The Influence Of PKC, JNK And NF-?B Signaling Pathways On The Promoter Activity Of The Prototype Foam Virus

The genome structure,life span and gene transcription regulation approach of foamy virus revealed great difference from other retrovirus.What's more,contradiction between the fact that foamy virus could cause obvious cytopathic effect and its ability to establish sustainable infection without any pathogenicity make foamy virus becoming a hotspot for all relevant researchers.At least two promoters were involved in foamy virus gene transcription regulation.The long terminal repeats could modulate expression of structural protein such as Gag,Pol and Env,the internal promoter could regulate expression of nonstructural protein such as Tas and bet.It has been established that a 40bp sequence at the 3 terminal of gag could interact with Tas and could also be activated by Tas,in this paper relevant research was performed to figure out whether this was also the case for prototype foamy virus.Recent research revealed that Tas could interplay with several kinds of proteins and this could affect cellular activities and at the same time affect the proliferation of foamy virus.After infection of foamy virus,immune system of host would adopt corresponding approach to react the infection.In this process,many signal pathway and their intermediate protein would also take action to react,for example,PKC,JNK,NF-KB signaling.Based on these background knowledge,experiments in the following were performed.(1)Effects of PFV Tas on different lengths of LTR,IP and GagPl sequence.Firstly,online informatics software such as JASPAR and TFSEARCH were used to analyze transcription factors binding sites which involve in NF-?B,JNK and PKC signal pathway.Secondly,take pHSRV13 as template,a series of different lengths of LTR,IP and GagPl luciferase expression vectors were constructed into pGL3-Basic luciferase reporting vector.Thirdly,transient transfection of the constructed vector was performed to analyze activity of LTR,IP and GagP1 promoter.Endotoxin free plasmids of all the constructed vectors were prepared.Then lipofectamine2000 was used for transfection.Deletion of two TREs on 5' terminal of LTR could attenuate 80 percent of its activated activity by Tas.This indicated that these two TREs play important roles in LTR promoter activity.We also found that deletion of 8379-8927nt of IP greatly affected IP promoter activated activity.When informational analysis of this sequence applying JASPAR and TFSEARCH,one perfect AP-1 binding site was found which was supposed to have the potential in enhancing IP promoter activity.(2)Research on PKC,NF-?B,JNK signal pathway modulation of LTR,IP promoter and GagP1 sequence.According to the bioinformatic analysis of LTR and IP promoter,LTR(1-1121),LTR(1-780),LTR(412-780),LTR(662-780),IP(8379-9195),IP(8980-9195)and IP(9019-9195)were used for signal pathway experiments and found that PKC,NF-?B,JNK signal pathway involoved in regulating LTR and IP promoter activity.(3)Function of AP-1 which is an important protein involved in PKC signal pathway and BAG3 which is important for NF-?B activation on LTR,IP and GagP1.Firstly,two subunit expression vector of AP-1,pcDNA-FOS and pcDNA-JUN were constructed.Another BAG3 expression vector pcDNA-BAG3 was also constructed.Secondly,pGL3-LTR(1-1121),pGL3-IP(8379-9438),pGL3-GagP1 were cotransfected with AP-1 subunit expression vector pcDNA-FOS,pcDNA-JUN and BAG3 expression vector pcDNA-BAG3.AP-1 was found to greatly elevated LTR,IP and GagP1 basal activity.Proper amount of BAG3 could exert positive effect on basal activity of LTR,IP promoter and GagP1.However,AP-1 was not essential for LTR,IP and GagP1 activated activity.What's more,BAG3 could downregulate LTR,IP and GagP1 activated activity.