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

ABSTRACT

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.