Discovery of Small Molecules Against Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting NendoU Activity

Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to animal health and causes substantial economic losses worldwide. The nonstructural protein 11 (NSP11) of the causative agent, PRRSV, contains a highly conserved uridylate-specific endoribonuclease (NendoU) domain essential for viral replication and immune evasion. Targeting NSP11 offers a novel approach to antiviral intervention. Through in silico virtual screening followed by a FRET assay, we identified A8-A2 as a promising candidate that effectively inhibits NendoU activity. Molecular docking and mutational analysis revealed that A8-A2 and its analogs target the key catalytic residues His144 and Thr217 of NSP11, located within the NendoU enzyme activity loop and pocket region, respectively. A8-A2 demonstrated dose-dependent inhibition of PRRSV replication in porcine alveolar macrophages (PAMs). Notably, the NendoU is conserved across PRRSV strains and other Nidoviruses, and A8-A2 exhibited antiviral activity against both type I and type II PRRSV strains, as well as the infectious bronchitis virus (IBV), a coronavirus in the order Nidovirales. Further investigations revealed that A8-A2 impedes viral replication early in infection and reverses NSP11-mediated suppression of Poly(I:C)-induced interferon production. However, this effect occurs independent of mRNA splicing inhibition. These findings indicate that A8-A2 could act as an effective antiviral agent against infections caused by diverse PRRSV strains and may serve as a broad-spectrum agent for other Nidoviruses.