Study On The Endoplasmic Reticulum Stress Caused By Foamy Virus Infection In Host Cells

Foamy virus(Foamy virus,FV)belongs to the Spumaretrovirinae,Retroviridae,is the only member of the Spumavirus genus.As indicated by their names,FVs possess highly fusogenic activities which induce infected cells in vitro to form foam-like multinucleated cells.Simian foamy viruses(SFV)have evolved the prototype foamy viruses(PFV)that can infect people by transmission of species.Different from normal retroviruses like human immunodeficiencyvirus(HIV),FVs appear to be infections with lifelong persistence without evident pathology.FVs have a long genome and a wide host range.These characteristics make it become focal point of vector for gene therapy.At the same time,the transmission from Simian immunodeficiency virus(SIV)to HIV and PFV infection of animal cells in vitro can cause cytopathic effect remind us the fact that the safety and potential pathogenicity of foam viral vectors still are the main problems that need to be solved.Numerous viruses such as influenza A and HCV could induce endoplasmic reticulum stress(ERS),which caused accumulation of unfolded or misfolded proteins.If ERS is severe or protracted,cell apoptosis may be induced.Therefore,ERS is closely related to some diseases caused by many factors,such as metabolic disease,nerve system disease,virus infectious diseases and so on.To alleviate the stress placed on endoplasmic reticulum(ER),three specific signaling pathways including PKR-like ER kinase(PERK),activating transcription factor 6(ATF6)and inositol-requiring enzyme 1(IRE1)were activated respectively.GRP78 is a member of the Hsp70 chaperone family and is considered the master regulator of the ERS.In unstressed cells,GRP78 binds to the lumenal domain(LD)of PERK,ATF6 and IRE1.During ERS GRP78 is released from ATF6,PERK and IRE1 because of competitive binding to the increasing levels of mis-folded proteins to decrease the accumulation of unfolded or misfolded proteins and recover the function of ER.It is a survival response.FVs viral particles bud from ER which mean the viral proteins synthesize and assemble in ER.In view of this,it is necessary to research the relationship between the ERS and FV,which provided the theoretical basis of the FV potential pathogenic mechanism.The results in this study were as follows:1.Real-time PCR had been carried out to examine transcription of the genes involving in the ERS pathways.And the results indicated that all these genes were significantly up-regulated in the HT1080 cells infected by PFV.2.The expression of the representative proteins in ERS pathways had been tested by Western blotting in the HT1080 cells infected by PFV for 48 h.GRP78 was significantly enhanced by PFV infection.The expression of ATF4,spliced-XBP1 and p50-ATF6 were observed only in the PFV infected groups.The expression levels of above proteins were gradually increased along with the infection time.3.Tauroursodeoxycholic acid(TUDCA)has been shown to reduce ER stress by decreasing phosphorylation of eukaryotic initiation factor 2a(eif2a)so that declined the transcription of gadd34.In the exploration of ERS influence on FV replication,MTT experiments were done to test the survival rates of cells treated by TUDCA.The survival rate of HT1080 cells were more than 80%when the concentration of TUDCA reach 125 ?g/mL,250 ?g/mL,500 ?g/mL,respectively.Then HT1080 cells were treated by TUDCA under the 3 concentrations,and 500 ?g/mL was the effective inhibition according to the expression level of gadd34 detected by real-time PCR.4.HT1080 cells pretreated for 12 h with TUDCA(500 ?g/mL)was as experimental group and HT1080 cells only as control group.The cells of two groups were infected by PFV.The viral genes,including gag and bet were examined by real-time PCR.The results revealed a significant decrease of gag and bet transcription in the experimental group compared with control group.5.Dealing with cells as above,PFV proteins including Gag,pol,IN and Bet were examined by western blotting.The results indicated that these proteins were decreased when cells were pretreated with TUDCA.To sum up,the mRNA expression and protein synthesis of ERS genes were examined and the results indicated that ERS can be induced by PFV in HT1080 cells.While in the pre-treated cells infected by PFV,the expression levels of PFV proteins were decreased as the ERS was reduced by TUDCA.This suggests that ERS plays a facilitating role in the replication cycle of the PFV.The study was the first report that revealed the relationship between FV and ERS,which provided the theoretical basis of the FV potential pathogenic mechanism.