorly understood. Aiming to comprehensively study the host factors involved in FMDV infection, we develop a porcine genome-scale CRISPR/Cas9 knockout library and successfully confirmed that the knockout of TOB1 (Transducer of ERBB2.1) could significantly inhibit FMDV infection. On one hand, TOB1-knockout could promote the expression of RIG-I and MDA5, and thus IFNB and ISGs (interferon stimulating genes), thereby inhibiting FMDV infection. On the other hand, TOB1-knockout inhibits FMDV entry mediated by EGFR/ERBB2 pathway. These findings reveal TOB1 as a key host factor mediating FMDV infection in pigs.