Contrasting Effects of Tagging Turnip Mosaic Virus Proteins.

Potyvirus rapae (turnip mosaic virus, TuMV) is widely used as a model system in plant-virus interaction studies. The TuMV RNA genome encodes 11 proteins, some of which remain poorly characterized, while the functions of others are well defined. Studying individual proteins in isolation may not recapitulate native expression levels, subcellular localization, and interaction with host factors during virus replication and movement. An alternative approach is to tag individual viral proteins in the context of an infectious clone. Epitope tags may alter protein functions and affect viral replication, movement, or a combination of essential steps, thus leading to changes in pathogenicity. Because they have central roles in viral infection, here we measured the effect of individually tagging the helper component proteinase (HC-Pro) and nuclear inclusion protein b (NIb) with a 6His-3xFLAG tag. Epitope tags were placed at the N-terminus of HC-Pro and the N- and C-termini of NIb within a TuMV infectious clone carrying coding sequences for the green fluorescent protein (TuMV-GFP). Constructs carrying a tagged HC-Pro displayed pathogenicity similar to that observed for TuMV-GFP in Nicotiana benthamiana and Arabidopsis thaliana plants. In contrast, infectivity of NIb-tagged clones became temperature sensitive and, even at the permissive temperature, showed reduced pathogenicity compared to TuMV-GFP. Providing a silencing suppressor in trans did not restore infection efficiency, suggesting reduced viral fitness due to structural or functional disruption caused by the epitope tags. Structural models generated using AlphaFold2 showed no effect of the tag on HC-Pro. In contrast, structural models illustrated tag interference with the NIb catalytic site. AlphaFold2 was further used to predict the structural impact of several tags on NIb and to predict the effect of a 6HIS-3xFlag tag on all other TuMV proteins. This study provides a broadly applicable framework for selecting suitable epitope tags to mark viral proteins and maintain function in the context of virus infection.