Abstract
BackgroundBluetongue 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.MethodsWe 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.ResultsOur 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.ConclusionsWe 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.
Highlights
Bluetongue virus (BTV) is an arbovirus that is responsible for ‘bluetongue’, an economically important disease of livestock
Aberrant cell division during BTV infection During initial studies of NS2 interaction with microtubules in Baby hamster kidney-21 (BHK-21) cells in the context of a BTV infection, we observed a substantial number of cell divisions that appeared abnormal
The most conspicuous feature of BTV infected cell cultures examined by confocal microscopy was the large number of rounded cells, apparently arrested in mitosis
Summary
Bluetongue virus (BTV) is an arbovirus that is responsible for ‘bluetongue’, an economically important disease of livestock. BTV is well characterised at the protein level, less is known regarding its interaction with host cells. Bluetongue virus (BTV) is an arbovirus that is transmitted between its ruminant hosts by species of Culicoides biting midge. The infection of ruminants with BTV can result in bluetongue (BT), an economically important disease of livestock. BTV is the type species of the genus Orbivirus, family Reoviridae, with a genome composed of ten segments of linear dsRNA (Seg-1 to Seg-10). Each genome segment encodes a distinct protein, with the exception of Seg-9 which encodes two proteins: the viral helicase VP6. The BTV particle is arranged as three concentric shells, composed of the structural proteins VP3 and VP7 (making up the virus inner-core and outer-core layers respectively), with an additional outer capsid layer comprising VP2 and VP5. NS4 has a nucleolar localisation and has been demonstrated to benefit virus replication in the presence of type I interferon [1]
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