Extended Data Fig. 1: Tagged BG505 sgp140 SOSIP.664 proteins largely retain their known immunogenic features and preferentially sample state-2-like conformations.
From: Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET
a, Schematics for wild-type (WT) BG505 Env and BG505 sgp140 SOSIP.664 with D7324 affinity tag; V1–Q3 peptide in green, V4–A1 peptide in red. b, Validation of tagged BG505 sgp140 SOSIP.664. Top, antigenic profile of 100% untagged (WT), 100% double-tagged V1V4 (V1–Q3 V4–A1), and 20:1 of untagged to double-tagged BG505 sgp140 SOSIP.664. Binding by the indicated VRC01, 17b, PG9, 19b, PGT151 and 902090 antibodies was assessed from two independent ELISA assays in hexaplets and displayed as percentage of 2G12 binding (mean ± s.d.). The epitope for the antibody 902090 was more exposed in the 100%-tagged BG505 sgp140 SOSIP.664 than in the untagged BG505 sgp140 SOSIP.664, although this was not the case for the 1:20 tagged:wild-type trimers used for our smFRET analyses. The insertion of the Q3 tag into all three V1 regions of Env may exert local effects on the V2 β-barrel that contains the 902090 epitope (residues 171–177). Bottom, reference-free negative-staining electron microscopy two-dimensional class averages with representative trimeric density map of the BG505 sgp140 SOSIP.664 (wild type:V1V4-tagged at a 20:1 ratio) used for smFRET imaging. A Fourier shell correlation is also provided. c, Antigenic characteristics of BG505 sgp140 DS-SOSIP.664 (left) and 100% V1V4-tagged BG505 DS-SOSIP.Mut4 (right), determined by MSD. Antibodies are labelled. CD4bs, CD4 binding site; CD4i, CD4-induced; V1V2, V1V2-directed; V3, V3 glycan site-directed; gp120/gp41, interface between gp120 and gp41. Antigenic profiles of BG505 DS-SOSIP.664 (left) and 100% V1V4-tagged DS-SOSIP.Mut4 (right) after V3-negative selection were assessed by a panel of CD4-induced antibodies (17 and 48b, with and without sCD4), CD4 binding site antibodies (VRC01, VRC03, b12 and weakly neutralizing F105), V1V2-directed antibodies (PGT145 and VRC26.25), V3 glycan site-directed antibodies (2G12, PGT121, PGT128) and weakly neutralizing V3-directed antibodies (447-52D, 3074 and 2557, with and without soluble CD4), gp41–gp120 interface antibodies (PGT151, 35O22, 8ANC195 and VRC34.01) and the negative-control antibody CR9114 (an influenza virus antibody that does not recognize HIV-1 Env). ECL, electrochemiluminescence. d, The indicated BG505 sgp140 SOSIP.664 variants exhibit predominantly state-2-like conformations. FRET histograms for V1V4-tagged BG505 sgp140 SOSIP.664 with molecules after V3-negative selection (left), and for the stabilized BG505 sgp140 SOSIP.664 variant DS-SOSIP.Mut420 (right) (see Methods). Histograms represent mean ± s.e.m., determined from three independent populations of smFRET traces.
