Structural and functional insights into the tubule assembly mechanism of the Cauliflower mosaic virus movement protein

Some plant viruses, with RNA and DNA genomes, move from cell to cell in a tubule-guided fashion. The movement protein (MP) is the essential structural component of these tubules crossing highly modified plasmodesmata (PD) upon removal of the desmotubule. Entire virus particles move throughout tubules to adjacent cells. While the ultrastructural properties of MP tubules have been largely studied, the molecular mechanism of assembly and the mechanistic aspects of virus particle transport through them remain completely unknown. The MP of Cauliflower mosaic virus (CaMV) forms tubules that guide the movement of encapsidated virus particles via coiled-coil interaction with the virion-associated protein (VAP)1. We used scanning deletion mutagenesis starting from the MP C-terminus to determine the shortest sequence required for tubule formation. Expression of the deletion mutants in fusion with the green fluorescent protein (GFP) in Nicotiana benthamiana protoplasts revealed that the C-terminal coiled-coil domain (32-terminal amino acids), mediating interaction with VAP, is involved neither in tubule assembly nor in their stabilization. All mutants longer than 280 aminoacids (full length MP, 327 amino acids) formed protruding tubular structures whereas deletion or mutation of the two leucines occupying the positions 279 (L279) and 280 (L280) abolished tubule formation but did not affect protein targeting to plasma membrane (PM). Noticeably, dileucine motifs are cargo selection and sorting signals for recruitment to clathrin-coated vesicles2. Consistently with this observation, we also demonstrated that tubule assembly of MP is hindered upon treatment of N. benthamiana protoplasts with Brefeldin A, which inhibits anterograde vesicle transport including recycling from endosomes back to PM. These observations support the hypothesis that CaMV MP may use vesicular trafficking not only to be correctly supplied and targeted to the PM3 but also specifically for tubule assembly. To gain more insight into the arrangement of MP molecules within the tubule structure, we examined a possibility of MP self-interaction other than via the coiled-coil domain1. To this aim, the N-terminal (MPa), central (MPb), and C-terminal (MPc) fragments of the MP sequence were expressed in fusion to GST and used in GST-pull down experiments to test their interaction with a full-length CaMV MP tagged with the Hemagglutinin epitope (MP-HA). This experiment revealed the presence of binding regions in the MPa and MPc fragments. Analysis of another mutant of the GST-C-terminal fragment (MPc?cc) confirmed that the domain of self-interaction in the MPc fragment was distinct from the coiled-coil domain. Further investigations contributed to demonstrate the homo-interaction character of the fragments and allowed to form a hypothesis on how CaMV MP monomers oligomerize in the tubule structure. Finally, we demonstrated that self-interaction of the MPc fragment is not hindered upon deletion of the two leucines (L279 and L280). This evidence probably precluded an important role for the dileucine motif in the structural assembly of MP tubules whereas their essential requirement for tubule formation supports and strengthens the involvement of vesicle traffic in this MP function.