Influence Of Extracellular HMGB1on The Virus Replication Of HTLV-1Infected T Cells

BackgroundHigh mobility group protein1(High mobility group box1, HMGB1) is a kind of DNA-binding protein, which is an important member of the damage-associated molecular patterns (damage associated molecular patterns, DAMPs). HMGB1is over expressed in multiple cancers and inflammatory diseases, and closely associated with the physiological or pathological processes of autophagy, DNA damage repair, inflammatory cytokine release and so on. As a product of cell stress, HMGB1is also closely related to pathogen infection, including viruses. Recent studies have shown that HMGB1is associated with the virus replication in the cell of HCV,HIV,HBV and many other viruses. Human T lymphocyte leukemia virus type1(HTLV-1) which can cause adult T-lymphoblastic leukemia (ATL), is a kind of T cell-interested retroviruses.Research shows, that virus load is a key factor in the malignant transformation of T cells. The previous study of our group showed that HMGB1is associated with the activation of infected T cells of the HTLV-1.But there had seldom reports about the research of HTLV-1virus load and HMGB1.ObjectiveInvestigate the influence of extracellular HMGB1on virus replication of HTLV-1infected T cells.Method1. Experimental groups:Four experimental groups were performed in this study:blank group, control group, treatment groupl, treatment group2and isotype control antibody group. Each group set3repeats; the experiment was repeated three times. 2. Cell culture:All the cells were cultured in RPMI1640in an incubator with atmosphere of5%CO2, including15%heat-inactivated fetal bovine serum(FBS),100U/mL penicillin,100μg/mL streptomycin,25mM Hepes and2mM L-glutamine. The culture medium was changed every three days and adjusted the cell density to not more than5×105/mL.3. Cell transfection:The HTLV-1long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into MT2cells in24cell culture plates by Tfx-50-mediated transfection,0.03,0.1,0.3μg/ml rhHMGB1and control PBS, and0.25,0.50,0.75μg/ml of rHMGB1polyclonalantibody(HMGB1pAb) and Rabbit IgG antibodies, were added into culture supernatant respectively, then luciferase activity was detected after48h.4. Agent treatment:Treated MT2cell with0.25μg/ml HMGB1pAb and the isotype control antibody,0.3μg/ml rhHMGB1and the control PBS respectively,after24hours, extracting RNA and detecting the gene expression.5.Measurement of indicators:ELISA detects the HMGB1level in culture supernatants of HTLV-1virus-negative cell and HTLV-1virus-positive cells.Real-time PCR detects effects of over expressing or blocking HMGB1on virus replication. Immunoblotting detects the specific protein of HTLV-1encodes.6. Statistics analysis:The data was analyzed by the SPSS12.0and the results were expressed with mean±tandard (x∧-±s). One-way analysis of variance and T-test were adopted for statistics. Statistical significance was set at P<0.05, significant difference was setat P<0.01.Results1. ELISA results showed that HMGB1level in culture supernatant of HTLV-1virus negative T cell lines and HTLV-1virus positive cell line had no obvious difference.2. Western Blot results indicated that MT2cells expressed Tax, p19protein which were encoded by virus, MT4cells only expressed Tax protein; p19protein was not MT4cells only express Tax protein, p19protein was not detected in MT4cells, HTLV-1virus negative cell Jurkat and MOLT4do not express Tax and p19protein.3. Using luciferin enzyme to report gene show that rhHMGB1obviously promoted the LTR transcription activity, while HMGB1pAb inhibited HTLV-1-LTR transcription activity.4. Real-timn PCR results showed that HMGB1pAb can obviously inhibit the expression of HTLV-1structural gene including gag, env, pol1, pol2, while the rHMGB1can obviously promote the expression of HTLV-1structural gene including gag, env, pol1, po12.ConclusionThe extracellular HMGB1can promote virus replication of HTLV-1infected T cells.