[Effects of different mutated sites in vpr gene of HIV on apoptosis of host cells: experiment with HeLa cells].
OBJECTIVE
To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host cells, and the possible mechanism thereof.


METHODS
Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3.1 (+) plasmid was extracted, the PCR purified product was double-cut by HindIII and BamH, and the cut products were ligated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after 24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument.


RESULTS
The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspase-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 cells were all lower than the cells transfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1-7 recombinant plasmids (all of the Vpr AE subtype) were all lower than those of the No 8-14 plasmids (of Vpr B, AB, C, and C/BC subtypes).


CONCLUSIONS
The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.
Zheng
Yu-huang
YH
AIDS Research Laboratory, Second Xiangya Hospital, Central South University, Changsha 410011, China. yhzyhz2006@yahoo.com.cn
Zhang
Chun-ying
CY
He
Yan
Y
Gong
Guo-zhong
GZ
Li
Hui
H
Chen
Zi
Z
Liu
Meng
M
Zhou
Hua-ying
HY
Li
Ying
Y
Liu
Chun
C
Li
Jing
J
Zhou
Guo-qiang
GQ
Yin
Wei
W
Yuan
Hong-li
HL
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D016334:Genes, vpr; D005822:Genetic Vectors; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D014162:Transfection; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D054325:vpr Gene Products, Human Immunodeficiency Virus; D053148:Caspase 3