HIV-1 entry requires the envelope glycoprotein (Env) trimer to bind the receptor CD4 and a chemokine coreceptor (CCR5 or CXCR4). The CD4-bound open trimer, in which the coreceptor binding site is exposed but the gp41 fusion peptide remains sequestered in an interprotomer pocket, has been structurally characterized; however, the structural and mechanistic basis by which coreceptor engagement catalyzes the subsequent gp41 rearrangements — release of the fusion peptide, its insertion into the target-cell membrane, and progression through the extended prehairpin intermediate to the final six-helix bundle — remains unresolved. The stoichiometry and geometry of coreceptor engagement on the trimer (one, two, or three coreceptor molecules, or coreceptor dimers bridging adjacent protomers) are likewise open, as are two related questions raised in the same work but not posed there as part of the triggering question: the breathing dynamics that would transiently expose the b12 epitope on native membrane-embedded trimers, and the fidelity of soluble SOSIP trimers to the native transition sequence. The coreceptor-triggering problem, stated explicitly by Ozorowski et al. (Nature, 2017), remains open despite the monomeric gp120–CD4–CCR5 ternary structure (Shaik et al., 2019) and CXCR4-tetramer/gp120 structures (Zhang et al., 2025).

Background

The HIV-1 envelope glycoprotein (Env) is a trimer of gp120–gp41 heterodimers that mediates viral entry by fusing the viral and target-cell membranes. gp120 is the receptor-binding subunit; gp41 is the membrane-fusion subunit containing a hydrophobic fusion peptide (FP) at its N-terminus, two heptad-repeat regions (HR1 and HR2, coiled-coil segments that refold against each other during fusion), a transmembrane region, and a cytoplasmic tail. Entry proceeds through a conformational cascade: (1) the closed, prefusion trimer; (2) the CD4-bound open state, in which CD4 binding rotates the gp120 subunits outward and displaces the V1/V2 loops, exposing the coreceptor binding site formed by the V3 loop and the bridging sheet of gp120; (3) coreceptor (CCR5 or CXCR4, seven-transmembrane G-protein-coupled chemokine receptors on T cells and macrophages) engagement at that site; and (4) gp41 refolding through the extended prehairpin intermediate, in which HR1 forms a trimeric coiled coil that bridges the two membranes with the FP inserted in the target membrane, to the postfusion six-helix bundle (three HR1 helices surrounded by three antiparallel HR2 helices) that pulls the membranes together. SOSIP trimers are soluble, engineered, stabilized gp140 Env ectodomain constructs (named for their disulfide and Ile-to-Pro stabilizing mutations) that mimic the closed prefusion native trimer and are the standard soluble substrate for structural and vaccine studies. b12 is a broadly neutralizing antibody targeting the CD4 binding site; 17b is an antibody whose epitope overlaps the coreceptor binding site. Cryo-EM (single-particle electron cryomicroscopy) and cryo-ET (electron cryotomography of objects in situ, such as Env on virion membranes) are the principal structural methods; single-molecule Förster resonance energy transfer (smFRET) and single-particle tracking total internal reflection fluorescence (SPT-TIRF) microscopy measure conformational dynamics.

