, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.