| Summary: | 博士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 96 === Enterovirus infection induces a marked shutoff of host protein synthesis which is mainly achieved through the cleavage of host eukaryotic initiation factors 4GI and 4GII (eIF4GI and eIF4GII, respectively) mediated by enterovirus 2A protease (2Apro). A fluorescence resonance energy transfer (FRET)-based assay was developed to investigate the real time dynamics of 2Apro activity on cleaving eIF4G in the context of enterovirus-infected cell. The specifically designed fusion substrate consists of the green fluorescent protein 2 (GFP2)-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif of eIF4GI or eIF4GII embedded within the peptide region. FRET can be readily visualized from cells expressing the fluorogenic substrate until a proteolytic cleavage by 2Apro from the input virus instead of activated caspases during apoptosis. Enterovirus 71 (EV71) infections inhibited cellular protein synthesis accompanied by the cleavage of eIF4G. In comparison with eIF4GI, the cleavage kinetics of eIF4GII revealed as FRET disruption closely correlated with protein synthesis shutoff, consistent with the results obtained from Western blot. Additionally, the FRET biosensors also responded well to other related enteroviruses. The FRET sensors provide sensitive and reliable tools for the study of 2Apro dynamics on host translation mechanism in living cells.
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