Expressed HIV-1 Vpr Mediated Apoptosis And Proteome Alteration Of Human T Cell Leukemia Virus Type 1 Transformed Cells

Aim:To construct the recombinant adenovirus carrying the HIV-1 vpr gene,to express HIV-1 viral protein R(Vpr) endogenously in the recombinant adenovirus rAd-vpr infected HTLV-1transformed C8166 cells.To investigate the effects of expressed HIV-1 Vpr induced cell cycle arrest and cell death.To characterize how intracellular HIV-1 Vpr protein expression modulated the protein expression profile of C8166 cells,and further to speculate the possible mechanisms of Vpr on C8166 cells via differentially expressed proteins identified on the basis of establishing the pivotal technological platform for studying the proteomics of C8166 cells.To evaluate the feasibility of employing the HIV-vpr gene,via targeted gene transfer,as a potential therapy to treat adult T-cell leukemia(ATL).Methods:The gene fragment of vpr was amplified by PCR and inserted into the shuttle vector pAdTrack-CMV to obtain the recombinant plasmid pAdTrack-CMV-vpr.Then,the pAdTrack-CMV-vpr was linearized with restriction endonuclease Pme I,and transformed into E.coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 for homologous recombination by electroporation.This generated recombinant plasmids, which were digested by Pac I and transfected into HEK293 cells with Lipofectamine 2000 to package and amplify the recombinant adenovirus rAd-vpr.The intracellular expression of HIV-1 Vpr in recombinant adenovirus rAd-vpr infected C8166 cells were detected by Western blotting.The efficiency of C8166 cell infected with recombinant adenovirus was detected by flow cytometry and observed under fluorescence microscopy.C8166 cell membrane integrity change,cell cycle arrest,apoptosis and mitochondrial membrane potential loss that induced by the recombinant adenovirus rAd-vpr were checked by FACS and observed by laser confocal microscopy.The whole cell proteins were prepared from mock-,rAd-vpr and rAd-vector infected C8166 cells respectively,and applied to two dimension gel electrophoresis(2-DE).The linear IPG dry strip(pH4-7,17cm) was used for isoelectric focusing of the first dimension and 10%SDS-PAGE was performed for the second dimension.The 2-DE patterns of the whole cell proteins from 3 group cells were analyzed with PDQuest 7.4 software.12 differentially expressed protein spots were selected for trypsin digestion,and the resulted peptides were subjected to LC/MS/MS analysis.MASCOT was employed to identify proteins from acquired MS spectra by searching against NCBInr database,and the mRNA levels of 14-3-3 and GSTP1 were detected by semi-quantitative RT-PCR.Results:The recombinant adenovirus rAd-vpr containing vpr gene was successfully constructed,which could effectively infect C8166 cells.The result of Western blotting confirmed Vpr expression endogenously in recombinant adenovirus rAd-vpr infected C8166 cells.G₂/M phase cell cycle arrest was observed and typical characteristics of apoptosis were detected in rAd-vpr infected cells,including sub-diploid peak exhibition in DNA content assay,Hoechst 33342 accumulation,apoptotic body formation,mitochondrial membrane potential and plasma membrane integrity loss.The 2-DE patterns of mock-, rAd-vpr and rAd-vector infected C8166 cells with high resolution and reproducibility were obtained.12 differentially expressed protein spots were successfully identified by liquid chromatography-tandem mass spectrometry(LC/MS/MS).By proteomic assay,the apoptosis mechanism was confirmed,exhibiting the regulation of caspase-3 activity indicator proteins(vimentin and Rho GDP-dissociation inhibitor 2),mitochondrial protein (prohibitin) and other regulatory proteins.In addition,the up-regulation of anti-inflammatory redox protein,thioredoxin,was identified in the rAd-vpr infected group. Finally,the mRNA levels of 14-3-3 and GSTP1 were higher the recombinant adenovirus rAd-vpr infected C8166 cells than those in the control and rAd-vector infected groups.Conclusion:The recombinant adenovirus rAd-vpr was successfully constructed by homologous recombination in bacteria.C8166 cells infected with the recombinant adenovirus expressed Vpr.Intracellular expressed HIV-1 Vpr can kill HTLV-1transformed cells effectively,and may avoid the risks of inducing severe host inflammatory responses through apoptosis-inducing and anti-inflammatory activities.These properties of recombinant adenovirus rAd-vpr shown in C8166 cells demonstrated its potential benefits in ATL therapy.