Metabonomic Studies Of Bioactivity Of HIV-1Tat Protein Based On Mass Spectrometry

As one of the six accessory proteins of HIV-1, Tat is critical for viral replication andplays an important role in viral dissemination and pathogenesis. Intracellular Tat, recruitscellular proteins to relieve the repression of the viral long-terminal repeat (LTR), andthereby the viral promoter can induce the expression of viral genes. Extracellular Tat,secreted from infected cells, was found to play an important role in the pathogenesis ofproliferation inhibition and apoptosis of CD4+T cells, viral dissemination, AIDSassociated Kaposi's sarcoma (KS) and HIV-1associated dementia. These findings supportthe notion that Tat acts as a "viral toxin" and obligate it as an "important target" for drugintervention and therapeutic vaccines.A number of chronic metabolic abnormalities including disorders of lipid metabolismwith or without lipodystrophy, insulin resistance, cardiometabolic syndrome, alteredphosphate metabolism, and an increased prevalence of impaired glucose tolerance arefound prevalent in HIV patients with or without receiving highly active antiretroviraltherapy (HAART). Although multiple risk factors have been proposed, the etiology ofthese metabolic abnormalities remains unclear. It would be interesting to see whetherHIV-1Tat plays a critical role in HIV metabolic abnormalities.Metabonomics is a top-down systems biology approach whereby metabolic responsesto biological interventions or environmental factors are analyzed and modeled.Metabonomics provides insights into the global metabolic status of the entire organism bymonitoring the entire pattern of low molecular weight compounds rather than focusing onan individual metabolic intermediate (MI). It has been widely applied into diseasediagnosis, pathophysiology, toxicology, pharmacodynamic assessment, gene function andquality control of Traditional Chinese Medicines. Compared to the transcriptome andproteome, the physical and chemical properties of the objects are more complex andmetabolome are diverse in nature (range from highly polar to non-polar) and concentration(range from mM to pM). An extensive metabonomics study needs non-discriminatoryanalytical techniques with each analytical instrument being selective to certain metabolites,and therefore parallel use of multiple analytical methods would be advantageous in identifying a broader spectrum of metabolites relevant to physiopathological alteration.The objectives of this study are to determine how HIV-1Tat influences metabolicenvironments both in mice and CD4+T cells to elucidate its complex pathogenicmechanism using gas chromatography-mass spectrometry (GC-MS), reversed-phase liquidchromatography–mass spectrometry (RPLC-MS) and hydrophilic interaction liquidchromatography-mass spectrometry (HILIC-MS)-based metabonomic methods. The maincontents are:1, Expression, purification and identification of bioactive Tat proteinThe DNA encoding for Tat protein (1–86aa) from the BH-10clone of the IIIB strainof HIV-1(clade B) was amplified by PCR and inserted into pET21b vector to constructprokaryotic expression plasmids tat-pET21b. The recombinant plasmid was transformedinto E.coli BL21(DE3) for expression. The fusion protein Tat was expressed with relativemolecular weight (MW)15kD under induction of IPTG, which accounts for about15%oftotal bacterial proteins. The expressed Tat protein was purified by Ni-NTA column and SPSepharose fast flow Ion-exchange chromatography, and the purity was about90%.Endotoxin level of purified recombinant Tat protein was determined using the Limulusamebocyte lysate test and the result showed the final concentration of endotoxin in theeluted Tat was approximately0.05EU/μg of protein which is within the acceptable limit.Then the purified endotoxin-free Tat protein was testified with specific anti-Tatmonoclonal antibody by Western-blot and was verified to be bioactive using theLTR-transactivation assay.2, Metabolic profiling in HIV-1Tat induced ICR mice serum using GC-MSThe HIV-1Tat protein is released by infected cells and has numerous biologicalactivities which might contribute either to the impairment of the immune response or toviral dissemination and pathogenesis. To date, the effects of Tat protein on metabolitesremain unclear. In this study, a metabolomic study on serum of HIV-1Tat-induced ICRmice was performed to research the pathologic mechanism of Tat protein by using gaschromatography coupled to mass spectrometry (GC-MS). NIST2002, a commercial massspectral database, was used for rapid identification of the detected metabolites. The procedures of sample preparation, derivatizative conditions and chromatographicconditions were optimized, and validation of the method was carried out. The resultsshowed that GC-MS analytical method was stable and reliable. It was capable to describethe biochemical composition of biological samples. The established GC-MS analyticalmethod was applied to the metabonomic study on Tat-induced mice serum. Principalcomponents analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) wereused