Qiao, Zhi-SongKim, MikyungReinhold, BruceMontefiori, DavidWang, Jia-huaiReinherz, Ellis L.2019-10-172005Qiao, Zhi-Song, Mikyung Kim, Bruce Reinhold, David Montefiori, Jia-huai Wang, and Ellis L. Reinherz. 2005. “Design, Expression, and Immunogenicity of a Soluble HIV Trimeric Envelope Fragment Adopting a Prefusion gp41 Configuration.” Journal of Biological Chemistry 280 (24). American Society for Biochemistry & Molecular Biology (ASBMB): 23138–46. doi:10.1074/jbc.m414515200.0021-92581083-351Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41555754The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e- gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e- gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e- gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.en-USJournal Article2019-10-1710.1074/jbc.M414515200