Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2010
Publication Date: 1/1/2011
Citation: Piccone, M.E., Diaz-San Segundo, F., Kramer, E., Rodriguez, L.L., De Los Santos, T.B. 2011. Introduction of tag epitopes in the inter-AUG region of foot and mouth disease virus: effect on the L protein. Virus Research. 155(1):91-97. Interpretive Summary: Understanding the factors and mechanisms that determine foot-and-mouth disease virus (FMDV) virulence and ability to cause disease in animals is important not only for understanding disease epidemiology and transmission but also in the development of affective countermeasures such as vaccines and biotherapeutics. Recently we have shown that the region in the FMDV genome located between the translation initiation codons (Inter-AUG) contains a virulence determinant. Viruses mutated in this region failed to cause disease in cattle and could potentially serve for developing novel stable attenuated vaccines. In an effort to determine the biological function of the inter-AUG region and its relationship to the pathogenesis of FMDV, we have introduced several “tag” sequences in this region to differentiate the leader (L) proteins originated at the first or the second AUG codon. Mutant viruses containing these tags were characterized in vitro and in cell culture showing the effect of the tag sequences on L functions. Results from this work provide an understanding of the molecular mechanisms for attenuation of inter-AUG mutant viruses. This information will be used in our continuing efforts to develop more effective FMDV vaccines.
Technical Abstract: Foot-and-mouth disease virus (FMDV) initiates translation from two in–frame AUG codons producing two forms of the leader (L) proteinase, termed Lab (starting at the first AUG) and Lb (starting at second AUG). In a previous study, we have demonstrated that acDNA-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 an attenuated 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 completely 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 vivo.