Introduction of tag epitopes in the inter-AUG region of foot and mouth disease virus: effect on the L protein.

Foot-and-mouth disease virus (FMDV) initiates translation from two in-frame AUG codons producing two forms of the leader (L) proteinase, Lab (starting at the first AUG) and Lb (starting at second AUG). In a previous study, we have demonstrated that a cDNA-derived mutant FMDV (A24-L1123) containing a 57-nucleotide transposon (tn) insertion between the two AUG initiation codons (inter-AUG region) was completely attenuated in cattle, suggesting that this region is involved in viral pathogenesis. To investigate the potential role of the Lab protein in attenuation, we have introduced two epitope tags (Flag: DYKDDDK and HA: YPYDVPDYA) or a small tetracysteine motif (tc: CCGPCC) into the pA24-L1123 infectious DNA clone. Mutant viruses with a small plaque phenotype similar to the parental A24-L1123 were recovered after transfection of constructs encoding the Flag tag and the tc motif. However, expression of the Flag- or tc-tagged Lab protein was abolished or greatly diminished in these viruses. Interestingly, the A24-L1123/Flag virus acquired an extra base in the inter-AUG region that resulted in new AUG codons in-frame with the second AUG, and produced a larger Lb protein. This N terminal extension of the Lb protein in mutant A24-L1123/Flag did not affect virus viability or L functions in cell culture.