Lettuce Big-Vein Associated Virus ORF3 Encodes a Functional 30K Movement Protein

Movement proteins (MPs) modulate the size exclusion limit of plasmodesmata—membrane-lined channels connecting plant cells—thereby allowing cell-to-cell movement and systemic spread of plant viruses. The largest and arguably best-studied group of MPs is the 30K superfamily. Its family members share little sequence similarity, with only a handful of residues being well conserved. Yet, all family members appear to adopt the same jelly-roll protein fold structure. Lettuce big-vein associated virus (LBVaV), a member of the Rhabdoviridae family, is closely associated with lettuce big-vein disease (LBVD). It appears to facilitate the long-distance movement of Mirafiori lettuce big-vein virus (MiLBVV) in plants through an unknown mechanism. Notably, enhanced MiLBVV spread correlates with severe LBVD symptoms. Despite LBVaV having been known for decades, its proteins have not been studied in detail thus far. By using a combination of Alphafold2 structure modelling and FoldSeek structure-based homology searches, we managed to annotate all LBVaV open reading frames (ORFs), with ORF3 clustering with the 30K superfamily. While ORF3 is the most conserved protein sequence among the LBVaV-encoded ORFs, it shares only 5%–11% protein sequence identity with related MPs in the same genus. Microscopy studies confirmed that ORF3 locates at plasmodesmata, and in planta expression of ORF3 allowed cell-to-cell movement of two movement-impaired plant viruses. Thus, the Alphafold2-FoldSeek strategy allowed successful annotation of a plant viral genome even when viral proteins show little sequence similarity.