The three mutants were tested in three independent experiments. Intracellular RNA was isolated at 14 h posttransfection, early enough to prevent spread of the wildtype control virus to non-transfected cells (®rst cycle analysis). Transfection ef®ciencies were determined by immuno¯uorescence assays (see Materials and methods) and varied between 10 and 23% (data not shown). Prior to RNA analysis, the amount of isolated intracellular RNA was corrected for the transfection ef®ciency of the sample, so that each lane in Figure 2 represents EAV-speci®c RNA from an approximately equal number of EAV-positive cells. Phosphoimager quantitation revealed that genomic RNA replication of mutants L4, B4 and LB4 varied by not more than 30% (Table I) . These differences could re¯ect, for example, a slight in¯uence of RNA secondary structure changes in the TRS regions on genomic RNA synthesis. Remarkably, however, the genomic RNA level of the leader±body TRS double mutant LB4 was not affected by more than 10%. In view of the results obtained with these pentanucleotide TRS mutants, we assumed that the amount of genomic RNA could indeed be used as an internal standard during the analysis of mutants containing only single nucleotide replacements in leader TRS and/or RNA7 body TRS. The regions of the genome specifying the leader (L) sequence, the replicase gene (ORFs 1a and 1b) and the structural genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2±7) is depicted below. The black boxes in the genomic RNA indicate the position of leader and major body TRSs. (B and C) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus strand (B) or minus strand (C) RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus strand template. For details see text. Northern analysis of EAV-speci®c RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from TRS pentanucleotide mutants (UCAAC to AGUUG). The results of two independent experiments are shown.