Scheme of the NL4-3 and NL4-3/ADA constructs and protein analysis by SDS-PAGE. (A) Scheme of gp140 expression constructs indicating cloning of the HIV-1 NL4-3 Env ectodomain with tPA leader sequence and deleted cleavage site. The hybrid construct NL4-3/ADA was generated by exchanging the V3 domain in conjunction with adjacent constant regions of the X4 tropic NL4-3 construct with the respective sequences of the R5 tropic HIV-1 ADA strain. The exchange of the extended V3 region contains two short linear elements of the discontinuous CD4 binding site (see also Additional file 1 and 2). (B, C) SDS-PAGE analysis of purified NL4-3 and NL4-3/ADA gp140 trimers under non-reducing (B) and reducing conditions (+50 mM DTT) (C).