Role Of NOC2L In HIV-1 Tat Protein Regulating Human Herpes Virus Type 8 Replication Cycle

Objective: To clarify the role of NOC2 L in HIV-1 Tat protein regulating HHV-8 replication,to investigate whether NOC2 L participates in regulation of HHV-8 replication by HIV-1 Tat through the P53-P21 axis.To provide few parts of evidences to reveal the role of NOC2 L in the occurrence and development of AIDS-Kaposi’s Sarcoma.Methods: 1.p CDH-GFP-NOC2 L lentivirus plasmid was constructed firstly by homologous recombination and verified by PCR,double-enzyme digestion and sequencing of the plasmid.Furtherly,the NOC2 L overexpression recombinant lentiviruses were packaged by transfection of 293 T cells with p CDH-GFP-NOC2 L lentivirus plasmids and package plasmids.Subsequently,the expression of NOC2 L recombinant lentivirus in 293 T cells was verified by Realtime quantities PCR(RT-q PCR).2.BCBL-1 cells were infected with Tat recombinant lentivirus following with confirmation of Tat protein expression with RT-q PCR.The expression levels of NOC2 L,P53 and P21 m RNA were also detected respectively.The expression levels of phosphate-P53,P53 and P21 protein were detected by Western blot.3.NOC2 L were firstly overexpressed in BCBL-1 cells with NOC2 L recombinant lentiviruses.Then the m RNA expression levels of ORF73 and ORF50 from HHV-8 were detected by RT-q PCR.Meanwhile,the m RNA and protein expression levels of phosphate-P53,P53 and P21 protein were detected by RT-q PCR and Western blot,respectively.Results: 1.p CDH-GFP-NOC2 L lentiviral transfer plasmid was successfully constructed by homologous recombination.The results of colony PCR and double digestion showed that the NOC2 L gene fragment was successfully inserted into the constructed plasmid.The plasmid sequencing results showed the insert fragment is 100% homologous to the NOC2 L gene registered in Gen Bank.2.After infection of BCBL-1 cells with Tat recombinant lentivirus,RT-q PCR detection showed that Tat expression can be detected.96 h after the Tat lentivirus infection,BCBL-1 cells showed the highest Tat expression level and the expressed Tat protein were detected with Western blot.NOC2 L,P53 and P21 m RNA expression levels of host cells were up-regulated 96 hours after BCBL-1 cells infected with Tat recombinant lentivirus.Western blot analysis showed that Phospho-p53,P53 and P21 protein levels were up-regulated.3.After infection of BCBL-1 cells with NOC2 L recombinant lentivirus,the expression of NOC2 L was documented by RT-q PCR.The data showed NOC2 L expression level was highest at 96 h after infection.Following NOC2 L overexpression in BCBL-1 cells,m RNA expression levels of P53 and P21 from cells,and ORF50,ORF73 from HHV-8 were up-regulated.Western blot analysis showed that Phospho-p53,P53,and P21 protein levels were up-regulated.Conclusion: 1.Following expression of Tat protein in BCBL-1 cells,NOC2 L m RNA expression level was up-regulated,as well as P53 and P21 m RNA expression levels and protein expression levels were up-regulated.The data indicates that NOC2 L,P53 and P21 participate in Tat regulation.2.ORF50 and ORF73 were up-regulated following NOC2 L overexpression in BCBL-1 cells.It was confirmed that NOC2 L probably participate in HHV-8 reactivation.P53 and P21 m RNA and protein expression were up-regulated after NOC2 L over-expression indicated that P53-P21 axis most possiblely is involved in promotion HHV-8 reactivation by NOC2 L.