Figure 3

BTV non-structural protein 2 interacts with microtubules and the condensed chromosomes, and can induce aberrant mitoses in the absence of other BTV proteins. (A) Plasmid pNS2-V5 was transfected into uninfected BHK-21 cells. After 24 hours the cells were fixed and immunolabeled using an antibody targeting the V5 epitope (green). Microtubules were revealed using alpha tubulin labelling (red). Small aggregates of NS2 were closely associated with the microtubules, suggesting that other BTV proteins are not required for the interaction to occur. Scale bar = 5 μm. (B) In BTV-16v infected mitotic BHK-21 cells, NS2 (red - arrows) was observed (using z-stack and deconvolution analysis) associated with the condensed chromosomes (blue) in locations suggestive of the kinetochores. Scale bar = 5 μm. (C) and (D) Uninfected mitotic cells transfected with pNS2-V5 (green) exhibited similar aberrant mitoses to those observed during BTV infection, including chromosomes localised parallel to the microtubule spindle (C) and multiple spindles (D). In these mitotic cells, NS2-V5 staining was diffuse, as opposed to the condensed VIB-like form seen in BTV infected cells. Scale bar = 10 μm.