Ozorowski et al. (2017) determined high-resolution cryo-EM structures of subtype B B41 SOSIP trimers in complex with CD4 and antibody 17b, and with antibody b12. They showed that both ligands induce large gp120 rearrangements and gp41 helix movements, and that in the CD4-bound open trimer the fusion peptide relocates into a newly formed interprotomer pocket at the gp120–gp41 interface, where it is buried, structured, and distant from the host membrane. They therefore interpreted the CD4/17b-bound state as a stable fusion intermediate that requires further triggering by coreceptor engagement, and noted that the disposition of the three coreceptor binding sites would allow up to three CCR5 molecules to bind the open trimer, while the crystallographic CCR5 dimer, when docked onto the trimer, could span two gp120 subunits and engage two coreceptor binding sites simultaneously. They further proposed that the trimer 'breathes' between closed and open states, which is required for the b12 epitope to become accessible, since b12 is sterically occluded from the fully closed trimer. Subsequent work substantially clarified the neighboring steps but not the coreceptor-dependent ones. Shaik et al. (2019) solved the first ternary complex of full-length monomeric gp120 with soluble CD4 and unmodified CCR5 (3.9 Å), revealing how the V3 loop inserts into the CCR5 chemokine-binding pocket and how the tyrosine-sulfated CCR5 N-terminus engages the gp120 bridging sheet — the recognition mode, but on a monomer without gp41 or a trimer context. Yang et al. (2019) captured asymmetric CD4-bound trimers with E51, a tyrosine-sulfated coreceptor-mimicking antibody, exposing two protomer conformations that differ in fusion peptide rearrangement. Dam et al. (2023) structurally defined the substoichiometric CD4-bound opening cascade (one-CD4 trimers largely closed; two-CD4 trimers open with the third protomer in an occluded-open state). Ladinsky et al. (2020) visualized the downstream extended prehairpin intermediate on virions by cryo-ET using fusion inhibitors as traps. Thakur et al. (2025) traced the fusion peptide trajectory during CD4-induced opening, and Cui et al. (2026) assembled a classification framework of Env states from closed to fully open. Zhang et al. (2025) determined CXCR4 tetramer structures bound to one or two HIV-2 gp120 molecules, and Quijada-Freire et al. (2026) showed that Env binding reorganizes CXCR4 clustering on the T-cell membrane. Quantitative entry-stoichiometry studies (Bonner et al. 2024; Katte et al. 2025) constrain how many CD4 molecules and Env trimers participate in a fusion event. No structure of the intact Env trimer (including gp41) in complex with CD4 and a bona fide coreceptor exists, and no accepted mechanistic account connects coreceptor engagement to fusion peptide release and gp41 refolding.

Problem Statement

Determine the structural and mechanistic basis by which binding of the coreceptor (CCR5 or CXCR4) to the exposed coreceptor binding site on the CD4-bound HIV-1 envelope glycoprotein trimer triggers the conformational transitions beyond the CD4-bound intermediate: how coreceptor engagement catalyzes the gp41 rearrangements that release the fusion peptide from its interprotomer pocket at the gp120–gp41 interface, drive its insertion into the target-cell membrane, and progress through the extended prehairpin intermediate to the final six-helix bundle. Integral to this mechanistic question is (i) the stoichiometry and geometry of coreceptor engagement on the trimer — whether one, two, or three coreceptor molecules (or coreceptor dimers or higher oligomers) engage the CD4-bound trimer, and whether such oligomeric arrangements span adjacent protomers and influence triggering. Two further questions raised in the source work bear on it, but are related open questions and a methodological caveat rather than required components of the triggering mechanism itself: (ii) the dynamics and kinetics of the transient trimer 'breathing' events that render the b12 epitope accessible (b12 is a broadly neutralizing antibody to the CD4 binding site; its epitope is sterically occluded in the closed trimer, so its accessibility reports on transient opening), i.e. how frequently native, membrane-embedded trimers on virions sample open states; and (iii) whether soluble SOSIP-stabilized trimers reproduce the native sequence of conformational transitions, including the coreceptor-driven steps, in the same membrane- and receptor-bound context.

