Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting pigs and belongs to the enveloped plus-stranded RNA virus family Arteriviridae. A unique feature of Arteriviruses is that the genes encoding the structural proteins overlap at their 3` and 5` ends. This impedes mutagenesis opportunities and precludes the binding of short peptides for antibody detection, as this would alter the amino acids encoded by the overlapping gene. In this study, we aimed to generate infectious PRRSV variants with separated genes encoding the minor glycoproteins Gp2, Gp3, and Gp4, accompanied by appended tags for detection. All recombinant genomes facilitate the release of infectious virus particles into the supernatant of transfected 293 T cells, as evidenced by immunofluorescence of infected MARC-145 cells using anti-nucleocapsid antibodies. Furthermore, expression of Gp2-Myc and Gp3-HA was confirmed through immunofluorescence and western blot analysis with tag-specific antibodies. However, after two passages of Gp2-Myc and Gp3-HA viruses, the appended tags were completely removed as indicated by sequencing the viral genome. Recombinant viruses with separated Gp2 and Gp3 genes remained stable for at least nine passages, while those with Gp3 and Gp4 genes separated reverted to wild type after only four passages. Notably, this virus exhibited significantly reduced titers in growth assays. Furthermore, we introduced a tag to the C-terminus of Gp4. The Gp4-HA virus was consistently stable for at least 10 passages, and the HA-tag was detectable by western blotting and immunofluorescence. • Most genes encoding the structural proteins are overlapping in the PRRSV genome. • We separated the genes encoding Gp2, Gp3 and Gp4 of PRRSV-2 and added small tags. • Although initially viable, recombinant Gp2-Myc and Gp3-HA viruses lose the tags. • Even viruses with separated genes rapidly reverted to the wild-type sequence. • ORF4 does not overlap with the subsequent genes and Gp4-HA virus is stable.