Research On The Interaction Between CVB3VP1and Host Protein MAT1

Objective:To validate interaction between VP1and MAT1;to analysis the activity of CAKafter the interaction between VP1and MAT1aiming to investigate the molecularmechanism of CVB3infection.Methods:1. HeLa cells were infected by CVB3(MOI=5) and the total cellular proteinswere collected after3h post infection, then VP1was pulled down by anti-MAT1andMAT1was pulled down by anti-VP1in collected proteins,Western blotting was usedto detect VP1and MAT1.2. HeLa cells were differently collected at0h,3h,6h,and9h post infection byCVB3and at12h,18h,and24h post transfection by pBudCE4.1-VP1.VP1wasdisplayed by using Rhodanmine donkey anti-mouse (red fluorescence) combined withmouse anti-VP1antibody and MAT1was displayed by using Alexa Flour488donkey anti-goat (green fluorescence) combined with goat anti-MAT1antibody withimmunofluorescence technique to observe the subcellular co-localization betweenVP1and MAT1by confocal microscopy.3. The cytoplasmic and nuclear proteins of HeLa cells were differently collectedat12h,18h,and24h post transfection by pBudCE4.1-VP1and at0h,3h,6h,and9h postinfection by CVB3,then the expression of MAT1, CDK7,and Cyclin H in thatproteins were detected by Western blotting.4. HeLa cells were transfected by pBudCE4.1-VP1and the total cellular proteinswere collected at12h,24h,and36h post infection, then CDK7was pulled down byanti-MAT1in collected proteins, finally the expression of CDK7in CAK complexwas analyzed by Co-IP.5. The cytoplasmic and nuclear proteins of HeLa cells were differently collectedat12h,18h,and24h post transfection by pBudCE4.1-VP1and at0h,3h,6h, and9h postinfection by CVB3,and the expression of RNA Pol II CTD and p-RNA Pol II CTD inthat proteins were detected by Western blotting. Result:1. VP1was coprecipitation with anti-MAT1and MAT1was coprecipitation withanti-VP1in the collecting precipitate. The result showed that VP1and MAT1couldinteract in the CVB3infected cells.2. VP1and MAT1had the subcellular co-localization in the viral infection groupand plasmid transfection group by confocal microscopy,and this phenomenon wasmore obvious at the6h or9h post infection.3. The expression of MAT1and CDK7in total protein gradually reduced and theexpression of Cyclin H had no obvious change trend with the prolongation of plasmidtransfection and viral infection time.4. The expression of CDK7in CAK complex gradually reduced with theprolongation of plasmid transfection time by Co-IP.5. The expression of RNA Pol II CTD and p-RNA Pol II CTD were det-ected in the nuclear protein and they gradually reduced with the prolongationof plasmid transfection and viral infection time by Western blotting.Conclusions:1. The interaction between CVB3VP1and host protein MAT1was furtherconfirmed.2. The expression of CDK7in CAK complex reduced and CDK7down-regulated the phosphorylation of RNA Pol II CTD resulting in the decrease ofCAK activity under the interaction between VP1and MAT1.