Screening Of Host Cell Methylation Genes Involved In Reactivation Of KSHV Replication By HIV-1 Tat Protein

Objective: To investigate the role of host cell genomic methylation in the reactivation of Kaposi sarcoma-associated herpesvirus(KSHV)life cycle by human immunodeficiency virus(HIV-1)transactivative transcription protein(Tat protein)and further to determinate the methylation sites of host cells involved in the reactivation of KSHV life cycle by HIV-1 Tat protein.Methods: 1.HIV-1 Tat recombinant lentivirus expression vector pCDH-GFP-Tat-F was constructed.The constructed pCDH-GFP-Tat-F and lentivirus packaging plasmids were co-transfected into 293 T cells to assemble HIV-1 Tat recombinant lentivirus.Meanwhile,lentivirus containing empty pCDH-GFP was assembled as control virus.2.BCBL-1 cells were infected with HIV-1 Tat recombinant lentivirus,following the evaluation of viral infection efficiency by fluorescence microscope.Western blotting was used to detect the expression of Tat protein in cells infected with recombinant lentivirus.Meanwhile,the activity of expressed Tat protein in BCBL-1 cells,which were infected with recombinant lentivirus and then transfected with pTZIII-CAT plasmid,was detected by CAT-ELISA kit.The pTZIII-CAT plasmid contains chloramphenicol acetyltransferase coding gene as reporter which was controlled by HIV-1 long terminal repeat(LTR).The activity of expressed Tat protein was documented by the expression of CAT in those BCBL-1 cells.3.Real time PCR was used to detect the expression of KSHV gene related to the lytic and latent stage of replication.According to the data,the effect of Tat recombinant virus infection on KSHV replication cycle was analyzed.4.The second generation high-throughput DNA methylation sequencing technique was used to screen the host genome methylation sites in BCBL-1 cells expressing Tat protein.Results: 1.The expression vector of lentivirus was successfully constructed,and the lentivirus was assembled in 293 T cells efficiently.2.The lentivirus infected BCBL-1 cells were observed with fluorescence microscope and the infection efficiency was more than 80%.The expression of Tat protein in lentivirus infected BCBL-1 cells was visuable by Western blotting.Meanwhile,the CAT-ELISA data showed that expression of CAT in BCBL-1 cells,which were infected with lentivirus and then transfected with pTZIII-CAT plasmid,was higher than that in control BCBL-1 cells.The data show that expressed Tat protein has trans-activation activity.3.The data of Realtime PCR indicated that expressed Tat protein in BCBL-1 cells up-regulated the lytic gene PAN,otherwise down-regulated the latent gene LANA of KSHV.4.The second generation high-throughput DNA(Deoxyribo nucleic acid)methylation sequencing technique with heavy sulfite treatment showed that Tat did cause methylation in many chromosomal regions of BCBL-1 cells.Nine up-regulated different methylation region(DMR)and eight down-regulated DMR were selected repectively.Go analysis showed that the methylation state of host cells induced by tat protein involved 6 cell signaling pathways and KEGG signaling pathway was annotated to screen out 15 signal pathways with statistical difference(q-value < 0.01).Conclusion: 1.This study proved that the Tat protein promote the replication of KSHV lytic replication.2.This study suggests that the methylazation of the chromosomal region of BCBL-1 cells might participated in reactivation of KSHV by Tat protein.