The Study On Comparative Changes Of Cell Function And Protein Profiles In T Cell Lines Expressed HIV-1 And SIV Nef Protein

AIM:Study on the comparative changes of cell function and proteomics in T cell line expressed HIV-1 and SIV Nef protein.METHODS:Constructions of pAd-HIV-1 nef-EGFP and pAd-HIV-1 nef-EGFP recombinant adenoviral plasmids.Use the HEK293T cell to produce and amply the adenovirus, detecting the adenovirus titers to infect the T cell lines as well as make which express the protein.Setting four experimental groups:Control,Ad-EGFP,Ad-HIV-1 nef-EGFP and Ad-SIVmac239 nef-EGFP group.Detecting the infection rate of the recombinant adenovirus by the flow cytometry;Observing the fluorescence expression by the fluorescene microscopy;Identify the expression of HIV-1 Nef and SIVmac239 Nef protein by western blotting;Detecting the influence on the cell proliferation,cell cycle,the suface molecules of CD4,CXCR4,HLA-I and HLA-DR after expression of HIV-1 Nef and SIVmac239 Nef protein respectively.Preparation the whole-cell protein of MT-2 cell from four experimental groups respectively,separating the proteins in four experimental groups by two-dimensional electrophoresis(2-DE),analyzing the results by ImageMaster 2D Elite 5.0 software, to find the differentially expressed proteins between Ad-HIV-1 nef-EGFP and Ad-SIVmac239 nef-EGFP groups.The differential expression proteins were identified by matrix-assisted laser resorption/ionization time of flight mass spectrometry (MALDI-TOF-MS),getting peptide mass fingerprint(PMF),and go on doing MALDI-TOF/TOF-MS,getting secondary PMF.Using the Mascot software and IPI Human database to define the protein. RESULTS:Constructions of pAd-HIV-1 nef-EGFP and pAd-HIV-1 nef-EGFP recombinant adenoviral plasmids successfully.After the package and amplification by the HEK 293T cell,the producing adenovirus titers was:pAd-EGFP group:3.56×10~6 infection union/mL(IU/mL),pAd-HIV-1 nef-EGFP group:5.40×10~6 infection union /mL(IU/mL),pAd-SIVmac239 nef-EGFP group:5.62×10~6 infection union/mL(IU/ mL);According to the adenovirus titers to infect the T cell line and detect the infection rate,confirming the MOI when infection rate is getting or beyond 95%.All the experiment after according to the MOI:pAd-EGFP:17.8 IU/cell,pAd-HIV-1 nef-EGFP:27IU/cell,pAd-SIVmac239 nef-EGFP:28.1 IU/cell.The HIV-1 and SIV nef gene expression in MT-2 cell were identified by western blotting.The HIV-1 and SIV nef gene expression could promote the proliferation of MT-2 and C8166,and increase the cell ratio in DNA synthesizing and dividing phase. HIV-1 Nef and SIVmac239 Nef protein expression could decrease the CD4,CXCR4, HLA-I and HLA-DR surface molecules on MT-2 cell,furthermore,the effect of decrease the CD4,CXCR4,HLA-I by SIVmac239 Nef protein is stronger than the HIV-1 Nef.Comparative analyzing the 2-DE patterns of Ad-HIV-1 nef-EGFPand Ad-SIVmac239 nef-EGFP group,the protein spots is 1002 and 949 respectively,the matching rate is 89.9%.Eight differential expression protein spots were selected to identify by the mass spectrometry,the result turned out that four protein were probably related to HIV pathogenicity.Proteasome 26S non-ATPase subunit 13 isoform 2 was not expressed in the Ad-SIVmac239 nef-EGFP group,which possibly related the regulation of proteasome function and the lower activation level of SIV infected T cell,HIV-1 Nef might lost this function and lead to the T cell activation level increase;CMPK cytidylate kinase was expressed only in Ad-HIV-1 nef-EGFP group,which probably related to the HIV replication;TPT1 tumor protein, translationally-controlled 1 was not expressed in Ad-SIVmac239 nef-EGFP group but strongly expressed in Ad-HIV-1 nef-EGFP group,STRAP Serine-threonine kinase receptor-associated protein was down-regulated expressed in Ad-SIVmac239 nef-EGFP group,which were possibly related to increase proliferation of T cells which infected with HIV.CONCLUSIONS:Constructions of pAd-HIV-1 nef-EGFP and pAd-HIV-1 nef-EGFP recombinant adenoviral plasmids successfully,the HIV-1 and SIV Nef protein expression in MT-2 cell were identified,the Nef could also decrease the CD4,CXCR4,HLA-I and HLA-DR surface molecules,furthermore,the effect of decrease the CD4,CXCR4, HLA-I by SIVmac239 Nef protein is stronger than the HIV-1 Nef.The result of comparative proteomics between MT-2 cell expressed HIV-1 and SIV Nef protein turned out to be four different proteins probably related to the increase of HIV replication support by the host,all of them were the highly conserved proteins.During the HIV evolution and zoonosis,HIV-1 Nef might reacted selectively on some conserved proteins in host and led to the increased pathogenicity.These conserved proteins probably could act as the medicine target to reduce the HIV pathogenicity in host.