FIG. 2.

Role of the VSV genomic termini in RNA synthesis and assembly of infectious particles. (A) Arrangements of the termini. A region of the T7 transcription plasmid, pWT, is shown to illustrate the sections of the VSV genome, the T7 promoter, the HDV ribozyme (δ), and the T7 terminator (TΦ). The negative-sense primary T7 transcripts transcribed from pWT are shown to illustrate the sequence of the wild-type 3′ Le and 5′ Tr. Numbers indicate nucleotides from the exact VSV genomic 3′ or 5′ terminus and do not include the two G residues (gg) present at the 5′ end of the primary transcripts. pWT was altered to generate three additional plasmids designed to generate RNAs with 5′ copyback (5′CB), 3′ copyback (3′CB), or inverted (Inv) termini. Substitution of the 3′ Le for 46 nt of TrC was performed such that the sequence and position of the nontranscribed Le-N junction (boxed) and N gene start site (underlined), which are important requirements for transcriptional initiation, remained as in the WT replicon. Substitution of the 5′ Tr by 46 nt of LeC was performed so that the size of Tr was unaltered. Sequences of these modified termini are shown to illustrate the nucleotide differences from WT. (B) RNA synthesis. Cells were infected with vTF7-3, transfected with cDNAs for the VSV genomic analog WT, 5′CB, 3′CB, or Inv, as indicated, and with plasmids encoding the VSV N, P, and L proteins, and exposed to [³H]uridine (33 μCi/ml) in the presence of ActD (10 μg/ml) as described in Materials and Methods. Cytoplasmic extracts were prepared, and total RNA was analyzed by electrophoresis on agarose-urea gels and visualized by fluorography, as described in Materials and Methods. (C) Infectivity assay. Shown is agarose-urea gel analysis of VSV-specific RNAs synthesized in BHK-21 cells that were transfected with the VSV N, P, and L support plasmids and infected with 0.5 ml of clarified supernatants from cells that were transfected 36 h previously with cDNAs encoding a VSV genomic analog (WT, 5′CB, 3′CB, or Inv) and the VSV N, P, L, M, and G support plasmids as described in Materials and Methods. Genomic analogs that produced RNAs that were assembled and budded infectious particles into the culture media were detected by the ability to initiate VSV-specific RNA synthesis in these infected cells. Rep and Tx represent replication and transcription (N/L mRNA) products; respectively.