PRKAG1 mediates the anti-Japanese encephalitis virus effect of Nitazoxanide through autophagy.

Nitazoxanide (NTZ) not only demonstrates broad-spectrum activity against various pathogens that infect both animals and humans, but also has the capacity to induce cellular autophagy. Previous studies have indicated that NTZ may interact with PRKAG1 (AMPKγ1 subunit); however, it remains unclear whether NTZ mediates autophagy through PRKAG1 to exert antiviral effects. To investigate the potential interaction and associated biological effects between NTZ and PRKAG1, we demonstrated that NTZ can regulate the PRKAG1 protein using Western blotting and Solvent-induced protein precipitation profiling (SIP). RNA interference experiments revealed that knockdown of PRKAG1 gene expression enhances both the autophagy level in cells and their ability to inhibit the replication of Japanese encephalitis virus (JEV), with this effect showing a synergistic interaction with NTZ treatment. Conversely, PRKAG1 overexpression reduces cellular autophagy and promotes JEV replication. Phosphorylation quantitative proteomic analysis of PRKAG1-silenced cells showed that the phosphorylation levels of several key functional proteins, including TP53 and GSK3B, were significantly increased. These findings suggest that PRKAG1 modulates multiple critical cellular signaling pathways involved in promoting autophagy and antiviral responses. In conclusion, our results indicate that PRKAG1 interacts with NTZ and plays a central role in NTZ-induced autophagy and anti-JEV activity, thereby providing novel insights into the molecular mechanisms underlying NTZ's antiviral efficacy.