Abstract
The 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.
Highlights
The 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
The structure of the ectodomain of gp41 (e-gp41) in its fusogenic state has been solved by both x-ray crystallography and NMR and consists of a central parallel trimeric coiled-coil of N-terminal helices surrounded by C-terminal helices of gp41 in an antiparallel hairpin fashion, forming a six-helix bundle (4 –7)
Design and Stepwise Modification of Topless gp140 Variants—To design gp140 variants in which e-gp41 is maintained in a prefusion state, C1 and C5 segments were retained in various gp140 constructs while the other gp120 components were eliminated
Summary
The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp subunits that form trimeric spikes on the virion surface. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure 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. This paper is available on line at http://www.jbc.org achieving this goal has been impeded in part, due to the extensive glycosylation, heterogeneous conformation, and lability of the gp120/gp complex To circumvent these issues, we have designed a truncated “topless” gp140 variant in which most parts of gp120 have been removed, while maintaining e-gp in a prefusion configuration
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