Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant's Superior Transmission

Abstract

Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta's spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spike's molecular interactions with syndecan-4 also involve syndecan-4's cell-binding domain that mediates cell-to-cell adhesion. Regardless of the complexity of these interactions, exogenously added heparin blocks Delta's cellular entry as efficiently as syndecan-4 knockdown. Therefore, a profound understanding of the molecular mechanisms underlying Delta infections enables the development of molecularly targeted yet simple strategies to reduce the Delta variant's spread.

Keywords: Delta variant; SARS-CoV-2; cellular entry; heparan sulfate proteoglycans; syndecan; viral transmission.

Figure 1

Cellular uptake of WT and Delta spike into 293T and ACE2 overexpressing 293T-ACE2 cells. The cells were incubated with WT and Delta spike proteins for 4 h at 37 °C. After incubation, the cells were washed, trypsinized, fixed, permeabilized and treated with AF 647-labeled antibodies specific for the His-tag of the recombinant spike proteins. Cellular uptake was then analyzed with imaging flow cytometry. (A–D) Representative flow cytometry histograms (A,C), brightfield (BF) and fluorescent cellular images (B,D) showing the intracellular fluorescence of 293T and 293T-ACE2 cells treated with WT or Delta spike proteins. Scale bar = 20 μm. (E) Detected fluorescence intensities were normalized to WT spike-treated 293T cells as standards. The bars represent the mean + SEM of nine independent experiments. Experimental data are presented as dots. Statistical significance was assessed with ANOVA. ** p < 0.05 vs. standards; *** p < 0.01 vs. standards; ns: not significant.

Figure 2

ACE2, HS, and SDC expression of 293T and 293T-ACE2 cells. ACE2, HS, and SDC expression of 293T and 293T-ACE2 cells was measured with imaging flow cytometry by using fluorescently labeled specific antibodies. (A,B) ACE2 and HS expression profile of 293T and 293T-ACE2 cells. The representative flow cytometry histograms and cellular images show the ACE2 and HS expression of 293T and 293T-ACE2 cells treated with the fluorescent ACE2 and HS antibodies. Scale bar = 20 μm. (C) SDC expression profile of 293T and 293T-ACE2 cells. The representative flow cytometry histograms and fluorescent cellular images show the SDC expression of 293T cells and 293T-ACE2 cells treated with the APC-labeled respective SDC antibodies. Scale bar = 20 μm. (D–F) Detected ACE2, HS and SDC expression measures were normalized to those of 293T cells as standards. The bars represent the mean ± SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Effect of ACE2 and proteoglycan inhibition on cellular uptake. 293T and 293T-ACE2 cells, preincubated with or without heparin (200 ug/mL) or DX600 (10 uM), were treated with the WT or Delta spike proteins. (A–F) Representative flow cytometry histograms (A,B) and fluorescent cellular images (C–F) show the fluorescence of 293T and 293T-ACE2 cells treated with either the WT or Delta spike proteins in the presence or absence of the inhibitors. Scale bar = 20 μm. (G,H) The effect of the inhibitors expressed as percent inhibition, calculated with the following formula: ((X − Y)/X) × 100, where X is the fluorescence intensity obtained on cells treated with the spike proteins in the absence of the inhibitor, and Y is the fluorescence intensity obtained on cells treated with proteins in the presence of the inhibitor. The bars represent the mean ± SEM of three independent experiments (experimental data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Cellular uptake of the WT spike and its Delta variant into WT K562 cells and SDC1, 4 transfectants. WT K562 cells, SDC1 and SDC4 transfectants were treated with the WT spike and its Delta variant. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spikes. (A,B) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT K562 cells, SDC1 and SDC4 transfectants treated with either of the spike proteins. Scale bar = 20 μm. (C) Detected fluorescence intensities normalized to WT-spike-treated K562 cells as standards. The bars represent the mean + SEM of six independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Detected fluorescence intensities normalized to WT-spike-treated cells as standards. The bars represent the mean + SEM of six independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

