The HIV-1 transmembrane envelope protein gp41 plays an important role in mucosal transmission and is a target during cell infection. The highly conserved membrane proximal external region is recognized by the broadly neutralizing 2F5 and 4E10 antibodies and is therefore an important component in subunit vaccine development against the virus.  The structure of the membrane proximal external region in its native conformation is unknown, presenting challenges for both vaccine development and a full understanding of its role in the viral infection process.  By expressing the membrane proximal external region within its native context (i.e. attached to the transmembrane domain and cytoplasmic tail of gp41, which we call dgp41), we aimed to determine the structure of this region as it is presented on the HIV-1 virus, as well as test the immunogenicity of the protein within its native context.  This was explored via two different constructs and expression systems.  The dgp41 protein was co-expressed with the HIV-1 Gag protein in Nicotiana benthamiana using a novel combination of transgenic and transient plant-expression systems, which, surprisingly, formed enveloped Gag virus-like particles that incorporated dgp41 within the lipid membrane.  Both Gag and dgp41 co-localized during density gradient ultracentrifugation, suggesting an interaction of the two proteins when co-expressed in N. benthamiana.  These interactions were further characterized by transmission electron microscopy and trypsin digestion in the presence or absence of detergent, the results of which further suggested that the two proteins are forming 100 nm enveloped virus-like particles that incorporate both Gag and dgp41. These particles were purified and found to be both systemically and mucosally immunogenic in mice.  In addition, dgp41 was expressed as a fusion protein via a Mistic membrane-protein expression vector in E. coli.  We have initiated structural studies of both the virus-like particles as well the E. coli-expressed fusion protein, for which we have diffracting crystals, to determine the native structure of the membrane proximal external region.