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. 2023 Oct 23;14(1):6710.
doi: 10.1038/s41467-023-42500-2.

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir

Jérémie Prévost #  1   2 , Yaozong Chen #  3 , Fei Zhou  4 , William D Tolbert  3 , Romain Gasser  1   2 , Halima Medjahed  1 , Manon Nayrac  1   2 , Dung N Nguyen  3 , Suneetha Gottumukkala  3 , Ann J Hessell  5 , Venigalla B Rao  6 , Edwin Pozharski  7   8 , Rick K Huang  9 , Doreen Matthies  4 , Andrés Finzi  10   11 , Marzena Pazgier  12
Affiliations

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir

Jérémie Prévost et al. Nat Commun. .

Abstract

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

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Conflict of interest statement

J.P., A.F., and M.P., are inventors on U.S. patent application No. 17/762,333 related to compositions and methods based on HIV gp120 LMHS mutants, in which there are no restrictions on the publication of this manuscript data. The remaining authors declare no competing interests. The views expressed in this manuscript are those of the authors and do not reflect the official policy or position of the Uniformed Services University, US Army, the Department of Defense, or the US Government.

Figures

Fig. 1
Fig. 1. Intrinsic resistance of HIV-1 CRF01_AE strains to neutralization by attachment inhibitor temsavir.
a, b The ability of temsavir to neutralize viral particles from a panel of 208 different strains was previously reported by Pancera et al.. These data were reanalyzed with a focus on (a) the clade or (b) the identity of the polymorphic residue 375 in the Phe43 cavity of the gp120 subunit of Env (n = 208 biologically independent viral strains). Horizontal lines indicate median values. c Recombinant HIV-1 pseudoviruses expressing luciferase and bearing wild type (WT) or mutant EnvJR-FL were used to infect Cf2Th-CD4/CCR5 cells in the presence of increasing concentrations of temsavir. Infectivity at each dilution of the compound tested is shown as the percentage of infection without the compound for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results obtained in n = 3 independent experiments. The error bars represent the standard deviations. Neutralization half maximal inhibitory concentration (IC50) were calculated by non-linear regression using the Graphpad Prism software. d Capacity of temsavir to compete with CD4 binding as evaluated by cell-surface staining of HEK293T cells transfected with a HIV-1JR-FL Env expressor WT or its mutated counterpart. Binding of soluble CD4 (sCD4) in the presence of temsavir (10 µM) or an equal amount of DMSO was detected with the anti-CD4 OKT4 monoclonal antibody (mAb). Shown are the mean fluorescence intensities (MFI) obtained in the presence of temsavir normalized to the MFI in the absence of temsavir (DMSO) from the transfected (GFP+) population for staining obtained in n = 3 independent experiments. MFI values were normalized to the values obtained with anti-Env 2G12 mAb for each Env mutant. Error bars indicate mean ± SEM. Statistical significance was tested using a two-tailed unpaired t-test. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Impact of gp120 inner domain Layer residues on temsavir neutralization sensitivity.
a The ability of temsavir to neutralize viral particles from a panel of 208 Env strains was previously evaluated by Pancera et al.. These data were reanalyzed with a focus on the identity of polymorphic residues (61, 105, 108, 474, 475, 476) in the inner domain of the gp120 subunit in Env (n = 208 biologically independent viral strains). Residues that co-evolved with Ser375 or His375 are depicted in black and blue, respectively. Horizontal lines indicate median values. Recombinant HIV-1 pseudoviruses expressing luciferase and bearing WT or mutated Env from CRF01_AE strains (b) 92TH023, (d) CM244 or (f) clade B YU2 were used to infect Cf2Th-CD4/CCR5 cells in the presence of increasing concentrations of temsavir. Infectivity at each dilution of the compound tested is shown as the percentage of infection without the compound for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results obtained in n = 3 independent experiments. The error bars represent the standard deviations. Neutralization half maximal inhibitory concentration (IC50) were calculated by non-linear regression using the Graphpad Prism software. Capacity of temsavir to compete with CD4 binding as evaluated by cell-surface staining of HEK293T cells transfected with Env expressors from WT or mutated CRF01_AE strains (c) 92TH023, (e) CM244 or (g) clade B YU2. Binding of sCD4 in the presence of temsavir (10 µM) or equal amount of DMSO was detected with the anti-CD4 mAb OKT4. Shown are the mean fluorescence intensities (MFI) obtained in the presence of temsavir normalized to the MFI in the absence of temsavir (DMSO) from the transfected (GFP+) population for staining obtained in n = 3 independent experiments. MFI values were normalized to the values obtained with anti-Env 2G12 mAb for each Env mutant. Error bars indicate mean ± SEM. Statistical significance was tested using a two-tailed unpaired t-test. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Structure of an engineered CRF01_AE SOSIP.664 Env trimer in complex with temsavir.
