A popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product.
Copyright 1997 Academic Press.