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Abstract
The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host–receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface–expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.
Highlights
The HIV-1 envelope glycoprotein (Env), or spike, is critical for virus entry into host CD4+ T cells and is the target of HIV-1–specific broadly neutralizing antibodies, a number of which have been identified with exceptional neutralization breadth and potency against diverse virus isolates [1–6]
For single-molecule Förster resonance energy transfer (smFRET) State 2, we selected a structure of HIV-1 BG505 SOSIP.664 prefusion Env trimer in complex with the small-molecule HIV entry inhibitor BMS378806 and human antibodies 3H109L, containing heavy and light chain intermediates of the glycan-V3-directed PGT121 broadly neutralizing antibodies (bnAbs) lineage, and the gp120-gp41 interface-directed bnAb 35O22 (PDB 6MTJ) (Fig. 1A) [31]
To determine antigenic differences between smFRETdefined States 1, 2, and 3, we made use of three ligands that preferentially enrich HIV-1 trimers in distinct States: the small-molecule HIV-1 entry inhibitor BMS-626529/temsavir that enriches trimers in State 1, PGT151 fragment antigen binding (Fab) that enriches State 2, and a cocktail of soluble CD4 (sCD4) and 17b Fab that enriches State 3 in the same manner as dodecameric CD4 (Fig. 1B) [6, 15]. 293T cells were transfected with plasmids encoding JR-FL.E168K.dCT Env glycoprotein and furin such that the cells expressed fully cleaved JRFL trimers on the cell surface (Fig. 1C)
Summary
The HIV-1 envelope glycoprotein (Env), or spike, is critical for virus entry into host CD4+ T cells and is the target of HIV-1–specific broadly neutralizing antibodies (bnAbs), a number of which have been identified with exceptional neutralization breadth and potency against diverse virus isolates [1–6]. The RMSDs of epitopes for different HIV-1 bnAbs between States 2 and 3 were determined from the State 2 and 3 structures and plotted against the ratio of ABRs of State 2 and State 3 binding for each antibody, and a linear relationship between the data was determined (Fig. 1E).
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