Analysis Of Genetic Diversity Of HIV-1 Tat In Patients With ADC And Non-ADC And Effect Of The Diversity On The Eliciting Of TNF-α And IL-1β By U87 Cells

ObjectiveADC is one of the most important neurological complications after the invasion of HIV-1 into the central nervous system(CNS). HIV-1 viral load in the CNS was not always related to the lesions of ADC, which indicated that the occurrence of ADC may also be associated with the genetic mutation and biological activity of HIV-1 itself. HIV-1 is highly variable, HIV-1 variation in the CNS is different from periphery because of the CNS-specific environment. The HIV-1 trans-activator (Tat) plays an important role in the pathognensis of ADC, Tat can stimulate the activated macrophages and glial cells to secrete various of cytokines, such as TNF-a and IL-1β, causing damage to neuronal cells. To search for the genetic diversity of HIV-1 tat in the central nervous system and peripheral tissues and relationship between the genetic diversity and the pathogenesis of ADC, our research analyzed the genetic diversity and biological functional sites of forty-five HIV-1 tat clones isolated from central nervous system and peripheral tissues of one ADC and one non-ADC patients, we also studyed effect of the genetic diversity on the eliciting of TNF-a and IL-1βby U87 cells, in order to provide basis for pathogenensis, prevention and treatment of ADC.Methods1. The tat exon 1 and surroudding gene was amplified with nested PCR from genomic DNA which was extracted from central nervous system (grey matter from frontal cortex, temporal cortex, meninges, basal ganglia, white matter from frontal cortex) and peripheral tissues (spleen and lymph node) of one ADC patient and one non-ADC patient. PCR products were cloned into the pGEM-T vector, after transformation and selection by ampicillin and blue/white spotting, five of the positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid sites were all done.2. Four HIV-1 tat clones isolated from spleen and basal ganglia tissues of the two patients were selected, then tat exon 1 gene were amplified with PCR. PCR products were cloned into the pMD19-T vector, after transformation into DH5αand selection by ampicillin and blue/white spotting, the positive clones were sequenced. The confirmed recombinant vectors were digested with EcoRⅠ,XhoⅠand the tat gene was then cloned into the MSCV-IRES-GFP(MIG) which was digested with the same restriction enzyme. After transformation into DHsa and selection by ampicillin, the combinant retroviral expressing vectors were constructed. Combinant retroviral expressing vector was cotransfected into 293T cells with pUMVC and pCMV-VSV-G vectors, after tested by fluorescent microscope, the recombinant retrovirus were collected and added into U87 cells in equal infection units, then the concentration of TNF-αand IL-1βin U87 cell supernatant were measured by ELISA, data was processed by SPSS software.Results1. Forty-five HIV-1 tat clones were obtained from central nervours system and peripheral tissues of the two patients, and they were all confirmed by sequencing and BLAST. Neighbor-joining trees showed that most of the HIV-1 tat sequences isolated from different tissues of the ADC patient were in their own branches while few in the same branch. The ten HIV-1 tat sequences isolated from two tissues of the non-ADC patient cross together. Which indicated that the mutation process of tat genes isolated from ADC patient suffered more compartmentalization than tat genes isolated from non-ADC patient.2. The analysis of genetic distances showed that the genetic distance of HIV-1 tat genes isolated from the same central nervours system or peripheral tissues was smaller while it is larger between central nervours system and peripheral tissues. Moreover, in the same patient the mutation of tat genes isolated from periphery was greater than tat genes isolated from CNS, it is thus clear that the environment of CNS and periphery can affect the genovariation of tat gene. In addition, the distance between HXB2 tat and tat genes isolated from central nervours system or peripheral tissue respectively of ADC patient was different significantly, but there was no difference in non-ADC patient, which illustrated that all the different situations such as patient,virus,lesions and body condition can affect the genovariation of tat gene.3. HIV-1 evolution was affected by the interaction of gene mutation and environmental selection, ds/dn is one important parameter to estimate evolutionism. The ds/dn of all HIV-1 tat sequences isolated from ADC patient was 3.6653±2.5555, the ds/dn of tat sequences isolated from periphery of the ADC patient was 3.8976±0.0000 while it was 1.9241±1.0838 of tat sequences isolated from CNS; The ds/dn of all HIV-1 tat sequences isolated from non-ADC patient was 4.5448±2.9407, the ds/dn of tat sequences isolated from periphery of the non-ADC patient was 3.2528±1.7273 while it was 5.5216±3.5461 of tat sequences isolated from CNS. The genovariation of tat sequences isolated from central nervours system and peripheral tissues of the two patients were all suffered from negative selection pressure, the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.4. Analysis of amino acid sites showed that the amino acid mutation sites of Tat isolated from the ADC patient and non-ADC patient were different, in the same patient the amino acid mutation sites and mutation frequency of Tat isolated from the central nervour system and peripheral tissues were different too, some mutation sites occurs only in the periphery while some occurs only in the CNS.5. According to HIV-1 tat sequence analysis, we choose four tat genes named Sa,Ba,Sn,Bn which were isolated from spleen and basal ganglia tissues of the two patients, later we successfully constructed four recombinant retroviral expressing vectors named Sa/MIG,Ba/MIG,Sn/MIG,Bn/MIG, then the retroviral expressing vectors were cotransfected into 293T cells with pUMVC and pCMV-VSV-G vectors, to package recombinant retrovirus, the infection units of recombinant retrovirus were from 5×106 to 6.33×106 IU/ml.6,After infection of U87 cells by equal recombinant retrovirus, TNF-α, IL-1βconcentration in supernatant of U87 cells were detected with ELISA, the results showed that there was no difference between the negative control group C and U87 cells control group U, which indicated that the recombinant retrovirus could not affect the eliciting of TNF-αand IL-1β. The TNF-αIL-1βconcentration in Tat experimental groups were all higher than negative control group C, which indicated that Tat could induce U87 cells to elicit TNF-a and IL-1β. Compared with the four Tat experimental groups(Sa,Ba,Sn,Bn), there was no difference in the concentration of TNF-a, the concentration of IL-1βin Sa,Ba,Sn groups were higher than Bn group, which indicated that the amino acid mutation sites of the four Tat sequences could not affect the eliciting of TNF-a, but could promote U87 cells to elicit IL-1β.Conclusion1. Genetic diversity of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between central nervours system and peripheral tissues in ADC patient were greater than the non-ADC patient, moreover, in the same patient the mutation of tat isolated from periphery is greater than tat isolated from CNS. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations. Besides, the amino acid mutation sites of Tat isolated from the ADC patient and non-ADC patient were different, in the same patient the amino acid mutation sites and mutation frequency of Tat isolated from the central nervous system and peripheral tissues were different too.2. Tat could induce U87 cells to elicit TNF-αand IL-1β. Furthermore, the concentration of IL-1βinduced by Tat isolated from spleen,basal ganglia tissues of ADC patient and spleen tissue of non-ADC patient were higher than Tat isolated from basal ganglia tissue of non-ADC patient, which indicated that some of the amino acid mutation sites of Tat could promote U87 cells to elicit IL-1β, this may be related to the damage of neurons in ADC patient.