for recognizing the different metabolic patterns between the Tat and control groups.Sixteen significantly changed metabolites in HIV-1Tat-induced mice were identified thatare involved in amino acid metabolism, tricarboxylic acid (TCA) cycle and lipidmetabolism, which contributed to the elucidation of the complex pathogenic mechanism ofTat protein and may shed new lights on future improvement of HIV-1therapy.3, Combined LC/GC-MS and quantitative real-Time PCR analyses reveal systemsmetabolic changes in Jurkat T-cells induced by HIV-1Tat proteinHIV-1Tat protein is released by infected cells and can affect bystander uninfected Tcells and induce numerous biological responses which contribute to its pathogenesis. Toelucidate the complex pathogenic mechanism, we conducted a comprehensiveinvestigation on Tat protein-related extracellular and intracellular metabolic changes inJurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS),reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilicinteraction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomicsapproach. Quantitative real-time PCR (qRT-PCR) analyses were further employed tomeasure expressions of several relevant enzymes together with perturbed metabolicpathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1Tat causedsignificant and comprehensive metabolic changes, as represented by significant changes of37metabolites and7relevant enzymes in HIV-1Tat-treated cells, which are mainlyinvolved in glycolysis, citrate cycle, lipid metabolism and amino acid metabolism. Theseresults provide metabolic evidence of the complex pathogenic mechanism of HIV-1Tatprotein as a "viral toxin", and would help obligate Tat protein as "an important target" fortherapeutic intervention and vaccine development. 4, Characterization of Tat antibody responses in Chinese individuals infected withHIV-1There are two converse conclusions about the relationship between the anti-Tatantibody in HIV infected patients and the development and progression of AIDS. Therefore,wider and deeper anti-Tat responses analyses are very important. To define theseimmunoprofiles, we investigated Tat antibody responses in plasma samples from326Chinese individuals infected with HIV-1as well as100samples from healthy blood donorsas controls. We first screened for anti-Tat-seropositive samples using ELISA withrecombinant full-length Tat protein. Next, the immunoprofiles of the anti-Tat antibodyresponses were determined by ELISA using a synthetic N-terminal domain of Tat (Tat1-21)and six recombinant Tat peptides as the analytic antigens. The neutralizing activities of theanti-Tat-seropositive samples were evaluated using an LTR-transactivation neutralizingassay. Out of326HIV-1-seropositive individuals, only42were found to be positive foranti-Tat antibodies and6for Tat-related antibodies. Among the anti-Tat-seropositive andTat-related samples we found six different immunological profiles of anti-Tat antibodyresponses: full-potential response, combined response, N-specific response, C-specificresponse, full-length Tat-specific response and Tat-related response. These responses weredefined based on differential reactivity with several analytic antigens, and they representtwo types of anti-Tat responses—the major complete response and the alternative C-proneresponse. The anti-Tat-seropositive samples showed significantly higher Tat-neutralizingactivities compared with samples from both anti-Tat-negative HIV-1patients or healthyblood-donor samples. Tat-neutralizing activities were found to significantly correlate withthe reactivities of antibodies against specific Tat antigens and the CD8cell counts. Siximmunoprofiles of anti-Tat responses in Chinese patients infected with HIV-1were definedand were found to have significant Tat-neutralizing activities. The data we present herecould contribute to a better understanding of the significance of anti-Tat responses inpreventing HIV pathogenesis and could be useful for designing more effective vaccines inthe future. It also provided important information for studying the role of Tat in HIVinfected patients using metabonomic approach.In conclusion, this is the first metabonomic study to determine HIV-1Tat-induced biochemical alterations both in ICR mice and Jurkat T-cells using combined LC-MS andGC-MS. We identified16and37significantly changed metabolites in Tat-induced miceserum and Jurkat T-cells, respectively, which are mainly involved in glycolysis, citratecycle, lipid metabolism and amino acid metabolism, which were helpful for revealing thecomplex pathogenic mechanism of HIV-1Tat as a "viral toxin" and may present importanttargets for therapeutic intervention. Blocking or modifying these points of HIV-1Tatperturbed pathways are attractive approaches to the treatment of HIV-related chronicmetabolic abnormalities. Further studies are needed to deepen the understanding about thebiological function and regulation mechanism of HIV-1Tat protein. In addition, ourfindings suggest that metabonomics based on integration of GC-MS and LC-MStechniques could be a potent tool for cell metabolomes.