Effect Of CD45 Isoforms On HIV-1 Gp120 Protein-mediated T Cell Apoptosis & Construction Of SIV Strain Carrying GFP

The depletion of CD4+ T lymphocytes is a central pathogenic featureduring HIV-1 infection,and the loss of CD4+T cells is associated with apoptosis,which represents the major mechanism of CD4+ T cell depletion.However,activation-induced cell death(AICD)played an important rolein the apoptosis of CD4+ T cells,and the molecular mechanism of AICD is remaining uncertain.The protein encoded by HIV,gp120,which was a part of Env can induced apoptosis by AICD.CD45 is regulated the apoptosis by induced the expression of Fas ligation.CD45 is a type I glycoprotein,containing a single trans-membrane domain.CD45 is expressed in multiple isoforms,such as CD45RABC,CD45RO,CD45RA,CD45RB and CD45RC.The CD45 isoforms modified by N-glycans and O-glycans on extracellular domain are reported to be involved in CD45-mediated apoptosis.Therefore,we decided to explorethe roles of CD45 isoforms on apoptosis.In our study,regulation of gp120-induced cell death was investigated in various transfectants with different CD45 isoforms.We detected the expression of CD45 isoforms on Jurkat cells and J45.01 cells.CD45RA,CD45RB,CD45RC,CD45RABC and CD45RO was expressed respectively on Jurkat but not J45.01 cells.We found that gp120 could induce the apoptosis of CD45+ Jurkat cells,but not CD45’ J45.01 cells,suggesting the key role of CD45 in regulation of gp120-induced death of Jurkat cells.Then we examined the effect of gp120 on J45.01 expressed with CD45 isoforms.CD45RO-J45.01 cells undergo a significant apoptosis,J45.01 expressed with other CD45 isoforms could not undergo apoptosis.CD45 is a heavily glycosylated protein,therefore,we next investigated roles played by the glycans in CD45 molecule in the regulation of gp120-induced death.J45.01 expressed with CD45RA,RB,RC and RABC were modified by O-glycans and N-glycans.J45.01 expressed with CD45RO only contained N-glycans.Since the difference of apoptosis among these cells implying the O-glycans may play some role during the apoptosis process,so we conferred that the O-glycans inhibited the apoptosis induced by gp120.Furthermore,our results showed that gp120-induced apoptosis of CD45RA-J45.01 cells and CD45RABC-J45.01 cells could be improved by BG,which can inhibit O-glycosylation of cells.Therefore,it can be suggested that O-glycans on CD45 inhibited apoptosis of Jurkat cells induced by gp120.Lck has suggested that play a key role in the TCR proximal signals leading to death of activated T cells.As the substrate of CD45,dephosphorylation of Lck Tyr505 reflect the activation of CD45.In our study,we compared the dephosphorylation of Lck Tyr505 among J45.01 cells expressed with CD45 isoforms.The result showed that Lck was most susceptible to CD45RO.And the dephosphorylation of Lck Tyr505 were increased when CD45RA-J45.01 cells pretreated by GB,namely,the activation of CD45RA was enhanced.Our result demonstrated that the activation of CD45 was increased by gp120,and the CD45RO is more susceptible to be activated.Lck Tyr394 is an activation site.When the phosphorylation of Tyr 394 increased,the activation of Lck are enhanced.The phosphorylation level of Tyr 394 was enhanced in CD45RO-J45.01 cells and Jurkat cells treated with gp120.Then,the cells were pretreated with P22(the specific inhibitor of Lck)and HBA.The apoptosis was inhibited by PP2 in CD45RO-J45.01 cells and Jurkat cells.In summary,our results illustrate that Lck kinase phosphorylates Tyr394 with resultant T cell activation upon gp120-induced cell stimulation.This would be the basis of the depletion of memory T cells in HIV infection process.Simian immunodeficiency virus(SIVmac)infection in macaques is the best characterized model of AIDS,andSIV-infected macaque has proven of enormous value in studying AIDS pathogenesis and evaluating vaccine strategies to prevent human immunodeficiency virus(HIV)infection.Inorder todetect the SIV-infected cells both in cell culture and in rhesus monkeys,we use enhanced green fluorescent protein(EGFP)as a reporter gene,which was engineered recombinant derivatives of SIV.In our study,we get the GFP DNA sequence from plasmid pEGFP by PCR,then,replacement of nef sequences with EGFP on p239PSE3’,afterenzyme by Sph I,p239SpE3’-GFP and p239SpSp5’ werelinked whichresulted in SIV infection molecule clone.The recombinant SIV infection molecule clone can be used to product a genome that produce recombinant SIV that is engineered to express EGFP,and the recombinant SIV infected cells can be detected by GFP.