Sequences at gene segment termini inclusive of untranslated regions and partial open reading frames play a critical role in mammalian orthoreovirus S gene packaging

Mammalian orthoreovirus (MRV) is a prototypic member of the Spinareoviridae family and has ten double-stranded RNA segments. One copy of each segment must be faithfully packaged into the mature virion, and prior literature suggests that nucleotides (nts) at the terminal ends of each gene likely facilitate their packaging. However, little is known about the precise packaging sequences required or how the packaging process is coordinated. Using a novel approach, we have determined that 200 nts at each terminus, inclusive of untranslated regions (UTR) and parts of the open reading frame (ORF), are sufficient for packaging each S gene segment (S1-S4) individually and together into replicating virus. Further, we mapped the minimal sequences required for packaging the S1 gene segment to 25 5′ nts and 50 3′ nts. The S1 UTRs alone are not sufficient, but are necessary for packaging, as mutations of the 5′ or 3′ UTRs led to a complete loss of virus recovery. Using a second novel assay, we determined that 50 5′nts and 50 3′ nts of S1 are sufficient to package a non-viral gene segment into MRV. The 5′ and 3′ termini of the S1 gene are predicted to form a panhandle structure and specific mutations within the predicted stem of the panhandle region led to a significant decrease in viral recovery. Additionally, mutation of six nts that are conserved in the three major serotypes of MRV and are predicted to form an unpaired loop in the S1 3′UTR, led to a complete loss of viral recovery. Overall, our data provide strong experimental proof that MRV packaging signals lie at the terminal ends of the S gene segments and offer support that the sequence requirements for efficient packaging of the S1 segment include a predicted panhandle structure and specific sequences within an unpaired loop in the 3′ UTR.