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. 2013 Oct 28:10:319.
doi: 10.1186/1743-422X-10-319.

Bluetongue virus infection induces aberrant mitosis in mammalian cells

Andrew E Shaw, Anke Brüning-Richardson, Ewan E Morrison, Jacquelyn Bond, Jennifer Simpson, Natalie Ross-Smith, Oya Alpar, Peter P C Mertens  1 , Paul Monaghan
Affiliations

Bluetongue virus infection induces aberrant mitosis in mammalian cells

Andrew E Shaw et al. Virol J. .

Abstract

Background: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division.

Methods: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2.

Results: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects.

Conclusions: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.

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Figures

Figure 1
Figure 1
BTV induces aberrant mitosis in cultured mammalian cells. Cells were cultured in the presence of growth medium containing 10% serum and either infected or mock infected with BTV. At 16–24 hours post infection cells were fixed with paraformaldehyde and prepared for confocal immunofluorescence microscopy as described in the Materials and Methods. (A) Uninfected BHK-21 cells showed highly organised and symmetrical microtubule spindles. (B) In contrast, BTV-16v infected mitotic cells had multiple, disorganised and asymmetric spindles (alpha tubulin labelling in green) that were disassociated from the condensed chromosomes (blue). (C) and (D) BTV-1 and BTV-8 were also able to induce aberrant mitosis in BHK-21 cells, with NS2 (red) associated with the chromosomes. Vero cells and bovine pulmonary aortic endothelial (BPAEC) cells infected with BTV-16v (F) and (H) also showed abnormal mitotic events compared to the uninfected controls (E) and (G). Scale bar = 10 μm.
Figure 2
Figure 2
BTV affects the microtubule organising centre (MTOC) and disrupts the interaction between condensed chromosomes and microtubules. BTV-16v-infected or mock infected BHK-21 cells were fixed and assessed for microtubule organising centre (MTOC) integrity using confocal microscopy. (A) Uninfected cells in interphase showed a loose array of microtubules (alpha tubulin labelling, green) with a single MTOC (gamma tubulin labelling, red). (B) Uninfected but mitotic cells contained a single highly organised microtubule spindle with an MTOC at each pole, associated with the condensed chromosomes (blue) arranged perpendicular to the spindle. Mitotic cells infected with BTV-16v lacked organisation and symmetry in the spindle and the MTOC appeared to be disrupted (C), with the MTOC also disrupted in infected cells in interphase (D). Centrin labelling was altered, from a largely punctate form in uninfected cells (E) to a more dispersed distribution in infected cells (F). NS1 (green) localised to the region of the MTOC. (G,H) In contrast, FOP (arrows) labelling remained punctate in both infected and uninfected cells. (I) In uninfected cells, p150glued (green) exhibited a diffuse cytoplasmic distribution in interphase and a spindle-like formation in mitosis (insert), but in infected cells (J) p150glued was more condensed and localised towards the nucleus in a region suggestive of the MTOC. (K) EB1 labelling of the growing tips of microtubules shows a close association with the condensed chromosomes in uninfected mitotic cells. (L) In contrast, EB1 labelling and the microtubule tips showed a wider distribution and less association with the condensed chromosomes in infected mitotic cells. Scale bar = 10 μm.
Figure 3
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.
Figure 4
Figure 4
Live cell imaging of HeLa-mCherry tubulin cells shows that transfection with EGFP-NS2 leads to prolonged mitosis and binucleation events. HeLa-mCherry cells mock transfected or transfected with plasmids expressing either EGFP or an EGFP-NS2 fusion proteins were followed using live cell imaging as described in Materials and methods. (A) Cells transfected with pEGFP-NS2 spent a significantly longer time in mitosis than cells transfected with pEGFP alone, or untransfected cells (p = 0.038). (B) Cells expressing EGFP-NS2 showed the lowest level of completed mitosis (p < 0.0001). (C) In cells that successfully completed mitosis, only those transfected with EGFP-NS2 displayed binucleation events (p < 0.0001), while mock transfected cells, or cells expressing EGFP alone showed a normal mitosis outcome.
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