FIG 5.

Analysis of subgenomic mRNA leader-body junctions produced by the S1b and S2b mutants. (A) Positive-sense RNA species produced during MHV infection. At the top is the genome (gRNA), comprising the replicase genes (rep 1a and 1b) and downstream genes for the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N), and for accessory proteins 2a, hemagglutinin-esterase (HE), 4, and 5a. Below the gRNA is the 3′ nested set of transcribed subgenomic mRNA species (sgRNA) 2 to 7. The leader RNA, which appears at the 5′ ends of the gRNA and all sgRNA species, is represented as a black rectangle. The positions and sizes of PCR primers L, A, B, and C are not drawn to scale; the exact positions of primers are given in Materials and Methods. The expected sizes of PCR products produced by primer pairs are indicated. (B) RT-PCR products crossing the leader-body junctions of sgRNA2, -4, -5, and -7 from total RNA purified from mock-infected cells (m) or from cells infected with wild-type MHV (wt) or the S1b or S2b mutants. PCR products were analyzed by agarose gel electrophoresis; std, DNA size standards. The star and circles indicate aberrant PCR products due to mispriming by downstream primers A and B, respectively. (C) Sequences of the PCR products for the leader-body junctions of sgRNA2 and sgRNA5. The sequence traces, obtained with downstream primers A and B, respectively, are negative sense. The corresponding positive-sense RNA sequences are given above each set of traces; the TRS for each is boxed.