Signal peptide cleavage and ectodomain regions of GP2 are required for PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically important pathogen of swine, yet the molecular mechanisms governing its entry into host cells remain incompletely understood. The minor envelope glycoprotein GP2, together with GP3 and GP4, forms an essential complex that engages the entry receptor CD163; however, the specific GP2 regions required for receptor association have not been fully defined experimentally. Here, we investigated GP2 processing, intracellular trafficking and interactions with viral glycoproteins and CD163. We demonstrate that the GP2 signal peptide (SP) is cleaved in both transfected and infected cells and is necessary and sufficient for ER localization. Removal of the SP disrupted GP2 maturation, impaired interactions with GP3, GP4 and GP5, and significantly reduced viral infectivity in infectious clone assays. Deletion of the SP also abolished GP2-CD163 association, indicating that proper SP-dependent processing is required for receptor engagement. Using co-immunoprecipitation and colocalization analyses, we identified two highly conserved regions within the GP2 ectodomain that associate with CD163. Deletion of either region completely eliminated PRRSV infection. Together, these findings define the structural determinants within GP2 required for association with CD163 and advance our understanding of the early steps of PRRSV entry.