The Influence Of The Translation Initarion Region And A Single Nucleotide Mutation Of2A Of Foot-and-mouth Disease Virus On The Translation Initiation And Cleavage Efficiency

FMDV (Foot and Mouth Disease Virus) is the pathogen of FMD, which is a highly contagiousdisease among cloven-hoofed animals. In FMDV genome, the internal ribosome entry site (IRES)element and the2A sequence encoding2A oligopeptides play an important role in translation initiationand cleavage, maturation process of FMDV polyprotein.There exist two functional AUG and two formsof L protein (Lab and Lb).The second AUG is preferred in FMDV and the mechanisms of AUGselection in FMDV has been a hotspot recently. The cracking site of2A/2B is located between theglycine and proline in C-terminal of2A sequence, which highlights the importance of glycine in2A.In this study, ninety-nine FMDV genomes of different serotypes were selected as study object. Byusing bioinformatics, the translation initiation sequence contexts surrounding the two AUG nucleotidetriplet downstream of IRES and the codon usage of2A sequence were analysed respectively. The resultsshowed that the translation initiation nucleotide environment of the two AUGs exhibits distinctdifferences and the superior translation initiation sequence (-TTTATGAAC-) and inferior translationinitiation sequence (-AAGATGGAA-) are determined corresponding to the first and second AUG startcodon contexts respectively. Sixty-six FMDV2A sequences were studied by a mathematical formulaestablished based on bioinformatics. The results showed that the cdon usage pattern of C-terminalglycine in2A sequence is conserved and tends to use the synonymous codon GGG.On the basis of bioinformatics, double report gene(Chloramphnicol acetyltransferase and Enhancedgreen fluorescent)expression system containing two specific translation initiation sequences and foursynonymous codon(GGA, GGT, GGC, GGG) encoding glycine in C-terminal of2A sequence wereconstructed respectively. The constructs were transfected into CHO and total RNA was extracted forreverse transcription to verify the expression of constructions, after which the expression wererecorded by fluorescence microscope in different periods. Finally, the cells were reclaimed for theconduction of Western-Blotting to detect and analyse the two reporter gene at the level of translation.The results showed that-TTTATGAAC-mediated by IRES has a significantly higher translationinitiation efficiency of EGFP compared with-AAGATGGAA-. In12,16,20,24,36h after transfection,the quantity of EGFP expression leaded by–AAGATGGAA-is7.3,4.45,3.16,2.3,1.63times of-TTTATGAAC-l.This suggests that oligonucleotides context surrounding the two AUGs is related tothe selection of translation initation site. Besides, the2A sequences with four synonymous codonencoding glycine in C-terminal performed different automatic cleavage efficiency and GGG（94%）、GGC(93.2%）、GGA(91.5%) is higher than GGT(86.8%)(0.01<P<0.05). The results suggest that FMDVtends to select GGG, which has a high base stacking strength, to encode glycine in C-terminal of2Asequence for cleavage of2A and2B. This study provides a new theoretical foundation for furtherexploration of the translation mode in FMDV host cell and the translation, replication of virus.