FIG. 4.

Reversion of mutant Loop2. (A) Sequences of the 3′ terminal region of the genome of the Loop2 revertant were determined by RT-PCR, followed by sequencing of nine individual clones. Of the sequenced clones, seven were found to contain a second-site mutation in the SL5 loop region (L-rev1), but two clones acquired mutations in the SL4 domain. The revertant sequences were introduced into the wild-type EAV cDNA clone. Virus replication was studied using IFA with EAV-specific antisera for nsp3 and N at different time points after transfection, and virus titration was performed using plaque assays. The sequence of the SL5 loop region is depicted in the 3′-to-5′ direction, whereas that of SL4 is depicted in the 5′-to-3′ direction. Base-pairing possibilities between the two sequences are indicated by dots. The mutations introduced in the Loop2 mutant are marked in italics; the acquired reversions are marked by a black box. (B) Hybridization analysis of the RNA synthesis of the loop2 revertants (see the legend to Fig. 2 for details).