a Overall structures of trimers complexed with chaperone Fabs of 10–1074 and 8ANC195 antibodies shown as cartoon with LMHS mutations highlighted in red within one gp120 promoter. Envelope sugars are shown as gray sticks. b Changes to overall trimer assembly, calculated as changes in position of gp120 relative to gp41 (referred to as trimer ‘opening’) of CRF01_AE_ T/F100 SOSIP.664 variants as compared to BG505 SOSIP.664 (PDB ID: 4ZMJ). The relative position for each gp120 in the trimer is calculated based on the α-carbon position for residue 375 at the base of the CD4 Phe43 binding pocket (shown as colored spheres for each structure) relative to the gp41 trimer center (gray sphere, Centr, calculated for all trimers aligned based on the α-carbon positions of the central gp41 α7 helices). The distances between Centr and the 375Cα of each protomer (a–c) and the 375Cα atoms of neighboring protomers (d–f) are shown to indicate the extent of the protomer rearrangement relative to gp41. The clockwise rotations of the gp120 subunits are calculated as angles relative to apo BG505 SOSIP (shown and labeled a-a’, b-b’, and c-c’). The BG505 SOSIP.664 bound to CD4 (PDB: 5THR) is shown as a reference to the ‘open’ CD4-triggered conformation of trimer. c Table summarizing a–f distances and a-a’, b-b’ and c-c’ rotation angles for CRF01_AE_ T/F100 SOSIP.664 complex variants relative to the unbound BG505 SOSIP trimer (PDB: 4ZMJ).
Fig. 4
Fig. 4. The LMHS mutations’ induced changes to the temsavir binding pocket.
a Insights into regions forming the temsavir binding pocket. The gp120/gp41 protomer of temsavir-SOSIP.664 LMHS complex with secondary elements colored yellow, red and green for β-strands, α-helices and loops respectively. Temsavir is shown as stick/surface representation and secondary elements forming or surrounding the pocket are as labeled. b Superimposition of the CRF01_AE_ T/F100 SOSIP.664 wild type (green), CRF01_AE_ T/F100 SOSIP.664 LMHS mutant (yellow) and its complex with temsavir (gray) with a blow-up view into the β20-β21 loop region. LMHS mutations introduced in this region are shown as red spheres/colored red. The inlet shows the chemical structure of temsavir. c Changes induced to the temsavir binding pocket by LMHS mutations. Inner domain Layers are colored blue, orange and cyan for Layer 1, 2 and 3 respectively, with the LMHS mutations shown within one gp120/gp41 promoter. The 7-stranded β-sandwich and N- and C-termini of the gp120 inner domain are colored magenta. The blow-up views show the network of interactions mediated by LMHS residues at the ‘entry’ of the temsavir binding pocket and the neighboring β20-β21 loop. Hydrogen bonds are shown as dashed blue lines.
Fig. 5
Fig. 5. Temsavir binding pocket.
a Temsavir in its binding pocket within the CRF01_AE_ T/F100 LMHS SOSIP.664. Cryo-EM density map of temsavir is shown (left panel) and residues lining the pocket shown as sticks (right panel). The LMHS introduced residues are labeled in red and H-bonds as blue dashes. b Superimposition of the temsavir binding pockets formed within CRF01_AE_ T/F100 LMHS SOSIP.664 (gray) and BG505-SOSIP.664. The inhibitor molecules are shown in ball-and-sticks while the pocket residues are shown in sticks with or without surface. The LMHS mutations within the pockets are highlighted in red. To the right and left are close-up views into the part of the pocket accommodating the acetyl-phenyl and methyltriazole moiety of the inhibitor, respectively. c The residue-resolved buried-surface-area (BSA) of gp120 contributing to the temsavir-protein interface, as determined by PISA. BSA values represent the average of the three copies in the trimer. The conservation of residues lining the temsavir pocket is shown at the bottom. The height of the residue at each position is proportional to its frequency of distribution among the HIV-1 isolates, as deposited in the Los Alamos HIV database (all clades are included). Residues are colored according to hydrophobicity: black - hydrophobic, green - neutral, blue - hydrophilic. d Close-up views of the extended tails on the thiazole ring from the two temsavir analogues whose structures have been determined (PDB: 6MU6 and 6MU7).
Fig. 6
Fig. 6. Comparisons of the 10–1074 and 8ANC195 binding interfaces formed with CRF01_AE_ T/F100 SOSIP.664 wild type and its LMHS mutant unbound or bound to temsavir.
a, c Buried Surface Area (BSA) contributed by individual residues of Env and the Fabs of 10–1074 (c) and 8ANC195 (b) in apo CRF01_AE_ T/F100 LMHS SOSIP.664 and CRF01_AE_ T/F100 LMHS SOSIP.664 bound to temsavir. The total Env glycan contribution to the interface is shown as a separate bar with the value of BSA shown at the top. BSA values represent the average of the three copies in the trimer. b Superimposition of the complexes with 10–1074 and 8ANC195 colored green, yellow and gray for wild-type CRF01_AE_ T/F100 SOSIP.664, and its apo and temsavir-bound LMHS mutant complexes, respectively. Blow-ups show similarities/differences between the complexes of how individual Fab/antibody residues interact with the Env antigen.
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