FIG. 2.

Deletion analysis of the M/SH gene junction in a dicistronic RNA replicon. (A) Schematic of the WT replicon-expressing plasmid pM/SH-B (top), along with the RNA species synthesized by this replicon (below, horizontal lines). This subgenomic RNA replicon contains two transcriptional units: the 704-nt NS1/M gene (gene 1) and 441-nt SH/L gene (gene 2). Locations in pM/SH-B of the M GE that was mutagenized (GE1*), the hepatitis delta virus ribozyme (Rz), RS virus leader (Le), restriction enzyme sites BglII and XhoI, virus trailer (Tr), and T7 promoter (T7) are shown. RNA species synthesized from this replicon include the negative- and positive-sense replication products (Rep; only the negative strand is shown), two monocistronic tran scripts (mRNA1 and mRNA2), and three polycistronic readthrough transcripts (rt1-2-Tr, rt1-2, and rt2-Tr). The 5′ caps (filled ovals) and 3′ poly(A) tails (AAAn) of mRNAs are indicated. (B) Deletions of regions of the M/SH gene junction. The genomic RNA sequence including part of the WT M/SH gene junction is aligned with sequences of replicons from which one of the three regions of the GE, or else 8 of 9 nt of the IG region, was deleted. Dashes indicate absence of any nucleotide at those positions. Conserved or nonconserved nucleotides are in bold capital or lowercase letters, respectively. The transcription initiation site for mRNA2 at the beginning of the GS signal is indicated by an arrow. CR, central region. (C) Analysis of RNA synthesis from RS virus replicons having deletion mutations. MVA-T7 infected HEp-2 cells were transfected with support plasmids expressing the N, P, L, and M2-1 proteins of RS virus, and with a plasmid encoding the indicated WT or deletion mutant replicon, as described in Materials and Methods. RNA synthesized by RS viral polymerase was metabolically labeled with [³H]uridine in the presence of actinomycin D, analyzed on agarose-urea gels, and detected by fluorography. RNA species are identified (right) using the same nomenclature as in Fig. 2A. Rep and rt1-2-Tr comigrated on this gel.