These results indicate that there are strong base preference constraints for some body TRS positions. To interpret these base preferences accurately, it is necessary to limit the analysis to the double mutants only, because in these mutants the down-regulation of sg RNA synthesis was only due to the sequence changes in the body TRS, and not to the disruption of the leader±body TRS duplex. There were strict preferences for positions 1, 3 and 4 of the body TRS: at position 1, only the U to A substitution allowed for a signi®cant RNA7 level (~40% of wild-type); and at positions 3 and 4, only the A to U mutants retained 15±20% of the wild-type level. For positions 2 and 5, the sequence constraints were less stringent (all substitutions allowed for >20% of wild-type level), but still only DC 2 A and DC 2 U reached >50%. At position 6 of the body TRS, only U to C was not allowed, whereas the other two double mutants still produced 50% or more of RNA7. In other words, the functional EAV RNA7 body TRS (based on the analysis of our single nucleotide substitutions) can be described as U 1 (C/u/a) 2 A 3 A 4 C 5 (U/a/g) 6 , with wild-type nucleotides shown in upper case and nucleotides that allowed for at least 50% of the wild-type RNA7 level shown in lower case. Remarkably, TRS nucleotides A 3 , A 4 and C 5 are conserved in the TRSs of all other arteriviruses (Snijder and Meulenberg, 1998) . Also the fact that DC 2 U retained 80% of RNA7 synthesis corresponded nicely to the presence of a U at this position in other arteriviruses.