The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120-gp41 interactions, whereas a "flat open" concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N-and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.T he human immunodeficiency virus envelope proteins gp120 and gp41 associate in a trimeric arrangement (1-3) and mediate membrane fusion through conformational rearrangement and fusion peptide insertion into the host membrane. The gp120 tertiary conformational change observed via X-ray crystallography has not been definitively characterized in the context of a quaternary structure. Several observations have been gleaned from tomography studies, though with inconclusive and sometimes contradictory results. Quaternary structural features reported previously include trimer morphology, with some groups reporting a mushroom-like shape and others a more rod-like arrangement, as well as the relative location of the primary epitope, hypervariable loops, and N-and C-termini, which have some variation among the different tomograms. Another point of conflict is the presence of a cavity at the threefold axis, capped by a density. One final feature is the arrangement of gp41, with some groups reporting a thin, stalk-like rod arrangement of gp41 and another group reporting a more splayed out, tripod-like conformation of gp41 proximal to the viral membrane. Taken together, these contrasting features portray a heterogeneous set of registers that seem to hinder a cohesive and thorough model of ligand-induced quaternary arrangement...
Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF165) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF165. Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF165 against thermal and enzyme degradation in vitro, and isolated VEGF165 from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n = 8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.Electronic supplementary materialThe online version of this article (10.1007/s10456-018-9622-9) contains supplementary material, which is available to authorized users.
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