Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state

Abstract

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

HIV-1 Envelope and MPER antibody structures in lipid assemblies

“…Thus, the compact three-helix bundle conformation likely represents the low-energy post-fusion conformation of the TMD and its formation is preferred in the minimal constructs used in the NMR and MD studies. More recently, a similar study of isolated MPER-TMD peptide was done in nanodiscs supporting our conclusions that in more native lipid environment stable, trimeric topology is unfavorable (Wang et al, 2019) Our data demonstrate how the ectodomain and MPER restrain the TMD in a crossed topology, particularly apparent in the MPER Fab -bound state. This topology of the TMD is consistent with the meta-stable prefusion state primed for the energetically downhill conformational changes associated with post-fusion conformation.…”

“…75 ° angle ( Figure 3B). The helices crossed in the micelle at the conserved R696, a residue previously shown to be important for modulating conformational changes of the TMD (Cooper et al, 2018;Hollingsworth et al, 2018;Wang et al, 2019).…”

Priming with DNA Expressing Trimeric HIV V1V2 Alters the Immune Hierarchy Favoring the Development of V2-Specific Antibodies in Rhesus Macaques

“…These constrained Env immunogens were achieved by several methods, including the use of (i) stabilized Env trimers (SOSIP) ( 57 – 60 ), (ii) gp120 with engineered outer domains ( 61 ), (iii) cleavage-independent native flexibly linked (NFL) Env trimers ( 62 ), and (iv) gp140 in a closed conformation displayed on ferritin nanoparticles ( 63 ). In addition, many alternative methods to focus the Ab response on protective epitopes have been used, including the use of distinct regions in Env that can be “isolated” from the remaining portions of Env while maintaining a native conformation; these include RC1, which facilitates the recognition of the glycan patch associated with the third variable region (V3) of gp120 ( 64 ); MPER peptides adjuvanted in liposomes, used in conjunction with a gp140 oligomer prime to induce Ab to the gp41 fusion intermediate ( 65 ); membrane-embedded formulations to induce MPER-targeting Abs ( 66 , 67 ); and use of a cross-linked V1 and V2 ( 68 ), and trimerized gp120 ( 69 ), glycan-stabilized V1V2 scaffold ( 26 , 27 ). Other approaches have been developed to specifically focus Ab responses on V2, including the design and use of (i) V1V2 scaffold protein immunogens together with gp120 DNA ( 25 , 28 , 29 ), (ii) a V2 loop peptide together with liposomal lipid A as an adjuvant ( 70 ), (iii) gp140 SOSIP or foldon trimers carrying signature-guided mutations of V2 broadly neutralizing monoclonal Abs ( 71 ), and (iv) a mutated V1V2 sequence which favors the β-strand conformation of the V2 C strand spliced onto the C terminus of murine leukemia virus gp70 ( 72 ).…”

Viral Membrane Fusion and the Transmembrane Domain

“…Inclusion of the TMD with an MPER peptide has been shown to increase the binding affinity of bNAbs to these peptides, although there is conflicting data on whether a trimeric TMD further increases this affinity. One set of binding assays completed in nanodiscs suggests that the addition of a trimeric-TMD recapitulates the bNAb binding of native-like Env protein [97], while another suggests inclusion of a single MPER-TMD peptide in each nanodisc increased the percentage of antibody bound to that peptide [99]. It has been shown by both NMR modeling [77] and crystallography with molecular dynamics simulations [100] that bNAbs targeting the MPER region of gp41 bind residues within the TMD as part of their epitope, explaining why the presence of the TMD increases binding affinity.…”

“…This may suggest that successful incorporation into particles relies in part on the TMD or CTD of RV G protein, indicating that signals in the TMD or CTD of RV may play a role in overall particle assembly. These regions may also have a role in the host immune response to viral infection, similar to the production of bNAbs specific to the MPER-TMD produced during HIV infection [77,99].…”