Cepharanthine inhibits African swine fever virus replication by suppressing AKT-associated pathways through disrupting Hsp90-Cdc37 complex

African swine fever (ASF) represents one of the most economically important viral infectious diseases in the swine industry worldwide. Presently, there is an absence of commercially available therapeutic drugs and safe vaccines. Cepharanthine (CEP), one of the naturally occurring bisbenzylisoquinoline alkaloids, has been approved as a drug to treat various diseases such as leukopenia, bronchial asthma, and snake bites for 70 years in Japan. Most recently, CEP was reported to inhibit ASFV replication by suppressing endosomal/lysosomal function although the specific molecular mechanisms were not elucidated. In this study, we demonstrate for the first time that ASFV infection promotes co-chaperone Cdc37 expression and its binding to Hsp90, leading to increased AKT phosphorylation to benefit viral replication. Notably, CEP disrupts the Hsp90-Cdc37 complex, subsequently decreasing p-AKT and inhibiting ASFV replication. Furthermore, our investigation reveals that enhanced AKT phosphorylation amplifies glycolysis, resulting in increased lactate production, while it upregulates the NF-κB signaling pathway, resulting in increased expression of IL-1β and other inflammatory cytokines. Elevated lactate enhances ASFV replication, and IL-1β acts synergistically on the proviral effect of lactate. CEP reduces ASFV replication by disrupting the formation of the Hsp90-Cdc37 complex and suppressing its downstream AKT/glycolysis axis and AKT/NF-κB pathway, leading to reduced lactate and IL-1β production. Our findings suggest that CEP could serve as a promising ASFV inhibitor, and the Hsp90-Cdc37 complex and glycolysis represent novel antiviral targets against ASFV infections, offering novel avenues for further exploration in antiviral therapeutic strategies. As the in vivo environment is largely complicated from ex vivo PAMs, anti-ASFV efficacy evaluation of CEP in pigs is the most imperative work in the future.