Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

Sequence changes in the apical loop of SL6. In mutants C12, C13, C14, C16 and C19 substitutions within the apical loop changed the nucleotide sequence without altering the overall conformation (Figures 3 and 4) . Four of these mutations C12, C13, C14 and C19 were introduced into the conserved hexanucleotide UGCCAA. Silent mutations C12, C13 and C14 changed plaque morphology; the C13 and C14 mutants that contain purine-to-pyrimidine substitutions also showed reduced growth characteristics and cpe (Table 1 and Figure 5 ). Silent substitution C16, located outside the conserved hexanucleotide in SL6, did not affect the virus phenotype. Two purines were changed for two pyrimidines in mutant C19, one in the conserved hexanucleotide. This mutant was highly attenuated in cell culture producing no cpe and a small turbid plaque phenotype (Figures 3-5 and Table 1 ). These two purine-pyrimidine substitutions resulted in the amino acid substitution M 33 !L that mimicked the corresponding natural amino acid in KFDV and AHFV (Figure 3) . Nevertheless, M 33 !L, mutant C15, produced by different nucleotide substitutions had only a moderate effect on virus replication (below). Therefore, the biological consequences of mutation C19 may be attributed, at least partially, to the nucleotide substitutions.