Preliminary Study On The Effect Of Foam Virus On Host Cell Factor C-Maf

Foamy viruses?FV?belong to the Retroviridae and have been found in numerous mammal species.As we know,Chimpanzees,felines,bovines,equines,sheep and humans are foamy viruses' hosts.Different from normal retroviruses just like HIV and HTLV,foamy viruses appear to be infections with lifelong persistence without evident pathology.However,the biomechanism of FV latency are poorly understood.In recent years,the demand for study on emerging zoonotic virus and new viral vector,promote the study on foamy viruses' latent infection mechanism,especially on how foamy viruses and cell factors of the host's immune system relate.c-Maf is encoded by v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog?c-maf?,and it is one typical member of the large Maf family.Recent studies show that c-Maf play a important role in latent infection of HTLV in CCR4+CD4+T cells by negatively regulating Tax activity.Besides that,c-Maf can cooperate with NFAT2 and NFkB p65 to augment HIV LTR transcription.Foamy viruses belong to retroviruses just like HIV and HTLV,thery are similar in transcriptional regulation,so c-Maf may play a important role in latent infection of FV.In addition,c-Maf can increase the expression of interleukin?IL?-4,interleukin?IL?-10 and interleukin?IL?-21.Also,it regulates the overexpression of Osteopontin,CCR1,Cyclin D2,Integrin and ARK5,which can drive T cells into malignant transformation.Thus,c-Maf has significant and distint functions in immune system.In this study,molecular biological techniques are used to determine how foamy viruses and cell factor c-Maf relate.Then the mechanism of this process is analyzed by all kinds of experiments.And this investigation will help to elucidate the mechanism of latent infection of FVs.The results of the research as follow:1.The gene expression array is analyzed by the company,then quantitative real-time PCR is used to add verification.Results of all the assays reveal that foamy viruses can increase c-Maf gene expression,and the process is associated with Tas.2.With bioinformatic technology,the genomic sequence of the c-Maf gene?Accession number NC₀00016.10?is retrieved from the database,and the c-Maf promoter region?-1067/+280?is analyzed using the onlione program.Surprisingly related cell factors are found in the promoter region of c-Maf,To select and define the c-Maf-TRE?The Tas response element of c-Maf?,we use sequence alignments by Clustalw2 with the known viral TRE sequences in the LTR and IP promoters and kip2-TRE?The Tas response element of p57kip2?.Then we find the c-Maf-TRE located in the c-Maf promoter region.3.HT1080 cells are infected with foamy viruses,and transcript levels of c-Maf in diferent times are tested by quantitative real-time PCR.The results show that foamy viruses induce c-Maf gene expression after 24 hours,and the significance level appears after 48 hours.These results suggest that foamy viruses can significantly induce c-Maf gene.4.HT1080 cells are transfected with viral protein expression plasmids,and transcript levels of c-Maf with 24 and 48 hours are tested by quantitative real-time PCR.We find that c-Maf gene expression level,whih is treated with Tas expression plasmid,is higher than the others.The results show that Tas perform its function during foamy viruses induce c-Maf gene expression.5.Human genomic DNA is isolated from normal cells.The 1348 bp region?-1067/+280?including the c-Maf promoter is amplified by PCR with designed primers.After the PCR products are double digested,clone them into pGL3-Basic.Then we get the reporter plasmid pGL3-Basic-cMAF within the c-Maf-TRE site and the reporter plasmid pGL3-Basic-TRE.del without c-Maf-TRE site.After bacteria liquid PCR and sequence alignments,the new reporter plasmids prove to be right.6.The new reporter plasmid pGL3-Basic-cMAF and pGL3-Basic-TRE.del are transient co-transfections with pCI-Tas into cells,then the activity of the luciferase is analysed.The results show that there is a little increase in relative luciferase activity of the group that c-Maf expression induced by Tas cotaining related transcription factors response elements without c-Maf-TRE.A significantly increased relative luciferase activity,however,is found in another group,the c-Maf expression induced by Tas with the c-Maf-TRE.Collectively,these data indicate that Tas perform its function directly throgh binding to the c-Maf-TRE,while other cell factors have little function in the process.All the results show that FVs significantly induce c-Maf gene expression via Tas and Tas perform its function directly throgh binding to the c-Maf-TRE.This investigation might help to study foamy viruses' latent infection mechanism.