In situ multimodal imaging reveals compartmentalization of reovirus replication factories

Avian reovirus (ARV) belongs to Reoviridae family of viruses with segmented dsRNA genomes. ARV is responsible for significant economic losses to the poultry industry by causing tenosynovitis and chronic respiratory disease in chickens. ARV virion exhibits an outer protein shell enclosing an inner core, which contains 10 unique dsRNA segments and multiple copies of viral polymerase. ARV assembly takes place in so-called viral factories (VFs). They are cytoplasmic membrane-less inclusions, which accumulate viral proteins and RNA and assist capsid assembly, genome packaging and replication. VFs also provide protection from the host cell defence mechanisms by sequestering viral RNA. Given the dense nature of VFs, little is known about ARV assembly and packaging intermediates. To determine the architecture of VFs across the different length scales and infection course, we employed a wide range of imaging methods. Our data reveal that spherical and fluid VF enriched in viral protein σNS become surrounded by σNS-depleted solid-like compartments at late stages of infection. We demonstrate that confocal Raman microscopy allows label-free imaging of VF composition. Volume electron microscopy, along with electron tomography (ET) and in situ cryo-ET, revealed that the inner phase of VF concentrates viral non-structural proteins and assists assembly of viral cores. The larger, RNA-filled and double-layered particles are assembled at the periphery of the inner fluid phase of VF, forming ordered virion arrays. This suggests that ARV VFs are functionally compartmentalized: the inner fluid phase assists RNA assortment and core particle assembly, whereas the peripheral phase is the site of RNA replication inside viral cores and virion completion.