Study on effects of G_2 arrest and apoptosis in Jurkat cell by HTV-1 Vpr / 中华微生物学和免疫学杂志

ABSTRACT

Objective To explore ability of the vpr gene of human immunodeficiency virus type 1 ( HIV-1 vpr) to induce cell G_2 arrest and apoptosis, and the influence when it mutated, the relationship between Vpr-induced G_2 arrest and apoptosis inductions. Methods Fourteen mutant vpr fragments selected from Chinese patients with HIV. Both eukaryotic expression vector pcDNA3.1( + ) and PCR products purified, double-cut by Hind Ⅲ and BamH Ⅰ and the cut products legated and transformed into competent cells JM109. The 14 reconstructed plasmids electronically transfected into Jurkat-cells, and established cells with pcDNA3. 1-vpr , pcDNA3. 1-vpr-Fs and pcDNA3. 1 blank cells, and without pcDNA3. 1 cell. Cells were harvested after 24 h. 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 G_2 percentage and apoptosis ratio. HIV-1 vpr induce cell cycle G_2 arrest and apoptosis, wherase Vpr Fs with a C-terminal end truncation, vector pcDNA3.1( + ) and the blank cells can not. The G_2 percentage and apoptosis ratio reduced when transfected with vpr expressing mutating of 70V, 85P, 86G, 94G compared to the wild type. Subtype AE has a weaker potential to induce cell cycle G_2 arrest and apoptosis. Preliminary, we find that the higher G_2 percentage followed the higher ratio of apoptosis. Conclusion HIV-1 vpr can induce cell cycle G_2 arrest and apoptosis, 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 cell cycle G_2 arrest and apoptosis, subtype AE of vpr in Chinese HIV-1 patients has a weaker potential to induce cell cycle G_2 arrest and apoptosis. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G_2 arrest correlated with the levels of apoptosis. And investigate the pathegenesis of HIV vpr. This can also make a good foundation for further study on gene therapy.