Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus-infected cell.
Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A(pro)). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET-based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP(2)) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real-time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A(pro) catalytic activity, irrespective of other viral-encoded protease, the activated caspases or general inhibition of protein synthesis in the EV-infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease-substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus-induced host translation inhibition.
Hsu
Yueh-Ying
YY
Department of Biotechnology, National Yang-Ming University, Taipei, Taiwan, R.O.C.
Liu
Yu-Ning
YN
Lu
Wen-Wen
WW
Kung
Szu-Hao
SH
D004770:Enterovirus / Q000201:enzymology* / Q000472:pathogenicity; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D039603:Eukaryotic Initiation Factor-4G; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010447:Peptide Hydrolases; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C113920:EIF4G1 protein, human; C491300:EIF4G2 protein, human