Role of a Synonymous Mutation at Codon 178 in P1 That Attenuates Potato Virus Y-Induced Tobacco Vein Necrosis and Its Application for Expressing Human Interferon.

Plant virus-based vectors are gaining significant attention as bioreactors for recombinant protein production due to their broad host range, robust expression levels and cost-effectiveness; however, their practical utility is frequently constrained by severe viral pathogenicity. Notably, the utility of potato virus Y (PVY), the type member of the genus Potyvirus, is severely hampered by its elicitation of tobacco vein necrosis (TVN). Here, we demonstrate that a single synonymous substitution (AAA to AAG) at the K178 codon of P1 causes almost no TVN. Mechanistically, the P1-AAG mutation attenuates the synergistic RNA silencing suppression (RSS) activity typically mediated by the P1/HC-Pro complex. This functional impairment abrogates the virus-induced upregulation of specific host microRNAs (miR6020, miR6164 and miR6021). Using this non-necrotic PVY-P1-AAG vector, we engineered an optimised bioproduction platform. By integrating a GFP-HRV3C fusion tag system with affinity purification and subsequent proteolytic cleavage, we achieved the high-yield synthesis of recombinant human interferons (IFN-α1b and IFN-α2b) in Nicotiana benthamiana and Nicotiana tabacum 'Xanthi'. Codon optimisation of the cargo genes further enhanced protein accumulation, achieving yields of up to 55.2 μg/g fresh leaf weight. Taken together, these results provide new insights into how synonymous substitutions influence potyviral pathogenicity and validate the symptomless P1-AAG vector as a robust system for biomanufacturing value-added proteins.