Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins µ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a µ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC µ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating µ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions. NON-TECHNICAL AUTHOR SUMMARY It is important to understand how viruses replicate and assemble to discover antiviral therapies and to modify viruses for applications like gene therapy or cancer therapy. Reovirus is a harmless virus being tested as a cancer therapy. Reovirus has two coats of proteins, an inner coat and an outer coat. To replicate, reovirus particles need only the inner coat, but to become infectious they require the outer coat. Strangely, inner and outer coat proteins are all made by the virus at once, so it was unknown what determines whether newly made viruses will contain just the inner coat to continue to replicate, or both coats to transmit to new hosts. Our experiments reveal that the inner coat proteins are located in a different area of an infected cell versus the outer coat proteins. The location therefore determines if the newly made viruses contain just the inner coat versus both coats. Reoviruses have evolved extravagant mechanisms to be able to efficiently take on the best composition required for replication and transmission. ### Competing Interest Statement The authors have declared no competing interest.