Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Interaction between HIV-1 Tat and EBV Zta favours immune escape of B cells by downregulating HLA-ABC expression

doi: 10.1007/s00018-025-06029-5

Figure Lengend Snippet: HIV-1 Tat and EBV Zta physically interact in B cells and human serum. (A) Immunoprecipitation of Tat in B cells. The RPMI8866 lymphoblastoid cell line inducibly expressing unconjugated HIV-1 Tat (RPMI8866 Tat i cell line) upon treatment with Doxycycline (Tat) was treated or not with 1 µg/ml recombinant Zta protein. Cell lysates were subjected to immunoprecipitation using anti-Tat antibodies. Input and immunoprecipitated fractions were analysed by western blotting staining for Tat, Zta, Galectin 9 (negative control) and β-actin (control of protein load). (B) Immunoprecipitation of Zta in B cells. The RPMI8866 lymphoblastoid cells were transfected with YFP (lane 1), YFP-Tat (Tat, lane 2), mCherry-Zta (Zta, lane 3), YFP-Tat and mCherry-Zta (lane 4), YFP-Zta and mCherry-Tat (lane 5), followed by immunoprecipitation of Zta using anti-Zta antibodies after 24 h. Input and immunoprecipitated fractions were analysed by western blotting staining for Tat, Zta, Galectin 9 (negative control) and GAPDH (control of protein load). ( C) A healthy donor (HIV-negative) serum sample supplemented with Tat and Zta (at 250 ng/ml and 1000 ng/ml, respectively, HIV-neg. + Tat + Zta) and a serum sample from an HIV-positive individual (HIV-pos.) were immunoprecipitated using Protein G magnetic beads cross-linked with anti-Zta or anti-Tat antibodies. Immunoprecipitated (IP) and non-immunoprecipitated flowthrough (FT) fractions were analysed by Western blotting staining for Zta, Tat, transferrin (negative control) and transthyretin (negative control). (D , E) Analysis of Tat and Zta interaction by an in vitro binding assay. Equal amounts of recombinant HIV-1 Tat protein linked to AminoLink agarose beads or BSA control agarose beads were incubated with increasing amounts of recombinant Zta protein. (D) Bound protein was separated on SDS-PAGE gel and bound Zta was then analysed by Western blotting staining. A representative image is shown. (E) The non-linear fit of Zta binding to Tat (densitometry analysis of western blotting band intensity of bound Zta) against Zta concentration in solution with one site-specific binding model. Zta binding at a concentration of 0.2 µM was set as 1 (fraction bound). (F , G) Tat and Zta interaction analysed by YFP reconstitution. (F) The modular composition of the fusion proteins used in this study (above). A representative image of HeLa cells after transfection with YFP1-Tat and YFP2-Zta plasmids, nuclei were counterstained with DAPI, scale bar 10 μm (below). (G) The percentage of YFP-positive cells in HeLa, RPMI8866 or Jurkat cells transfected with YFP1-Tat + YFP2-Zta or YFP1-ERGIC-53 and YFP2-MCFD2 (positive control) as analysed by flow cytometry. (H-J) The analysis of Tat and Zta interaction by FRET with the acceptor photobleaching method. (H) Representative images of RPMI8866 cells, transfected with different combinations of CFP, YFP, Tat-CFP and YFP-Zta. Scale bar 10 μm. (I-J) FRET efficiencies for each combination in RPMI8866 (I) and HeLa (J) cells. Data are presented as mean ± SEM

Article Snippet: Human cervical carcinoma cell line HeLa (American Type Culture Collection), Human Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line RPMI8866 (ECACC General Cell Collection), freshly EBV-transformed B lymphoblastoid cell line from healthy donor AS (BLAS, established by EBV (B95-8) immortalization of mature B cells and characterized by Genethon (Evry, France)), human immortalized T cell line Jurkat (American Type Culture Collection) and their derivatives were used in the study.

Techniques: Immunoprecipitation, Expressing, Recombinant, Western Blot, Staining, Negative Control, Control, Transfection, Magnetic Beads, In Vitro, Binding Assay, Incubation, SDS Page, Concentration Assay, Positive Control, Flow Cytometry