Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements

Abstract

30The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of 31 conformational transitions, with allosteric elements in each protomer orchestrating host 32 receptor-induced exposure of the co-receptor binding site and fusion elements. To 33 understand the molecular details of this allostery, we introduced Env mutations aimed to 34 prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis 35 performed on the soluble ectodomain BG505 SOSIP Env trimers, cell-surface expressed 36 BG505 full-length trimers and single-molecule Förster Resonance Energy Transfer 37 (smFRET) performed on the full-length virion-bound Env confirmed that these mutations 38 prevented CD4-induced transitions of the HIV-1 Env. Structural analysis by single-39 particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins 40 revealed rearrangements in the gp120 topological layer contacts with gp41. Specifically, 41 a conserved tryptophan at position 571 (W571) was displaced from its typical pocket at 42 the interface of gp120 topological layers 1 and 2 by lysine 567, disrupting key gp120-43 gp41 contacts and rendering the Env insensitive to CD4 binding. Vector based analysis 44 of closed Env SOSIP structures revealed the newly designed trimers exhibited a 45 quaternary structure distinct from that typical of SOSIPs and residing near a cluster of 46 Env trimers bound to vaccine-induced fusion peptide-directed antibodies (vFP Mabs). 47These results reveal the critical function of W571 as a conformational switch in Env 48 allostery and receptor-mediated viral entry and provide insights on Env conformation that 49 are relevant for vaccine design.50 51 52 53 54Host cell entry of HIV-1 is accomplished by the envelope glycoprotein (Env) spike. HIV-1 55Env is a trimer of heterodimers comprised of gp120 and gp41 protomers, and exists in a 56 metastable conformation capable of transitioning from a prefusion closed configuration to a 57 fusion-competent open state upon triggering by CD4. 1,2 The C-terminal gp41 domain contains a 58 single transmembrane helix and the membrane fusion elements of the trimer. 3-5 The gp12059 segment binds the primary receptor CD4, triggering conformational changes leading to the 60 binding of co-receptor CCR5/CXCR4 that causes global rearrangements in the trimer structure 61 leading to viral and cell membrane fusion and gp120 shedding. 6-8 While many studies have 62 sought to understand the nature of the communication between the CD4 binding site, the 63 coreceptor binding site and the fusogenic elements of the HIV-1 Env, a complete understanding 64 of the allosteric mechanism and metastability in HIV-1 Env remains lacking. 65The design of a soluble, stabilized ectodomain Env (SOSIP), containing an engineered 66 gp120 to gp41 disulfide (SOS) and an HR1 helix breaking I to P mutation, has revealed 67 structural details regarding broadly neutralizing antibody (bnAb) epitopes and as an immunogen 68 has induced autologous neutralizing antibodies. 2,9 The...

Env Exceptionalism: Why Are HIV-1 Env Glycoproteins Atypical Immunogens?

“…A different but related question is whether reducing CD4 binding or CD4induced conformational changes qualitatively improves the immunogenicity of Env by preventing responses to previously cryptic and potentially distractive non-NAb epitopes. Multiple SOSIP trimer variants have been designed and tested for exploring this hypothesis, but although some reductions in non-NAb responses have been observed, they have not been accompanied by major increases in autologous or heterologous NAb titers (Chuang et al, 2017;de Taeye et al, 2015;Henderson et al, 2020;Joyce et al, 2017;Kulp et al, 2017;Ringe et al, 2017;Torrents de la Peñ a et al, 2017;Zhang et al, 2018). Additional research in this area, including attempts to better suppress the immunogenicity of non-NAb neo-epitopes at the base of soluble trimers (Kulp et al, 2017;Ringe et al, 2020), is ongoing.…”

Ability of nucleoside-modified mRNA to encode HIV-1 envelope trimer nanoparticles

“…Strategies have been proposed either to stabilize the Env trimer protein in the prefusion closed conformation or to prevent CD4-triggered structural rearrangements (de Taeye et al, 2015;Guenaga et al, 2015; Henderson et al, 2020; Kong et al, 2016; Zhang et al, 2018). We studied nine stabilization designs in CH848 10.17DT Env for expression as modified mRNAs ( Table S1 ).…”

“…The Env open conformation exposes non-neutralizing antibody (nnAb) epitopes that can create competition for Env antigen between nnAb and bnAb precursors (Havenar-Daughton et al, 2017; Lee et al, 2021; McGuire et al, 2014). To address the problem of Env trimers opening and the exposure of non-neutralizing epitopes, multiple strategies have been designed to stabilize Env trimers in native-like conformations (de Taeye et al, 2015; Guenaga et al, 2015; Henderson et al, 2020; Kong et al, 2016). We hypothesized that the inclusion of optimal stabilizing mutations will be critical for modified mRNAs to express antigenic and immunogenic Envs, since delivering immunogens directly as mRNA-LNP does not allow for immunogen purification.…”

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates

“…The gp41-chimeric SOSIP gp140s were further stabilized by the addition of E64K and A316W mutations 68 . CH505 SOSIP gp140 Envs were expressed as previously described 71 mRNA production. mRNAs were produced as previously described 72 LNP formulation of mRNA.…”