The SARS-CoV-2 spike protein binds and modulates estrogen receptors

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ ¹⁸ F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

Figure 1.. S binds ERα with high affinity.

(a) [¹²⁵I]S saturation and (b) competition binding to recombinant ACE2. (c) Schematic of ProtoArray® experimental design. (d) Positive control ProtoArray® autoradiograms showing total and non-specific (NS) binding of [³H]estradiol (E2). (e) ProtoArray® autoradiograms showing total and NS binding of [¹²⁵I]S. (f, g) Representative array blocks showing total and NS [³H]E2 and [¹²⁵I]S binding. Red rectangles show location of ERα proteins. (h, i) Quantification of total and NS [¹²⁵I]S binding at ERα and BSA (control). Data are representative of three independent experiments. (j-l) Representative SPR sensorgrams showing kinetic and equilibrium binding analyses of immobilized S exposed to increasing concentrations of ACE2, NRP1 and ERα protein (K_on=2.03 ×10⁵, K_off= 1.96 × 10⁻³, K_D= 9.7 nM). In a-b, data are represented as mean ± SEM. In h, i, data are presented as median ± min and max limits.

Figure 2.. S and ER interact at conserved LXD nuclear receptor coregulatory (NRC) motifs.

(a) ER interaction network showing known and predicted protein associations. (b) LXD-like patterns in the S sequence. The LXXLL motif and a homologous region are highlighted in blue and red boxes, respectively, with dark grey background. −1 and −2 positions are reported in italic and light grey background. (c) The LPPLL and IEDLL residues of the two motifs are shown in the 3D X-ray S structure (pdb id 6VYB) with blue and red colors, respectively. The three S chains are shown in yellow, cyan and green. (d) S-ER motif-oriented docking. The best 3D docking hypothesis is shown. The ER dimer is in orange and gray, S is green. (e) Alignment between the best-pose and the 3OLL model tied with NCOA1. The region occupied by S’s alpha-helix interacts in the area where the NCOA fragment was crystallized. (f) S protein peptides and their location with respect to the S 3D structure. (g) The SP7 peptide containing the LPPLL motif significantly increased ERα activation (F (1,48) = 30.38; **P<0.01; two-way ANOVA, peptide treatment main effect). Data are mean ± SEM.

Figure 3.. S modulates ER-dependent biological functions.

S inhibits (a) E2-induced ERα DNA binding in MCF-7 nuclear extracts and (b) transcriptional activation in an ERα reporter cell line (F (1, 28) = 21.73, *P=0.01; two-way ANOVA, S treatment × E2 concentration interaction effect). (c) Immunofluorescent staining of S and endogenous ERα in MCF-7 cells transfected with empty vector, wild-type (WT) or the furin cleavage site mutant S^{(R682S, R685S)}. Scale bar = 16 μm. (d) S increases MCF-7 cell proliferation in an ER-dependent manner (**P<0.01, ***P<0.001 versus control; ^###P<0.001 versus E2; ^&&&P<0.001 versus S; one-way ANOVA with post hoc Tukey test). (e) S decreases osteoclast differentiation in an ER-dependent manner (***p<0.001 versus control w/o RANKL; ^###P<0.001 versus control w/ RANKL; one-way ANOVA with post hoc Tukey test). (f) S and E2 increase ACE2 protein levels in MCF-7 cells in an ER-dependent manner (***P<0.001 versus control; ^###P<0.001 versus E2; ^&&&P<0.001 versus S, one-way ANOVA with post hoc Tukey test). (g, h) E2 and S increase ACE2 mRNA (***P<0.001, one-way ANOVA with post hoc Tukey test) and (i, j) protein in the Calu-3 lung cell line in an ER-dependent manner. Scale bar = 30 μm. All data shown as mean ± SEM.

Figure 4.. SARS-CoV-2 infection increases cytoplasmic ERα accumulation and S-ERα colocalization in pulmonary macrophages.

(a) Schematic showing experimental design of SARS-CoV-2 hamster studies and BSL-3 imaging compartment. (b) CT, [¹⁸F]FES PET and AUC heatmap overlay images from hamsters at pre-infection (Day −1) and infection (Day 7) (MIP; maximum intensity projection, SUV; standard uptake value; AUC, area under the curve). (c) Time activity curves showing SUV ratio (SUVr; tissue/blood [¹⁸F]FES content) in each experimental group. n = 45/group. ***P<0.001 versus pre-infection (d) [¹⁸F]FES uptake expressed as area under the curve ratio (AUCr; tissue/blood [¹⁸F]FES content). F (3, 32) = 12.15; ***P<0.001, *P<0.05 (e) [¹⁸F]FES uptake expressed as % injected dose (ID)/g body weight in postmortem hamster lung (harvested 110 min post-injection; n = 3–4 /group). F (2, 7) = 7.161; *P<0.05. (f) Hamster lung immunohistochemistry showing colocalization of S and ERα immunoreactivity. (g) Immunogold EM showing SARS-CoV-2 particles (red arrowheads) and ERα-bound gold nanoparticles (blue arrowheads) in a hamster alveolar macrophage (scale bar = 200 nm). Yellow arrowheads correspond to cytoplasmic ERα accumulation. (h) S and (i) ERα immunostaining in SARS-CoV-2-infected human lung showing S-ERα colocalization in macrophages (black arrowheads). Scale bar (low mag = 100 nm; high mag = 25 nm). All data shown as mean ± SEM.