A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of res piratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observ ed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive wh en directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mu tant P protein was unable to produce genome, antigenome, or mRNA at the res trictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis d irected by a wild-type P plasmid. Binding of the mutant P protein to N prot ein in the absence of other viral proteins was unaffected by temperature, i ndicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in R SV polymerase components in RNA synthesis.