In 1995 we described an intracellular transcription and replication system for respiratory syncytial virus (RSV) based on cDNA-encoded components. Tissue culture cells were transfected with (i) a plasmid encoding a helper-dependent RNA analog of either negative-sense genomic RNA or of positive-sense replicative intermediate (antigenome) RNA (called an RSV-CAT """"""""minigenome"""""""" or """"""""mini-antigenome"""""""", respectively), and (ii) various plasmids which each encode one of the RSV proteins. Expression of the plasmids was directed by T7 RNA polymerase supplied by a vaccinia virus recombinant. Three proteins, the nucleocapsid N, phosphoprotein P and large polymerase subunit L proteins, were necessary and sufficient for RNA replication (the synthesis of genome and antigenome). Unexpectedly, transcription (synthesis of subgenomic mRNA) by these three proteins alone yielded prematurely terminated mRNA. The coexpression of """"""""catalytic"""""""" amounts of the 5'-terminal ORF of the M2 mRNA restored authentic transcription. Thus, contrary to expectations, the RSV """"""""replicase"""""""" and """"""""transcriptase"""""""" are not identical: the latter requires a processivity factor. The naturally-occurring M2 mRNA contains a second, internal ORF which slightly overlaps the first and lacks a known protein product. Expression of ORF2 in the presence of L, N and P (with or without M2 ORF1) inhibited RNA replication and transcription. The NS1 protein also was a very potent inhibitor of RNA synthesis, whereas the M, SH, G and F proteins lacked detectable effect on RNA synthesis. The relative level of transcription versus RNA replication was insensitive to increase in the level of intracellular N and P protein. This showed that the shift from transcription to RNA replication is not mediated by increase in the intracellular accumulation of these proteins, in contrast to an accepted (but unproven) model for RNA replication of nonsegmented negative strand viruses. Preliminary experiments indicated that transmissible RSV-CAT particles were produced when the mix of complementing proteins included the four envelope-associated proteins, M, SH, F and G, in addition to N, P, L and M2 ORF1.
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