FIG. 2.

Effect of replacing the 3′ 34 nt of Le with increasing lengths of TrC. (A) Structures (not to scale) of minigenome C41, representing wild-type RSV, and minigenomes A36 to A147, which are designated according to the length of the added TrC sequence. GS or GE signals are indicated with open or solid boxes, respectively. (B and C) Northern blots of positive-sense RNAs synthesized by the reconstituted RSV polymerase. HEp-2 cells were infected with vaccinia virus vTF7-3 and simultaneously transfected with plasmids that encode minigenome C41 (lanes 1, 2, and 3) or minigenomes A36 to A147 (lanes 4 to 9, as indicated) together with pTM1 support plasmids expressing N, P, and M2-1 (lane 2) or N, P, M2-1, and L (lanes 3 to 9) proteins. Lane 1 received empty pTM1 expression plasmid. Forty-eight hours later, the cells were processed directly for RNA purification (B), or lysates were prepared and treated with MCN to destroy unencapsidated RNA (C). The blots were hybridized with a negative-sense, CAT-specific riboprobe. (D and E) Northern blot analyses of plasmid-supplied minigenome template. HEp-2 cells were infected with vTF7-3 and transfected with plasmids that encode minigenome C41 (lanes 1 and 2) or minigenomes A36 to A147 (lanes 3 to 8, as indicated) together with plasmids expressing N, P, and M2-1 proteins (lanes 2 to 8) or with empty pTM1 plasmid (lane 1). L plasmid was omitted from all reactions, and hence the only source of minigenome RNA was plasmid. Forty-eight hours later, the cells were processed directly for RNA purification (D) or lysed and treated with MCN followed by RNA purification (E). The RNAs were detected with a positive-sense, CAT-specific riboprobe.