PSV-mediated gene transduction into WT K562 cells, SDC1 and SDC4 transfectants. The cells were incubated with SARS-CoV-2 PSV-RFP (either the WT or the Delta variant). RFP expression was analyzed 72 h later with imaging flow cytometry. (A) Representative flow cytometry histograms showing RFP fluorescence of WT K562 cells, SDC1 and SDC4 transfectants incubated with either the WT or the Delta SARS-CoV-2 PSV. (B) Cellular images of SARS-CoV-2 PSV-treated WT K562 cells and SDC transfectants as detected with imaging flow cytometry. Scale bar = 20 μm. (C) Detected cellular RFP intensities normalized to WT PSV-treated WT K562 cells as standards. The bars represent the mean + SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01. (D) Detected RFP intensities normalized to WT PSV-treated cells as standards. The bars represent the mean ± SEM of three independent experiments (data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Figure 6

Cellular uptake of the WT and Delta spike proteins into SDC4 deletion mutants. WT SDC4 transfectants and CBD, HSA and Si4 mutants (all tagged with GFP) were treated with the Delta spike. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spike proteins. (A) Representative flow cytometry histograms showing the intracellular fluorescence of WT SDC4 transfectants, CBD, HSA and Si4 mutants treated with the Delta spike protein (along with specific fluorescent antibodies). Scale bar = 20 μm. (B) Detected fluorescence intensities were normalized Delta spike-treated WT SDC4 transfectants as standards. The bars represent the mean ± SEM of four independent experiments (experimental data are represented as dots). Statistical significance vs. standards was assessed with ANOVA. *** p < 0.001. (C) Cellular images showing the intracellular fluorescence of WT SDC4 and CBD, HSA and Si4 mutants treated with the Delta spike protein. Scale bar = 20 μm. The indicated BDS values of SARS-CoV-2 and SDCs represent the mean + SEM of four independent experiments.

Figure 7

Comparative expression analysis of ACE2, HS and SDCs in 293T, Caco-2 and Calu-3 cells. ACE2, HS and SDC expression profile of the cell lines were analyzed with flow cytometry using fluorescently labeled antibodies. Detected expression levels were normalized to those of 293T cells. The bars represent the mean ± SEM of three independent experiments (data are represented as dots). Statistical significance vs. 293T expression levels was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Figure 8

Internalization of the WT and Delta spike into Calu-3 cells. Calu-3 cells were treated with the WT spike and its Delta variant. The cellular uptake was then analyzed with imaging flow cytometry by using fluorescently labeled specific antibodies against the His-tag of the recombinant spike proteins. (A) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of Calu-3 cells treated with either the WT or Delta spike. (B) Detected intracellular fluorescence levels normalized to those treated with the WT spike as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. 293T expression levels was assessed with ANOVA. * p < 0.05. (C) SDS-PAGE showing the WT or Delta spike immunoprecipitated with SDC4 antibodies from extracts of untreated control or spike-treated Calu-3 cells. Lanes 1–2: 0.5 ug of WT and Delta spike proteins; Lanes 3–4: immunoprecipitate of WT or Delta spike-treated Calu-3 cells; Lane 5: immunoprecipitate of untreated Calu-3. Standard protein size markers are indicated on the left. The signal of spike proteins was detected with UVITEC Alliance Q9 Advanced Imager, and the intensity of bands were analyzed with the NineAlliance^© software. (D) Detected band intensities were normalized to WT spike-treated Calu-3 cells as standard. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05.

Figure 9

Effect of SDC4 knockdown (KD) or GAG inhibition on Delta spike internalization into Calu-3 cells. SDC4 KD in Calu-3 cells was performed using a lentiviral vector specific to human SDC4. (A) SDC4 expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Detected SDC4 levels of KD cells were normalized to WT Calu-3 cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05. (B,C) SDC4 KD and WT Calu-3 cells were treated with either the Delta spike protein (B) or the Delta PSV (C). For GAG inhibition, some of the WT Calu-3 cells were preincubated with heparin (200 ug/mL, 30 min before Delta treatment). Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT or SDC4 KD Calu-3 treated with Delta spike protein or PSV in the presence or absence of heparin. Scale bar = 20 μm. The effect of SDC4 KD or heparin treatment was expressed as percent inhibition, calculated with the following formula: ((X − Y)/X) × 100, where X is the fluorescence intensity obtained on controls treated with the Delta spike or Delta PSV without SDC4 KD or heparin, and Y is the fluorescence intensity obtained on cells receiving Delta spike or Delta PSV treatment, along with SDC4 KD or heparin. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. controls was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.