| ObjectiveThe obvious factors that affected the pathogenic mechanism of AIDS dementia complex (ADC) include virus'intrinsic pathogenic properties, the neurosusceptibility of the infected individuals and the level of immune suppression caused by HIV-1. Studies have shown that HIV-1 viral load in the brain and cerebrospinal fluid was not always related to the pathopoiesis and extent of ADC, which indicated the occurrence and extent of ADC may be associated with the genetic and biological activity of HIV-1 itself. HIV-1gp120 plays an important role in the virus'binding and fusion with host cells. To search for the relationship between genetic diversity and biological functional site of HIV-1 gp120 and the pathogenesis of ADC, the first part in our study planned to analyze the genetic diversity and biological functional site of sixty HIV-1 gp120 clones isolated from every 6 tissues of two ADC patients.Although the pathogenic mechanism of ADC was unclear, it was certain that HIV-1 could stimulate the glial cells or macrophages secrete cytokines such as TNF-a, IL-1βwhich can make damage to neuronal cells. Since viral load of HIV was not always associated with the occurrence of ADC,the second part of our study were to explore whether gene differences of HIV-1 gp120 could affect their ability to elicit secretion of TNF-a and IL-1β,in order to provide new perspectives to mechanism and new ideas to prevention and control of ADC. MethodsPrimers were designed according to the DNA sequence of HXB2 gp120 by Primer 5.0. Nested PCR was used for amplifying HIV-1 gpl20 from 6 different tissues of each patient, then PCR products were cloned into the PGEM-T vectors, after transformation and selection by ampicillin and blue/white spotting, positive clones were sequenced. Five HIV-1 gp120 positive clones were processed with biological softwares. With the softwares, neighbor-joining tree, N-glycosylation sites, values of ds/dn, and analysis of V3 loop were all done.Two HIV-1 gp120 clones isolated from two different tissues of every patient with ADC (one from the peripheral tissue and the other one from the central tissue) were amplified from TA vectors and inserted into new TA vectors. After transformation into DH5αand selection by ampicillin and blue/white spotting, new TA vectors were digested with EcoR I and Xho I and ligated with MSCV-IRES-GFP digested with the same restriction enzyme. After transformation into DH5αand selection by ampicillin, the retroviral expressing vectors were constructed. After being confirmed by enzyme digestion and sequence analysis, one of retroviral expressing vectors were cotransfected into 293 T cells with pUMVC and pCMV-VSV-G. After assayed by fluorescent microscope and IHC, recombinant retrovirus were collected and added to U87 cells in equal infection units. Infected U87 cells were also assayed by fluorescent microscope and IHC, and concentration of TNF-a and IL-1βin cell supernatant were determined by ELISA.Results1. Sixty HIV-1gp120 clones were obtained, and confirmed by sequencing. All the HIV-1 gp120 clones were identified as HIV-1B strains with BLAST.2. Neighbor-joining trees of HIV-1 gp120 isolated from each ADC patient were drawn with MEGA4.0 software. Some HIV-1 gpl20 clones from different tissues were in the same branch while some clones isolated from different tissues were in different branches.3. Compared to standard HXB2 gp120 sequence, ds/dn of HIV-1gp 120 clones isolated from the first ADC patient was 1.1744±0.0348(p<0.001) while it was 0.8222±0.2086(p<0.001) in the HIV-1 gp120 clones from the second patient with ADC. There was statistical difference between the ds/dn of HIV-1 gp120 clones isolated from the two patients(p<0.001).4. Compared to N-glycosylation sites of HXB2gp120, N-glycosylation sites of HIV-1gp120 clones from the first ADC case were relatively conservative, while N-glycosylation sites of HIV-1 gp 120 clones from the second ADC case varied greatly.5. HIV-1 virus that had infected the brain of the two ADC patients were both predicted as CCR5 usage, macrophage-tropism and nonsyncytium-inducing strains with the present data.6. Four HIV-1gp120 retroviral expressing vectors were constructed and defined as A1/MIG, B1/MIG, A2/MIG, B2/MIG, which were identified by digestion of double restriction enzyme into fragments of 1.44kb and 6.8kb and confirmed by sequencing. Difference existed in four HIV-1 gp120 and V3 gene of A1/MIG was obviously different from the other three.7. Green fluorescent signal existed in both the 293T cells that cotransfected with MSCV-IRES-GFP, pUMVC and pCMV-VSV-G and U87 cells that infected by recombinant retrovirus under fluorescence microscopy. Infection unit of recombinant virus were about 106/ml.8. HIV-1gp120 positive cells appeared in both 293T cells transfected with Al/MIG, B1/MIG, A2/MIG, B2/MIG retroviral expressing vector and U87 cells infected by HIV-1gp120 positive recombinant retrovirus with immunohistochemistry experiments.9. There were obvious differences in TNF-a, IL-1βconcentration of supernatant between the HIV-1gpl20 positive recombinant retrovirus infected cells and HIV-1 gp 120 negative recombinant retrovirus infected cells. Concentrations of TNF-a, IL-1βin Al/MIG recombinant retro virus-infected cell supernatant were higher than the other three recombinant HIV-1gp120 positive retro virus-infected cells supernatant(p< 0.001) while there were no difference in the other three HIV-1gpl20 positive retrovirus-infected cell supernatant(p>0.10).Conclusion1. Genetic diversity and biological functional sites of HIV-1gp120 may be associated with the pathogenic mechanism of ADC. 2. HIV-1gp120 can enhance the ability of glial cells to secret cytokines TNF-a and1L-1β.3. Gene of HIV-1gp120, especially mapped to V3 loop, could affect its ability to elicit secretion of TNF-αand IL-1β.
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