Recently, we have established the pivotal role of an interaction between sense and antisense RNA sequences in the life cycle of EAV (van Marle et al., 1999a) . In that study, the role of TRS nucleotides C 2 and C 5 was tested by substituting them with G. It was concluded that base pairing between the sense leader TRS and the antisense body TRS plays a crucial role in nidovirus sg RNA synthesis. We now took a more systematic approach and performed an extensive site-directed co-variation mutagenesis study of the entire leader TRS and RNA7 body TRS, which directs the synthesis of the most abundant EAV sg RNA. Every nucleotide of the TRS (5¢-UCA-ACU-3¢) was replaced with each of the other possible nucleotides. As in the study of van Marle et al. (1999a) , every mutation was introduced into leader TRS, RNA7 body TRS and both TRSs, resulting in 54 mutant constructs. Each mutant was given a unique name: e.g. BU 1 A refers to a mutant in which a U has been changed to A at position 1 of the body TRS; LU 1 A refers to the same substitution in the leader TRS; and DU 1 A means that these two substitutions were combined in one double mutant construct. The amount of sg RNA7 was quantitated by phosphoimager scanning of hybridized gels and was corrected for the amount of genomic RNA in the same lane (as outlined above). Figure 3 shows the relative sg RNA7 level of the 54 mutants, compared with the RNA7 level of the wild-type control. For a selection of 11 interesting mutants (see below), the analysis was repeated three times (Figure 4 ), without observing signi®cant variations in sg RNA synthesis.