RK-33 inhibits the OC43 coronavirus and induces stress granules via DDX3X-independent mechanisms.

DDX3X is a human DEAD-box RNA helicase with multiple functions in RNA metabolism. Previous studies have suggested that DDX3X is an important proviral host factor for numerous RNA viruses, including HIV, HCV, and SARS-CoV-2, and may be targetable with inhibitors such as RK-33 for therapeutic benefit. In exploring the role of DDX3X and its homolog DDX3Y in coronavirus replication, we found that the DDX3X inhibitor RK-33 inhibits propagation of the OC43 coronavirus through a DDX3X/DDX3Y-independent mechanism. Knockdowns of DDX3X or DDX3X and DDX3Y had little effect on OC43 growth in multiple cell lines, yet RK-33 treatment reduced OC43 replication in the presence or absence of DDX3 proteins. We observed that RK-33 stimulates the integrated stress response independently of DDX3 proteins to cause stress granule formation, although this is not the primary mechanism by which RK-33 suppresses OC43. Together, our results show that DDX3 proteins are likely not general pro-coronaviral host factors, and caution should be used in interpreting results with RK-33 given its off-target activity.