ABSTRACTPositive-sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as “viral factories” to coordinate diverse viral processes, such as genome translation and replication. It is also becoming clear that enclosing viral RNA (vRNA) complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the familyPotyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the vRNA to plasmodesmata for delivery into adjacent noninfected cells. The viral protein 6K2is responsible for the formation of these vesicles, but how 6K2is involved in their biogenesis is unknown. We show here that 6K2is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12-amino-acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation experiments showed that 6K2interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in anArabidopsis thalianamutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K2, which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a.IMPORTANCEMany plant viruses remodel the endoplasmic reticulum (ER) to generate vesicles that are associated with the virus replication complex. The viral protein 6K2of turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K2is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K2physically interacts with the COPII coatomer Sec24a and that anArabidopsis thalianamutant line with a defective Sec24a shows a delay in the systemic infection by TuMV.
Analysis of tobamovirus multiplication in Arabidopsis thaliana mutants defective in TOM2A homologues
