Figure 1.

eEF1A interacts with HIV-1 Nef protein in vitro and in vivo. (a) Binding of HIV-1 Nef to eEF1A was measured in GST pull-down assays using U937 cells as a source of lysates. β-actin detection represents input loading controls of the lysates that were used in binding reactions. Results are representative of three independent experiments. The right panel represents coomassie staining of expressed proteins that were used in the binding reaction of GST pull-down. (b) Cytoplasmic and nuclear extracts from several cell lines (Vero, MRC5, and U937 cells), PBLs, and MDMs treated with rNef (100 ng/ml) for 0.5 h were immunoprecipitated with an anti-eEF1A antibody, or Nef (100 ng/ml)-treated total cellular lysates were immunoprecipitated with an isotype control antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody. Recombinant Nef-treated total cell lysates were used as a positive control. Results are representative of three independent experiments. (c) Cellular extracts of U937 cells transfected for 48 h with a nef-expressing plasmid (pNef) were immunoprecipitated with an anti-eEF1A antibody, anti-Nef antibody, or isotype control antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody. Results are representative of two independent experiments. (d) Cellular extracts of PBMCs infected in vitro with HIV-1_89.6 or mock infected were immunoprecipitated with an anti-eEF1A antibody or anti-Nef monoclonal antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody or anti-eEF1A antibody. Results are representative of two independent experiments. (e and f) eEF1A and HIV-1 Nef interact in vitro in a mammalian two-hybrid assay. (e) Schematic representation of expression constructs used in co-transfection experiments in the mammalian two-hybrid model. (f) Mammalian two-hybrid analysis with eEF1A fused to the VP16 activator domain and HIV-1 Nef fused to the GAL4 domain. Luciferase assays were conducted on total extracts from U937 cells transfected with the luciferase expression construct pG5-Luc, pBIND-Nef, pACT-eEF1A, or control plasmids. As a positive control, two plasmids, pACT-MyoD and pBIND-Id, were co-transfected, and co-transfection of empty vectors was used as a negative control. Results represent the mean of three independent experiments. ***P<0.001