References

  1. Ozorowski G, Pallesen J, de Val N, Lyumkis D, Cottrell CA, Torres JL, Copps J, Stanfield RL, Cupo A, Pugach P, Moore JP, Wilson IA, Ward AB. "Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike." Nature 547:360-363 (2017). DOI: 10.1038/nature23010. https://doi.org/10.1038/nature23010
  2. Shaik MM, Peng H, Lu J, Rits-Volloch S, Xu C, Liao M, Chen B. "Structural basis of coreceptor recognition by HIV-1 envelope spike." Nature 565:318-323 (2019). DOI: 10.1038/s41586-018-0804-9. https://doi.org/10.1038/s41586-018-0804-9
  3. Yang Z, Wang H, Liu AZ, Gristick HB, Bjorkman PJ. "Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody." Nature Structural & Molecular Biology 26:1167-1175 (2019). DOI: 10.1038/s41594-019-0344-5. https://doi.org/10.1038/s41594-019-0344-5
  4. Ladinsky MS, Gnanapragasam PN, Yang Z, West AP, Kay MS, Bjorkman PJ. "Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate." eLife 9:e58411 (2020). DOI: 10.7554/eLife.58411. https://doi.org/10.7554/eLife.58411
  5. Carlon-Andres I, Malinauskas T, Padilla-Parra S. "Structure dynamics of HIV-1 Env trimers on native virions engaged with living T cells." Communications Biology 4:1228 (2021). DOI: 10.1038/s42003-021-02658-1. https://doi.org/10.1038/s42003-021-02658-1
  6. Dam KA, Fan C, Yang Z, Bjorkman PJ. "Intermediate conformations of CD4-bound HIV-1 Env heterotrimers." Nature 623:1017-1025 (2023). DOI: 10.1038/s41586-023-06639-8. https://doi.org/10.1038/s41586-023-06639-8
  7. Bennett AL, Edwards R, Kosheleva I, Saunders C, Bililign Y, Williams A, Bubphamala P, Manosouri K, Anasti K, Saunders KO, Alam SM, Haynes BF, Acharya P, Henderson R. "Microsecond dynamics control the HIV-1 Envelope conformation." Science Advances 10:eadj0396 (2024). DOI: 10.1126/sciadv.adj0396. https://doi.org/10.1126/sciadv.adj0396
  8. Bonner X, Sondgeroth A, McCue A, Nicely N, Tripathy A, Spielvogel E, Moeser M, Ke R, Leiderman K, Joseph SB, Swanstrom R. "Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1." mBio 15:e00321-24 (2024). DOI: 10.1128/mbio.00321-24. https://doi.org/10.1128/mbio.00321-24
  9. Gasser R, Colin P, Jacquemard C, Montagné T, Zhou Z, Pons V, Kostenis E, Galés C, Kellenberger E, Lagane B. "Targeting of specific CCR5-G protein complexes underlies biased signaling by HIV-1 envelope glycoproteins." Cell Communication and Signaling 23:505 (2025). DOI: 10.1186/s12964-025-02508-1. https://doi.org/10.1186/s12964-025-02508-1
  10. KatteRH, Xu W, Han Y, Hong X, Lu M. "Inter-protomer opening cooperativity of envelope trimers positively correlates with HIV-1 entry stoichiometry." mBio 16:e02754-24 (2025). DOI: 10.1128/mbio.02754-24. https://doi.org/10.1128/mbio.02754-24
  11. Thakur B, Katte RH, Xu W, Janowska K, Sammour S, Henderson R, Lu M, Kwong PD, Acharya P. "Conformational trajectory of the HIV-1 fusion peptide during CD4-induced envelope opening." Nature Communications 16:4595 (2025). DOI: 10.1038/s41467-025-59721-2. https://doi.org/10.1038/s41467-025-59721-2
  12. Zhang Z, Zhang H, Zheng L, Chen S, Du S, Xiao J, Patel DJ. "CXCR4 mediated recognition of HIV envelope spike and inhibition by CXCL12." Nature Communications 16:8653 (2025). DOI: 10.1038/s41467-025-63815-2. https://doi.org/10.1038/s41467-025-63815-2
  13. Cui J, Lin ZJ, Ghosh S, Du J, Sadeesh R, Weiner DB, Pallesen J. "Conformational landscape of HIV-1 Env from closed to fully open." Nature Communications 17:4273 (2026). DOI: 10.1038/s41467-026-69921-z. https://doi.org/10.1038/s41467-026-69921-z
  14. Quijada-Freire A, Santiago CA, García-Cuesta EM, Soler Palacios B, Ayala-Bueno R, Gardeta SR, San Sebastian E, Armendariz-Burgoa E, Puertas MC, Villares R, Garaigorta U, González-Granado LI, Rodriguez Frade JM, Chojnacki J, Martinez-Picado J, Mellado M. "HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane." eLife 15:RP110354 (2026). DOI: 10.7554/eLife.110354. https://doi.org/10.7554/eLife.110354