FIG. 4.

Insertion of Tr at an internal site of replicon 3′CB. (A) 3′CB genomic analogs with Tr insertions. The 5′ terminal 51 nt of the genomic Tr were introduced in both orientations at a unique AflII site 51 nt from the 5′ end of mutant 3′CB, to yield two subgenomic replicons, 3′CB/Tr and 3′CB/TrC. The structures of 3′CB/Tr and 3′CB/TrC are shown, together with the sequence of the inserted nucleotides. The nucleotides that correspond to the AflII compatible ends are underlined. (B) RNA synthesis, determined by agarose-urea gel analysis of VSV-specific RNAs generated from 3′CB replicons with inserted Tr sequences. RNAs were labeled with [³H]uridine in the presence of ActD and analyzed as described in Materials and Methods. The products of replication and transcription by the VSV polymerase are indicated by Rep and Tx, respectively. (C) Infectivity assay. Shown is agarose-urea gel analysis of RNAs generated by infectious particles recovered from genomic analogs containing inserted Tr sequences. Supernatants were generated and assayed for infectious particles by using the infectivity assay described in Materials and Methods. WT and 3′CB replicons are shown as positive and negative controls, respectively.