Porcine reproductive and respiratory syndrome virus ORF5a protein is essential for virus viability

► RNA sequences in the overlapping region between ORF5 and ORF5a was essential for virus viability. ► Inactivation of ORF5a expression was lethal for the production of infectious virus. ► Viable PRRSV could be recovered by expressing ORF5a protein in trans. ► ORF5a protein could be putatively extended to 63 aas. It has been shown that ORF5a protein in EAV is important but not essential for virus infectivity. In this study, we found that RNA changes in the overlapping region (1–104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for virus viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for virus viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.