An alternative reverse genetics system for PRRS virus and its application to define the role of endocytic sorting signal in GP3 protein intracellular trafficking.

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 3 (GP3) forms a heterotrimeric complex with GP2 and GP4, which is essential for viral entry and assembly. However, the intracellular trafficking mechanisms governing GP3 localization and incorporation into virions remain incompletely understood. Here, we identified two highly conserved tyrosine-based sorting signals (YxxΦ) within GP3, motifs that mediate adaptor protein-dependent trafficking through the secretory and endocytic pathways. To define the functional roles of these motifs, we established a Linear Overlapping Infectious Polymerase Amplicon (LOIPA)-based reverse-genetics system for PRRSV. This system enabled precise reconstitution of full-length viral genomes from overlapping cDNA fragments and facilitated rapid introduction of site-specific mutations without bacterial cloning. Using LOIPA, we generated a set of recombinant PRRSV mutants carrying targeted substitutions within the two GP3 YxxΦ motifs. Mutation of Y108A in the YAWL motif at positions 108-111 disrupted GP3 sorting to downstream ER-Golgi intermediate compartments (ERGIC) and markedly reduced infectious virion production. In contrast, mutations in the YVDI motif did not alter GP3 trafficking patterns but exerted limited effects on viral replication, suggesting an indirect regulatory role. Interestingly, the ectopic monomeric expression of GP3-Y108A showed similar trafficking patterns to those of GP3-WT. These results provide novel insights into the molecular interplay between PRRSV envelope proteins and host trafficking machinery, contributing to a deeper understanding of PRRSV assembly, virion morphogenesis, and secretory dynamics. Our study also established LOIPA as a rapid and bacteria-free reverse genetics system for PRRSV, which is readily applicable to other member viruses in the family Arteriviridae, enabling functional interrogation of viral genes and rational engineering to produce mutant viruses.