Amita, H.; Dudhe, P.; Yadav, M.; Basu, B.; Vrati, S.; Dhanasekaran, K.

Flaviviruses contribute significantly to the global disease burden and are known to remodel host organelles extensively to their advantage. Centrosomal microtubule-organizing centres (MTOCs) having established role in cell division and signalling are also targeted by viruses. However, it remains uncertain whether they act as bystanders or actively engage in viral processes. Here, we elucidate the centrosome and cytoskeletal involvement during Japanese Encephalitis Virus (JEV) infection. Virus-free expression studies have identified a centrosome-targeting region within the C-terminal helicase domain that mediates the association of virus-derived structures with host MTOCs. When expressed exogenously, JEV-NS3 formed pericentriolar aggresomes resembling the distribution pattern of helicase-containing viroplasm in infected cells. Centriole depletion assays revealed the proviral role of centrosome facilitating JEV replication, where the viroplasm organization depends on centrosomal MTOCs, and vimentin cages. Additionally, microtubule disruption and dynarrestin blockade assays emphasized the roles of microtubules and dynein in concentrating NS3-containing vesicular packets towards centrosomes, highlighting the centrosome-cytoskeleton axis as a potential target for flaviviral intervention.