The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1,2,3,4,5,6,7,8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8,9,10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11,12,13,14,15,16,17,18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies—and thus of interest for the design of immunogens—remains unknown.
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The data that support the findings of this study are available from the corresponding authors upon reasonable request.
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We thank A. B. Ward, R. W. Sanders and P. J. Bjorkman for discussions, R. Blakemore for assistance with molecular modelling, D. Burton and M. Feinberg for reagents including PGT121, PGT122, PGT145, PGT151 and NC-Cow1, NC-Cow8, NC-Cow9 and NC-Cow10 antibodies, and the AIDS Research and the Reference Reagent Program (Division of AIDS, NIAID, NIH) for the antibodies 3BNC117, 10-1074, PG9 and PG16. This work was supported by NIH grants RO1 GM116654 and UM1 AI100645 to W.M., RO1 GM098859 to S.C.B., RO1 AI124982 and RO1 AI100645 to J.G.S., K22 AI116262 to J.B.M., CRC Tier 2 RCHS0235 and a CIHR foundation grant 352417 to A.F., PO1 GM056550 to W.M., J.G.S., S.C.B, A.B.S. and C.A., by a Brown Coxe Fellowship to M.L., a fellowship from the China Scholarship Council-Yale World Scholars to X.M., by the International AIDS Vaccine Initiative’s (IAVI’s) Neutralizing Antibody Consortium to P.D.K. and by the Intramural Research Program of the Vaccine Research Center (NIAID, NIH) to P.D.K. and A.B.M., and by the SFB1129 and the Emmy-Noether programme (project number 317530061) of the German Research Foundation to E.A.L. and I.N.-S., respectively.
Nature thanks David Millar, Alexandra Trkola and the other anonymous reviewer(s) for their contribution to the peer review of this work.