Mutual association of Broad bean wilt virus 2 VP37-derived tubules and plasmodesmata obtained from cytological observation.

The movement protein VP37 of broad bean wilt virus 2 (BBWV 2) forms tubules in the plasmodesmata (PD) for the transport of virions between cells. This paper reports a mutual association between the BBWV 2 VP37-tubule complex and PD at the cytological level as determined by transmission electron microscopy. The generation of VP37-tubules within different PD leads to a different occurrence frequency as well as different morphology lines of virus-like particles. In addition, the frequency of VP37-tubules was different between PD found at different cellular interfaces, as well as between single-lined PD and branched PD. VP37-tubule generation also induced structural alterations of PD as well as modifications to the cell wall (CW) in the vicinity of the PD. A structural comparison using three-dimensional (3D) electron tomography (ET), determined that desmotubule structures found in the center of normal PD were absent in PD containing VP37-tubules. Using gold labeling, modification of the CW by callose deposition and cellulose reduction was observable on PD containing VP37-tubule. These cytological observations provide evidence of a mutual association of MP-derived tubules and PD in a natural host, improving our fundamental understanding of interactions between viral MP and PD that result in intercellular movement of virus particles.

Cell-to-cell trafficking, subcellular distribution, and binding to coat protein of Broad bean wilt virus 2 VP37 protein.

Broad bean wilt virus 2 (BBWV 2) is a member of the genus Fabavirus of the family Comoviridae. To date, a movement protein (MP) of BBWV 2 has not been described. Here we demonstrate that the green fluorescent protein (GFP)-VP37 fusion protein can move from initial bombarded cells to neighboring cells in Nicotiana benthamiana epidermal leaves. In addition, the GFP-VP37 fusion protein localizes as a halo around the nucleus and as punctate spots on the cell periphery in N. benthamiana epidermal leaf cells and BY-2 suspension cells. Fluorescence near the nucleus also was co-localized with the endoplasmic reticulum in BY-2 cells. Fibrous networks were found in GFP-VP37 agro-infiltrated N. benthamiana epidermal leaf cells. Deletion analyses indicated that the C-terminal region of the VP37 protein is essential for localization at the cell periphery. Using a blot overlay assay and bimolecular fluorescence complementation assay, the purified 6xHis-tagged VP37 protein was shown to bind specifically to the small coat protein of BBWV 2. The above results indicate that VP37 is a movement protein.

In vivo accumulation of Broad bean wilt virus 2 VP37 protein and its ability to bind single-stranded nucleic acid.

The VP37 protein encoded by the RNA2 of Broad bean wilt virus 2 (BBWV2) was overexpressed in Escherichia coli. The protein was purified and a polyclonal antibody specific for the protein was produced. Time course studies by Western blot assays in BBWV2-infected Chenopodium quinoa leaves showed that the VP37 protein was present in cells of the inoculated leaves by 12 h post inoculation and in cells of systemically-infected leaves by 2 days post inoculation. The protein was able to accumulate to a high level in infected leaves at the late infection stage. Gel retardation and UV cross-linking assays demonstrated that the VP37 protein bound preferentially single-stranded (ss) RNA and DNA in a non-sequence-specific manner. The VP37 protein-RNA complex was stable in solutions containing less than 400 mM NaCl, but became fully dissociated in the solutions containing 800 mM NaCl. Sequence analysis of the VP37 protein and its ability to bind ssRNA and ssDNA suggest that the protein may play a role similar to the movement proteins reported for other plant viruses.