<i>Citrus psorosis virus</i> (CPsV) and <i>Mirafiori lettuce big-vein virus</i> (MiLBVV), members of the <i>Ophioviridae</i> family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution. Upon transient expression in <i>Nicotiana benthamiana</i> epidermal cells the 54K protein, and also its 54K counterpart protein of MiLBVV, localize to plasmodesmata and enhance GFP cell-to-cell diffusion between cells. Both proteins, but not the coat proteins (CP) of the respective viruses, functionally <i>trans</i>-complement cell-to-cell movement-defective <i>Potato virus</i> X (PVX) and <i>Tobacco mosaic virus</i> (TMV) mutants. The 54K and 54K proteins interact with the virus-specific CP in the cytoplasm, suggesting a potential role of CP in ophiovirus movement. This is the first study characterizing the movement proteins (MP) of ophioviruses.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecular