Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles

ARTICLE https://doi.org/10.1038/s42003-023-04916-w OPEN Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles 1234567890():,; Kunyu Wang1,2,11, Shijian Zhang3,4,11, Eden P. Go5, Haitao Ding6, Wei Li Wang1, Hanh T. Nguyen3,4, John C. Kappes6,7, Heather Desaire5, Joseph Sodroski 3,4,8,12 ✉ & Youdong Mao 1,2,9,10,12 ✉ During virus entry, the pretriggered human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer initially transits into a default intermediate state (DIS) that remains structurally uncharacterized. Here, we present cryo-EM structures at near-atomic resolution of two cleaved full-length HIV-1 Env trimers purified from cell membranes in styrene-maleic acid lipid nanoparticles without antibodies or receptors. The cleaved Env trimers exhibited tighter subunit packing than uncleaved trimers. Cleaved and uncleaved Env trimers assumed remarkably consistent yet distinct asymmetric conformations, with one smaller and two larger opening angles. Breaking conformational symmetry is allosterically coupled with dynamic helical transformations of the gp41 N-terminal heptad repeat (HR1N) regions in two protomers and with trimer tilting in the membrane. The broken symmetry of the DIS potentially assists Env binding to two CD4 receptors—while resisting antibody binding—and promotes extension of the gp41 HR1 helical coiled-coil, which relocates the fusion peptide closer to the target cell membrane. 1 State Key Laboratory for Mesoscopic Physics, School of Physics, Peking University, Beijing, China. 2 Peking-Tsinghua Joint Center for Life Science, Peking University, Beijing, China. 3 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA. 4 Department of Microbiology, Harvard Medical School, Boston, MA, USA. 5 Department of Chemistry, University of Kansas, Lawrence, KS, USA. 6 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. 7 Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL, USA. 8 Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA. 9 Center for Quantitative Biology, Academy of Advanced Interdisciplinary Studies, Peking University, Beijing, China. 10 National Biomedical Imaging Center, Peking University, Beijing, China. 11These authors contributed equally: Kunyu Wang, Shijian Zhang. 12These authors jointly supervised this work: Joseph Sodroski, Youdong Mao. ✉email: ; COMMUNICATIONS BIOLOGY | (2023)6:535 | https://doi.org/10.1038/s42003-023-04916-w | www.nature.com/commsbio 1 ARTICLE T COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04916-w he entry of human immunodeficiency virus type-1 (HIV1), the etiologic agent of AIDS, into host cells is mediated by the envelope glycoprotein (Env) trimer1,2. In infected cells, Env is synthesized as a gp160 precursor in the endoplasmic reticulum, where trimerization, disulfide bonding and the addition of high-mannose glycans occur3,4. During transport through the Golgi apparatus, a subset of the Env glycans are modified to complex carbohydrates and the Env precursor is cleaved by host furin-like proteases3,4. The resulting mature Env, which consists of a gp120 exterior subunit and a gp41 transmembrane subunit in each of the three protomers, is incorporated into budding virions. During virus entry, gp120 engages the target cell receptors, CD4 and CCR5 (or CXCR4), and gp41 fuses the viral and cell membranes1,2. Both spontaneous and CD4-induced transitions of virion Env conformations have been characterized by singlemolecule fluorescence resonance energy transfer (smFRET)5. Binding to the host receptors drives the metastable (State-1) pretriggered Env trimer into lower-energy conformations along the entry pathway. CD4 binding initially induces the default intermediate state (DIS), an asymmetric trimer in which the CD4-bound protomer is in State-3 and the unbound protomers are in State-25–8. To an extent dependent on HIV-1 strain, Envs can spontaneously sample conformations resembling the DIS in the absence of CD45. Destabilization of the pretriggered (State-1) Env trimer by particular Env amino acid changes or disruption of membrane anchorage can also lead to the DIS-like conformations6,9–13. Binding of multiple CD4 molecules generates the State-3 prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) regions form an extended coiled coil that relocates the hydrophobic gp41 fusion peptides in the target cell membrane2,14,15. CCR5/CXCR4 binding triggers further conformational transitions in the gp41 HR1 and HR2 regions leading to the formation of a six-helix bundle that mediates the fusion of the viral and cellular membranes2,16–18. HIV-1 establishes persistent infections in humans and has evolved mechanisms to avoid host immune responses. As the only virus-specific protein on the surface of the virion, Env serves as a target for neutralizing antibodies elicited in infected individuals19. Conformational flexibility, heavy glycosylation, high mutability and strain variation of HIV-1 Env contribute to antibody evasion20. Most antibodies generated during natural HIV-1 infection are poorly neutralizing, as they fail to bind conserved regions accessible on the cleaved pretriggered (State-1) Env trimer19–23. Broadly neutralizing antibodies (bNAbs) generally recognize this pretriggered (State-1) Env conformation but are only sporadically generated during natural infection19–23. Soluble stabilized (SOSIP) Env trimers retain many bNAb epitopes, allowing structural characterization of bNAb–Env trimer interactions20–22,24. However, to date, SOSIP trimers have not efficiently elicited bNAbs in animals or humans25–28. Potentially contributing to this difficulty are differences in antigenicity and glycosylation between SOSIP trimers and membrane Envs24,29–32. An smFRET study found that the conformations of individual protomers of the SOSIP trimers resemble State-2 more than the pretriggered (State-1) Env conformation32. These results indicate that a detailed structure of the pretriggered (State-1) Env conformation is currently lacking. As State-1 Env represents the major target for bNAbs5,23, this gap in knowledge could slow the development of successful vaccines. Moreover, the State-1-to-State-2 transition is modulated by both major classes of gp120-directed small-molecule entry inhibitors, BMS-806 and CD4-mimetic compounds (CD4mcs)5,12,14. More detailed information on the relationship between the pretriggered (State-1) Env conformation and the DIS would fill gaps in understanding the process of HIV-1 entry and assist the rational design of entry inhibitors and vaccines. 2 Here we used cryo-electron microscopy (cryo-EM) to solve the structures of two cleaved full-length HIV-1 Env trimers. We employed several measures designed to preserve the conformations of the native membrane Env during solubilization and (...truncated)