ABSTRACT
Human immunodeficiency virus-1(HIV-1)is the causative agent of acquired immunodeficiency syndrome(AIDS), which can invade the central nervous system and cause a series of neurological disorders such as HIV-associated neurocognitive disorders(HAND). Nef protein is one of the early proteins of HIV-1 virus, it is found that the neurotoxicity of HIV-1 mutant lacking nef gene is less neurotoxic than that of wild strain with nef gene, indicating that Nef protein plays an important role in HIV-1 neuropathogenicity.In this paper, neurotoxicity and mechanism of Nef are reviewed from three aspects, including the effects of Nef on the blood-brain barrier permeability and the direct and indirect damage to neurons.
ABSTRACT
Objective@#To investigate the toxic effect of HIV-1 Vpr protein on neurons.@*Methods@#HIV-1 vpr gene was amplified by nested PCR in four parts of peripheral spleen (SPL) and central nervous tissue meninges (MG) of HIV-associated dementia (HAD) patients and non-HAD patients. Eukaryotic expression vector pEGFP-N1-vpr was constructed. The gene sequence and key amino acid sites were analyzed by BLAST and MEGA6. The expression of Vpr protein in N2a cells was detected by Western-blotting. The effects of Vpr proteins from different sources on the activity and cell cycle of N2a cells were studied by flow cytometry.@*Results@#HIV-1 vpr gene was successfully amplified by PCR. Sequence analysis showed that the vpr gene sequence belonged to HIV-1B subtype. There were amino acid mutations at C-terminal 84, 86 and 87 sites of central Vpr protein from HAD and non-HAD patients. Vpr protein could inhibit the activity of nerve cells, leading to G2 phase arrest. Different sources of Vpr had different intensity of action. Compared with other groups, Vpr protein from the meninges of HAD patients showed stronger inhibition of cell activity and G2 phase arrest ability.@*Conclusions@#Variations in key amino acid sites of Vpr protein could cause significant changes in its biological functions, and its significance in the pathogenesis of HAD remains to be further studied.
ABSTRACT
Objective@#BG-derived HIV-1 Tat protein from an HIV-associated dementia (HAD) patient was expressed in E. coli BL21(DE3) and purified in order to research the effects on human umbilical vein endothelial cells (HUVECs) activity.@*Methods@#The recombinant plasmid pGEX-KG-tat with HIV-1 tat stored in our laboratory was amplified by PCR. The PCR product was cloned into pET-32a-tat. The recombinant plasmid pET-32a-tat was transfected into E. coli, and Tat protein was expressed in BL21(DE3), which was induced by IPTG. Then it was purified by Ni-chelating chromatography column and gel filtration preloaded column, and identified by SDS-PAGE and Western blot(WB). The concentration was determined by BCA Kit. Different concentrations of Tat were added into HUVECs to detect their effects on cell activity by cck-8.@*Results@#The Tat with high purity was efficiently expressed in BL21 (DE3) and obtained by using the Ni-chelating chromatography column and gel filtration preloaded column. The concentration was 0.47 mg/ml by using BCA Kit. As the concentration of Tat increased, HUVECs activity decreased. There was no significant difference in cells viability between negative control with 100 ng/ml and 200 ng/ml group (P>0.05). There was significant difference in cells viability between negative control with 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group (P<0.05). But the difference between 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group was not statistically significant (P>0.05).@*Conclusions@#The HIV-1 Tat with biological activity was efficiently expressed in BL21 (DE3), and the activity of HUVECs was significantly decreased when the concentration reached 300 ng/ml.
ABSTRACT
Objective@#To study the sequence characteristics and variation of HIV-1 Vpr gene in different parts of an AIDS dementia complex (ADC) patient and provide basis for the study of the neurologic pathogenesis of HIV-1-assciatd dementia.@*Methods@#Genomic DNA was extracted from peripheral samples (lymph nodes, spleen, liver) and central nervous system (meninges, frontal lobe, temporal lobe gray matter, frontal white matter, basal ganglia cortex) of an ADC patient, The Vpr gene was amplified with nested polymerase chain reaction (PCR). PCR products were cloned into the pMD19-T vector. After transformation into DH5α competent E. coli, five positive clones were sequenced. The phylogenetic tree was built and genetic distance was calculated through MEGA6, and the values of ds/dn was calculated through SNAP, then the changes of the amino acid sites were analyzed.@*Results@#HIV-1 Vpr genes isolated from different tissues of the ADC patient had variations. Vpr HIV-1 gene sequences from central nervous system and peripheral tissues were intercrossed together in the phylogenetic tree. Central nervous system and peripheral HXB2 Vpr had no significant differences in genetic distance. The ds/dn of all the HIV-1 Vpr gene sequences were 3.3749.@*Conclusions@#The HIV-1 Vpr sequences were different in the ADC patient, and there were different variations in different parts of the peripheral and central regions. Whether these variations are related to the pathogenesis of ADC remains to be further studied.