RNA Distribution During Flock House Virus Infection

The mechanisms involved in the specific packaging of multipartite genomes by (+) RNA viruses remain poorly understood. In order to gain insight into the location of genomic RNA encapsidation, we employed fluorescence in situ hybridization (FISH) to visualize the sub‐cellular distribution of (+) vRNA in cells infected with the model nodavirus, Flock House virus (FHV). During FHV assembly, genomic RNAs 1 and 2 are encapsidated into a T = 3 icosahedral capsid. Previously it has been shown that while vRNA replication and CP synthesis are initially targeted to different cellular locations (mitochondria and ER, respectively), these regions converge into perinuclear clusters late in infection. Our analysis of the cellular distribution of FHV RNA during infection, however, suggests that the vast majority of (+) RNA is trafficked towards the periphery of the cell relatively early during the infectious cycle (within 3 hours post infection) and does not relocate to the perinuclear region during mitochondrial condensation (15 hours post infection). In addition, the (+) RNA fits well within the region occupied by CP at the time points we tested. Our findings show that RNA transport precedes genome packaging and virus assembly. This suggests that genomic RNA is likely released from vRNA replication sites fairly soon after synthesis and trafficked to peripheral sites prior to encapsidation. Our analysis in mammalian cells also revealed that in the absence of B2, a small virus‐encoded protein known to bind dsRNAs, RNA2 is stalled in cytoplasmic aggregates while RNA1 trafficking is largely unaffected.