JEV helicase targets host MTOC and participates in the organization of pericentriolar viroplasm

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. SummaryThis study demonstrates that JEV helicase is directed to centrosomes via its CTHD domain, using them to initiate pericentriolar viroplasm formation. It also highlights the unappreciated function of centrosomes as a proviral hub, orchestrating cytoskeletal remodelling and viral factory organization. Graphical abstractJEV helicase is targeted to centrosomes to form viroplasm with the aid of microtubule and motor proteins. A) JEV lifecycle from receptor binding (Step 1) to endocytic internalization (Step 2) followed by uncoating and release of viral genome (Step 3) followed by replication within the ER derived Vesicular packets (Step 4). This vesicle packets harbors viral proteins that form replication complex, like helicase and replicase and the replicative intermediate, dsRNA. These vesicle packets with the aid of retrograde motor protein gets targeted towards the centrosome (Step 5). Ultimately multiple vesicle packets accumulate in the pericentrosomal region, where it forms a separate compartment enclosed by vimentin and tubulin cage (Step 6). Inset A, B and C depict the effect of cytoskeletal perturbations on Viroplasm formation. B) Disruption of microtubules using Nocodazole completely abolishes pericentriolar viroplasm organization. C) Centrinone mediated centrosome depletion markedly reduces both the number and size of pericentriolar viroplasm. D) Dynarrestin mediated retrograde microtubule transport significantly decreases the reorganization of vesicle packets to form the viroplasm in the pericentriolar region. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/740276v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@17a6354org.highwire.dtl.DTLVardef@f208eorg.highwire.dtl.DTLVardef@1224fcaorg.highwire.dtl.DTLVardef@1a48bab_HPS_FORMAT_FIGEXP M_FIG C_FIG