Inhibition Of Host Cell Protein Synthesis By EMCV3C Proteinase

Objectives To study the impact on eukaryotic cell protein synthesis by EMCV3C proteaseMethods Construct the eukaryotic coexpression plasmid EMCV3C proteinase:pcDNA3.1-3C, pEGFP-C3-3C. Locating expression of the fusion protein was identified by Laser Scanning Confocal Microscope.After Encephalomyocarditis virus3C gene was linked in eukaryotic expressing vector to form pcDNA3.1-3C vector, plasmid pEGFP-N1which expressed cap-dependent Green fluoresce protein (GFP) protein co-transfected with pcDNA3.1-3C into BHK-21cells, GFP protein were detected by Western blotting and GFP mRNA was detected by RT-PCR. PGL3vector expressing cap-dependent Fire luciferase protein were co-transfected with the pcDNA3.1-3C into BHK-21cells, activity of Flue were detected. The change of eukaryotic translation initiation factor4G (eIF4G I) and the poly(A)-binding protein (PABP1) were respectively detected by Western Blotting after EMCV virus infection or EMCV3C transfectionResults Eukaryotic expression plasmid EMCV3C was successfully constructed and the fusion protein were observed and expressed in the nucleus by Laser Scanning Confocal Microscope. EMCV3C inhibited cap-dependent translation of both GFP expression and activity of F-luc. Cleavage of PABP1was detected after both EMCV infection and EMCV3C protease but change of eIF4GI was not observed in this study.Conclusions Eukaryotic expression plasmid EMCV3C was successfully constructed and expressed in the nucleus. EMCV-3C protease inhibited synthesis of host cell protein by inhibition of mRNA transcription and cap-dependent protein translation.