Carboxypeptidase A6 contributes to bovine enterovirus F production through apoptosis-associated PI3K/AKT/FOXO1 signaling

Bovine enterovirus (BEV), a member of the family Picornaviridae, is an understudied pathogen associated with bovine enteric and respiratory disease, and host factors supporting its infection remain poorly defined. Here, a genome-wide CRISPR/Cas9 loss-of-function screen in Madin–Darby bovine kidney cells identified carboxypeptidase A6 (CPA6) as a candidate host factor for BEV-F infection. CPA6 deficiency impaired productive BEV-F infection, as shown by reduced viral titers, extracellular viral RNA, and viral protein expression, whereas CPA6 complementation restored these phenotypes. Stage-specific analyses indicated that CPA6 did not significantly affect viral attachment or internalization but contributed to late-stage infection associated with extracellular viral accumulation. BEV-F infection induced CPA6 expression and apoptosis, while CPA6 deficiency significantly attenuated virus-induced apoptosis. Further analyses showed that CPA6 loss enhanced PI3K/AKT signaling, increased FOXO1 phosphorylation, and reduced nuclear FOXO1 localization during infection. Pharmacological inhibition of PI3K in CPA6-knockout cells partially restored FOXO1 nuclear accumulation, apoptosis, and viral production, indicating a functional association between CPA6 and the PI3K/AKT/FOXO1 axis. Rescue experiments using wild-type CPA6 and a catalytic mutant further showed that CPA6 enzymatic activity contributed substantially to viral production, FOXO1 regulation, and apoptosis. Finally, inhibition of carboxypeptidase-associated activity with 2-benzylsuccinic acid reduced BEV-F infection in vitro and lowered intestinal viral burden and tissue injury in mice. Collectively, these findings identify CPA6 as a previously unrecognized host factor for BEV-F infection, support a model in which CPA6 is functionally associated with PI3K/AKT/FOXO1 signaling related to apoptosis, and suggest that carboxypeptidase-associated pathways may represent potential avenues for antiviral intervention.