Figure 5
Fine mapping of the influence of VP2 amino acids 65–114 on localisation. A) Schematic showing the alignment of BTV-10 VP2 protein sequence to a consensus sequence generated from alignment of 13 BTV serotypes. Mutants generated (M1-M5) which disrupt conserved amino acid positions are indicated. Amino acids which are completely conserved or conserved in charge are indicated by (*) or (:) respectively. Weakly conserved amino acids are indicated by (.). Amino acids targeted for mutagenesis are shown in bold in the consensus and BTV-10 VP2 sequences. Sequences predicted in silico to form β-sheet are underlined. B) Kyte and Dolittle mean hydrophobicity profile[45], generated using a scan window size of 13 on BTV-10 VP2 1–114 and M5 VP2 1–114, as indicated. C) Fluorescence microscopy images of cells transfected with plasmid expressing VP21–118GFP or M1-M5 as indicated. Intracellular localisation of VP2 is abolished in M5. D) Co-localisation of VP21–118GFP and M5 with vimentin (red). Vimentin distribution is unaffected in cells expressing M5 but the VP2 mutant is found throughout the cell. E) Trimerisation of full-length VP2 carrying the M5 mutation. Western blot of non-denatured samples for unmodified (left panel) and M5 (right panel) VP2 variants. Note that trimerisation is unaffected in the M5 mutant.