Aminoquinoline surfen inhibits pseudorabies virus attachment by preventing the binding of glycoprotein C to heparan sulfate

Pseudorabies virus (PRV) is an important herpesvirus that infects pigs and many other animals. PRV can also infect humans, but the relevant reports are still very rare. PRV is not only a serious concern in veterinary medicine, but also a potential threat to public health. Thus, the development of effective antiviral agents against PRV is needed for its prevention and control. PRV utilizes viral glycoprotein C (gC) to bind heparan sulfate (HS) on target cells to complete virus attachment. Here, we identified aminoquinoline surfen, a small-molecule antagonist of HS, as a novel and potent antiviral agent against PRV. We demonstrated that surfen not only effectively inhibited PRV infection in multiple permissive pig, human, and mouse cell lines, but also significantly elevated mice survival after lethal PRV challenge. Surfen pre-treatment significantly reduced PRV attachment and infectious virus production in target cells. Enzymatic removal of HS chains by heparinase or addition of exogenous HS counteracted PRV inhibition by surfen, demonstrating that its antiviral activity is dependent on HS. Notably, surfen lost its ability to inhibit the attachment of PRV gC-deficient virus, which enters cells in an HS-independent manner, indicating that the binding of surfen to HS interferes with the interaction between PRV gC and HS on the cell surface. Cumulatively, these findings demonstrate that surfen inhibits PRV attachment by preventing the binding of PRV gC to HS on target cells and reveal that targeted disruption of PRV gC-HS interaction is an effective strategy to develop antiviral drugs to defend PRV infection.IMPORTANCEPseudorabies virus (PRV) causes severe respiratory, reproductive, and neurological disorders in pigs, and its infection in humans is also reported occasionally. PRV is not only a serious concern in veterinary medicine, but also a potential threat to public health. Therefore, the development of effective antiviral agents against PRV is needed to prevent its infection and spread. Here, we demonstrated that aminoquinoline surfen, a small-molecule antagonist of HS, effectively inhibited PRV infection in multiple permissive cell lines <i>in vitro</i> and significantly elevated mice survival after lethal PRV challenge <i>in vivo</i>, revealing it as a novel and potent antiviral agent against PRV. Mechanistic studies demonstrated that surfen inhibited PRV attachment by preventing the binding of PRV viral glycoprotein gC to heparan sulfate on target cells. Cumulatively, these findings reveal that targeted disruption of PRV gC-HS interaction is an effective strategy to develop antiviral drugs to defend PRV infection.