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Figure 1. Schematic of the mature BTV particle. Organisation of the major structural proteins VP2, VP5, VP3 and VP7 in the architecturally complex BTV particle. On entry into cells the outer capsid proteins VP2 and VP5 are lost, releasing a transcriptionally active core particle.
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Figure 2. Distribution of VP2 within infected and transfected Vero cells by fluorescence microscopy. A) Cells infected with BTV-10, B-E) transfected cells expressing B) VP2, C) VP2-GFP, D) GFP-VP2 and E) GFP only. VP2 in A and B were detected with anti VP2 monoclonal antibody (rabbit) and either FITC (A) or TRITC (B) conjugated secondary antibody. C-E were visualised based on GFP fluorescence. Expression of full-length, tagged VP2 variants was confirmed by western blot using an anti-GFP antibody (F).
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Figure 3. VP2 segregates with vimentin in infected and transfected cells. A) Distribution of VP21–118GFP (left), VP2-GFP (centre), and VP2-GFP with ubiquitin (red, right) in transfected cells. Amino acids 1–118 give similar localisation to full-length protein. There is no evidence of co-localisation of ubuquitin with the punctuate distribution of VP2. B) Fractionation of untreated cells and cells transfected with plasmid expressing GFP or VP21–118GFP. Cells were solubulised in the presence of 1% Triton X-100 and fractionated into detergent insoluble (DI) and detergent soluble (DS) fractions. Western blot was used to detect fractionation of the cytoskeletal marker proteins actin, tubulin and vimentin and the nuclear envelope marker lamin A. Fractionation of VP21–118GFP and GFP was followed using anti-GFP antibody. VP21–118GFP co-fractionated with vimentin but none of the other proteins. C) Imunofluorescence microscopy co-localisation of VP21–118GFP with vimentin, lamin, actin and tubulin, as indicated. D) Distribution of vimentin (red) and untagged VP2 (green) in virus infected cells at 8 hours (top row) and 24 hours (bottom row) post infection. Note co-localisation of VP2 and vimentin even at early times post infection.
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Figure 4. Deletion anlysis of VP21–118GFP. A) Schematic showing the position of deletions generated in the VP21–118GFP. B) Fluorescence images showing the distribution of VP21–118GFP deletion mutants from A. Deletion of 65–92 and 93–114 was sufficient to abolish intracellular localisation of the N terminus of VP2.
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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.
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Figure 6. Disrupting vimentin inhibits virus release in BTV infected cells. A) Co-localization of untagged VP2 with vimentin in transfected cells. Top, untreated cells; Middle, cells treated with colchicine to disrupt microtubules; bottom, cells treated with acrylamide to disrupt vimentin. In each row VP2 and vimentin localisation are shown in green and red respectively. As expected, disruption of microtubules with colchicine causes a rearrangement in the localisation of vimentin. B) Effect of treating cells with colchicine on the release of virus from infected cells. Cells and culture medium were harvested at 4, 8 and 24 hours post infection with BTV-10 in untreated and colchicine (5 μg/ml) treated cells. Control samples are labelled 4c, 8c and 24c, respectively. The titre of cell associated and released virus for each timepoint normalised to 100% for untreated cells. Colchicine treatment resulted in a time dependent increase in the titre of cell associated virus and a time-dependent decrease in the titre of virus in the culture medium. D) As C but for cells treated with acrylamide (50 μM) to directly disrupt the vimentin network. Effect of acrylamide was similar but much faster and more dramatic than that observed by indirect disruption of vimentin through disruption of microtubules using colchicine. For C and D error bars indicate the standard error of three replicates of the experiments.
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