Design of gp41-derived immunogens. (A) Three genes based on gp41 were constructed in this study containing the CHR and TM regions (“gp41CTM”). Resulting proteins are directed by a TPA signal peptide towards the cell membrane and can be detected by a C-terminal HA tag. Two of these are stabilized in a trimeric conformation by an N-terminal heterologous zipper motif (GCN4- or H3-derived). (B) Schematic representation of truncated gp41 proteins on the surface of cellular membranes. The amino acid sequence is derived from isolate 96ZM651 (clade C). (C) The optimal fusion site of the GCN4 zipper domain and the gp41 CHR part was modeled by comparative modeling with the existing structure GCN4-NHR, and free energy was calculated with aid of DOPE score. (D) Correct cleavage of heterologous signal peptide, protein topology and membrane incorporation were predicted for the gp41CTM (based on 96ZM651) protein (data shown) and zipper-stabilized derivatives (data not shown).