Study On The Impacts Of CVB32A Protease On Protein Translation

Objective:To investigate the effects of CVB3virus protease2A on cap-dependent mechanism protein translation pathways and internal ribosome entry site (IRES) translation pathways on molecular cloning techniques.Methods:By molecular cloning techniques, pcDNA3.1-CVB3-2A and the four deletion mutant from plasmids were constructed, and pcDNA3.1-2A together with the deletion mutant from plasmids were transfected into the cells that were compared with CVB3infected cells. In addition, the impact of CVB3-2A protein on the mechanism of eukaryotic cell translation was also analyzed on the following protocols:1. Western blot was used to detect the expression of pcDNA3.1-CVB3-2Aplasmid and impact of CVB3on eIF4GI and PABPI expression;2. Western blot was used to examine the effects of pcDNA3.1-CVB3-2A deletion mutant from plasmids on eIF4GI expression in cells;3. pcDNA3.1-CVB3-2A and deletion mutant from plasmids, co-transfection with pEGFP-N1plasmids and expression of green fluorescent protein (GFP) were observed with inverted microscope to measure the GFP expression quantity;4. Co-transfected pGL3-F-luc with pcDNA3.1-CVB3-2A and Luciferase enzyme was examined with micropage photometer for the enzyme expression;5. pcDNA3.1-CVB3-2A and deletion mutant from plasmids were respectively co-transfected with pIRES-GFP plasmid, and the inverted microscope was used to dectect the GFP expression quantity;6. pcDNA3.1-CVB3-2A and deletion mutant from plasmids were used to transfect cells to prepare the specimens using immunofluorescence technique, and confocal microscope was used to examine the subtype2A virus and its mutants import.Results:1. Wester blotting revealed that both pcDNA3.1-CVB3-2A plasmid and CVB3had led to degradation of intracellular eIF4GI.Although CVB3acted a role in PABPI degradation, yet, pcDNA3.1-CVB3-2A was free;2. pcDNA3.1-CVB3-2A and the four deletion mutant from plasmids failed to produce any effects on intracellular eIF4GI by Western blotting detection; 3. Inverted microscopic findings revealed that pcDNA3.1-CVB3-2A had resulted in decreased expression quantity of pEGFP-N1plasmid in expressing GFP, yet the four mutant plasmids caused no effect on the expression of GFP in quantity;4. Count by microplate photomer indicated that pcDNA3.1-CVB3-2A had led to down-regualted expression of Luciferase enzyme in tranfected pGL3-F-luc plasmids;5. Inverted microscopic examination demonstrated that pcDNA3.1-CVB3-2A had increased pIRES-GFP plasmid expression, whereas the four mutant plasmids produced no effect on the expression of GFP in quantity;6. Confocal microscopic examination revealed nucleus import at the deleted c-terminal in pcDNA3.1-2A, and that pcDNA3.1-CVB3-2A and three other deletion mutant plasmids were retained in the cytoplasms.Conclusions:1. CVB32A protease can digest eIF4GI in eukaryotic cells to finally inhibit the translation pathways of cap-dependent protein;2. CVB32A protease is capapble of promoting the translation pathways for internal ribosome entry site (IRES);3. Nucleus import doesn’t occur in CVB3-2A, wherease occurs at the deleted c-terminal in pcDNA3.1-2A.