Study Of Effect On G2 Arrest And Apotosis In Jurkat And Hela Cells By HIV-1 Vpr

Objective The viral protein R(Vpr) of HIV-1 is a small basic protein(14 kDa) of 96 amino acids,and is well conserved in HIV-1, HIV-2 and SIV.The role of Vpr in the pathogenesis of AIDS is undeniable,but its genetic diversity and real functions during the natural course of infection are still subject to debate.The Vpr role in the patho-physiology of AIDS has been believed very important. Experimental animals infected with some defective vpr mutants or a vpr null mutant showed decreased virus replication and delayed disease progression,displayed a very low virus burden and did not develop immunodeficiency disease.Vpr has been shown to play kinds of functions during virus replication,including an effect on the accuracy of the reverse-transcription process,the nuclear import of the viral DNA as a component of the pre-integration complex(PIC),cell cycle progression, regulation of apoptosis,and the trans-activation of the HIV-LTR as well as host cell genes.This study is focused on the following topics:1 whether the capability of vpr induced cellcycle arrest changed when its amino acid occurred mutation.2 the possible relationship between vpr induced cellcycle arrest and apotosis.Methods We selected representative 14 mutants through statistical methods.Both eukaryotic expression vector pcDNA3.1(+) and PCR products purified,double-cut by Hindâ…¢and BamH and the cut products legated and transformed into competent cells JM109.The 14 reconstructed plasmid was confirmed right by sequencing,and then transfected into Jurkat-cells and Hela-cells by electronic and liposome transiently respectively,established Vpr_wt,Vpr-Fs,blank and pcDNA3.1 blank.Cells were harvested after 48 hours,mRNA expression was detected by RT-PCR,The DNA content and percentage of apoptosis were monitored by flow cytometry.Results Transfected with 14 mutant HIV-1 Vpr protein,cells display different G2 precentage and apotosis ratio.HIV-1 vpr induce cellcycle G2 arrest and apotosis,wherase Vpr Fs with a C-terminal end truncation,vector pcDNA3.1(+) and the blank cells can not.The G2 precentage and apotosis ratio reduced when transfected with vpr expressing mutationg of 70V,85P,86G,94G compared to the wild type.Subtype AE has a weaker potential to induce cellcycle G2 arrest and apoptosis.Preliminary,we find that the higher G2 percentage followde the higher ratio of apotosis.Conclusions HIV-1 vpr can induce cellcycle G2 arrest and apotosis,wherase Vpr Fs with a C-terminal end truncation can not.We firstly found that mutated sites of 70V,85P,86G,94G may reduce the ability of Vpr to induce cellcycle G2 arrest and apotosis,Subtype AE of vpr in Chinese HIV-1 patients has a weaker potential to induce cellcycle G2 arrest and apoptosis.Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis.