Subcellar Localization And Nucleic Acid Binding Ability Of VP37 Protein Of Broad Bean Wilt Virus 2

The 53kD and 37kD proteins (named VP53 and VP37, respectively) encoded by the 5'-terminal genes of RNA2 of Broad bean wilt virus 2 (BBWV-2) are C-co-terminally overlapping proteins results from two potential translation sites in RNA2. VP53/VP37 genes have similar positions in RNA2 to 58-kD/48-kD movement protein (MP) genes of Cowpea mosaic virus (CPMV) and encoded conserved motif of '30K super group' MP.Thin sections of host leaf cells infected by BBWV-2 isolate B935, which were gold-labeled by antibodies of BBWV-2 coat protein (CP) and VP37, respectively, were prepared to elucidate the locations of VP37 in cell and possible function of VP37 and CP in cell to cell movement. Observation in electron microscope showed that virus particles were presented not only in cytoplasma but also in chloroplast, while VP37 was existed only in cytoplasma and associated with tubular structure through the cell wall. Western blot assay using purified chloroplast confirmed that only CP was existed in it.In order to know whether VP37 has the ability to combine nucleic acid, which was thought as the basic characteristic of plant virus MP, gel-retardation and UV-crosslinking assays were employed. Results show that VP37 protein can bind single strand nucleic acid cooperatively and nonspecificallty, and the VP37-ssRNA complex was stable at high salt concentrations, suggesting VP37 is a possible MP. VP37 is the only protein characterized so far showing RNA-binding ability in genus Fabavirus.VP53 and VP37 genes were fused with green fluorescent protein (GFP) in their C-termini by overlapping PCR, and the fusion genes were successfully inserted into expression vector pRTL2. The construction of VP53/GFP and VP37/GFP genes will help us for further understanding of the functions of VP53/VP37 in cell-to-cell and long-distant movement in plant.Different hybridoma cell lines secreting monoclonal antibodies (Mabs) againstBBWV-1 and BBWV-2 were used and TAS-ELISA were applied to detect isolates of each virus, it is proved that TAS-ELISA with produced MAbs can be used for differentiation of two BBWV species.