We previously developed an experimental """"""""rescue"""""""" system for respiratory syncytial virus (RSV) based on a cDNA-encoded """"""""minigenome"""""""", called RSV- CAT RNA, bearing the foreign marker gene for chloramphenicol acetyl transferase (CAT). When synthesized in vitro and transfected into RSV- infected cells, RSV-CAT RNA was competent for RSV-specific transcription, replication and incorporation into virions (accompanying report). Here, we modified this system such that RSV as the source of complementing proteins was replaced by RSV proteins expressed from transfected cDNAs. Thus, the mix and relative amounts of complementing proteins could be varied. Three proteins, N, P and L, were necessary and sufficient for RNA replication (the synthesis of genome and antigenome [positive-sense replicative intermediate] RNAs). However, transcription (synthesis of subgenomic mRNA) by these three proteins alone yielded prematurely terminated mRNA. The coexpression of catalytic amounts of the M2 mRNA restored authentic transcription. Thus, the RSV """"""""replicase"""""""" and """"""""transcriptase"""""""" are not identical; the latter requires an additional elongation factor. At high concentrations of input M2 cDNA (yielding levels of protein comparable to those observed late during RSV infection), both transcription and RNA replication were inhibited. The M2 mRNA contains two overlapping orfs: the upstream orf encodes the M2 protein, and the second, internal orf lacks a known protein product. The effect of transcriptional elongation mapped to the former; the effect of inhibition of RNA synthesis to the latter. Coexpression (with N, P and L) of the NS1 protein resulted in inhibition of the synthesis of all RNAs, whereas coexpression of NS2 inhibited the synthesis of positive- sense RNAs. The M, SH, G and F proteins lacked detectable effect on RNA synthesis. Preliminary experiments indicated that transmissible particles were made 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.
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