FLAG and V5 L2 DNA sequences.

Reoviruses coordinate their replication and assembly through intricate spatial and temporal compartmentalization within host cells. In this study, we elucidate the dynamics of mammalian orthoreovirus (reovirus) core replication and viral particle assembly. Using high-resolution immunofluorescence confocal microscopy, we tracked input cores and de novo cores, revealing that input cores initially form peripheral, OC-negative factories that migrate inward while seeding independent peripheral factories. Over time, these input factories transition into intermediate core-plus-outercapsid (OC) factories, which are essential for full virion assembly in the perinuclear region. Notably, de novo core proteins predominantly form independent peripheral factories that can merge or mix with others, resulting in interconnected networks. We further demonstrate that microtubules are dispensable for early core movement and factory formation but are crucial for the transition of mature, assembled virions into perinuclear deposits and for timely virion production. Disruption of microtubules delays full virus assembly, reducing progeny yield. Our findings reveal a complex, regulated interplay between spatial organization and cytoskeletal components during reovirus infection, providing insights into mechanisms that could be targeted for antiviral interventions.