Respiratory Syncytial Virus: Quasi-Templated Initiation at Position 1 of the Encoded mRNA

strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were comple-mented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3*-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by “saturation ” mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5*-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3*-CCCUGUUUUA