FIG. 8.

The NS1 protein inhibits RNA synthesis from the antigenome promoter. MP123, a copyback-type minigenome in which the 3′-leader region was replaced by the complement of the last 57 nucleotides of the trailer region, which corresponds to the 3′ end of the antigenome, was constructed (Fig. 1). The TrA7C trailer mutation was included to block minigenome amplification by the RSV polymerase. MP123 was analyzed in parallel with MP96, which is a version of the prototype C2 minigenome which also contains the TrA7C point mutation. HEp-2 cells were infected with vTF7-3 and transfected with 100 ng of the MP96 (A and B) or MP123 (C and D) minigenome; the N, P, and L support plasmids in the amounts as described in the legend to Fig. 3; 50 ng of M2-1 plasmid; and the indicated amount (0 to 100 ng [lanes 2 to 6]) of NS1 plasmid. Lane 1 is a negative control in which the L plasmid was omitted. Cells were harvested 40 h postinfection, and lysates were prepared and processed for RNA purification directly (A and C) or following treatment with micrococcal nuclease (B and D). RNA was analyzed by Northern blot hybridization with a negative-sense CAT riboprobe.