title: [Effects of different mutated sites in vpr gene of HIV on apoptosis of host cells: experiment with HeLa cells].
abstract: 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.
authors: AIDS Research Laboratory, Second Xiangya Hospital, Central South University, Changsha 410011, China. yhzyhz2006@yahoo.com.cn
authors: Yu-huang
authors: YH
authors: Zheng
authors: Chun-ying
authors: CY
authors: Zhang
authors: Yan
authors: Y
authors: He
authors: Guo-zhong
authors: GZ
authors: Gong
authors: Hui
authors: H
authors: Li
authors: Zi
authors: Z
authors: Chen
authors: Meng
authors: M
authors: Liu
authors: Hua-ying
authors: HY
authors: Zhou
authors: Ying
authors: Y
authors: Li
authors: Chun
authors: C
authors: Liu
authors: Jing
authors: J
authors: Li
authors: Guo-qiang
authors: GQ
authors: Zhou
authors: Wei
authors: W
authors: Yin
authors: Hong-li
authors: HL
authors: Yuan
mesh_terms: 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*
chemical_list: D054325:vpr Gene Products, Human Immunodeficiency Virus; D053148:Caspase 3