ABSTRACTPorcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, multisystemic inflammation, and reproductive failure. Heme oxygenase-1 (HO-1), a stress-inducible enzyme, exerts protective functions by converting heme into carbon monoxide (CO), biliverdin (BV), and iron. However, the effects of HO-1 and its metabolites on PCV3 replication remain unknown. In this study, experiments involving specific inhibitors, lentivirus transduction, and small interfering RNA (siRNA) transfection revealed that active PCV3 infection reduced HO-1 expression and that the expression of HO-1 negatively regulated virus replication in cultured cells, depending on its enzymatic activity. Subsequently, the effects of the HO-1 metabolites (CO, BV, and iron) on PCV3 infection were investigated. The CO inducers (cobalt protoporphyrin IX [CoPP] or tricarbonyl dichloro ruthenium [II] dimer [CORM-2]) mediate PCV3 inhibition by generating CO, and this inhibition is reversed by hemoglobin (Hb; a CO scavenger). The inhibition of PCV3 replication by BV depended on BV-mediated reactive oxygen species (ROS) reduction, asN-acetyl-l-cysteine affected PCV3 replication while reducing ROS production. The reduction product of BV, bilirubin (BR), specifically promoted nitric oxide (NO) generation and further activated the cyclic GMP/protein kinase G (cGMP/PKG) pathway to attenuate PCV3 infection. Both the iron provided by FeCl3and the iron chelated by deferoxamine (DFO) with CoPP treatment failed to affect PCV3 replication. Our data demonstrate that the HO-1-CO-cGMP/PKG, HO-1-BV-ROS, and HO-1-BV-BR-NO-cGMP/PKG pathways contribute crucially to the inhibition of PCV3 replication. These results provide important insights regarding preventing and controlling PCV3 infection.IMPORTANCEThe regulation of host protein expression by virus infection is the key to facilitating self-replication. As an important emerging pathogen of swine, clarification of the interaction between PCV3 infection and the host enables us to understand the viral life cycle and pathogenesis better. Heme oxygenase-1 (HO-1) and its metabolites carbon monoxide (CO), biliverdin (BV), and iron have been demonstrated to involve a wealth of viral replications. Here, we, for the first time, demonstrated that HO-1 expression decreases in PCV3-infected cells and negatively regulates PCV3 replication and that the HO-1 metabolic products CO and BV inhibit PCV3 replication by the CO- or BV/BR/NO-dependent cGMP/PKG pathway or BV-mediated ROS reduction, but the iron (the third metabolic product) does not. Specifically, PCV3 infection maintains normal proliferation by downregulating HO-1 expression. These findings clarify the mechanism by which HO-1 modulates PCV3 replication in cells and provide important targets for preventing and controlling PCV3 infection.