SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies

Abstract

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403-405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the α_vβ₃ integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with α_vβ₃ integrin or pretreatment of cells with a monoclonal antibody against α_vβ₃ integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.

Keywords: BNT162b2 vaccine; RGD motif; SARS-CoV-2 variants; endothelial cell dysfunction; αvβ3 integrin.

Figure 1

Surface expression of α_vβ₃ and α₅β₁ integrins on HL-mECs and A549 ACE2⁺ cells. (A) Immunofluorescence visualization of α_vβ₃ and α₅β₁ integrins on HL-mECs and A549 ACE2⁺ cells. Cells were fixed with 4% PFA and incubated with a monoclonal antibody against α_vβ₃ or α₅β₁ integrins, followed by Alexa Fluor 488-conjugated anti-mouse IgG (green) or Alexa Fluor 594-conjugated anti-rabbit IgG (red). Nuclei were counterstained with DAPI (scale bar, 20 µm). (B) Flow cytometric analysis of α_vβ₃ and α₅β₁ expression on HL-mECs and A549 ACE2⁺ cells. Cells were fixed with 4% PFA and incubated with a monoclonal antibody against α_vβ₃ or α₅β₁ integrins and followed by Alexa Fluor 488-conjugated anti-human mouse IgG or Alexa Fluor 488-conjugated anti-rabbit IgG. Histograms show the rightward shift for the BB515-A + signal (red) compared to isotype control (light blue).

Figure 2

The RGD motif mediates SARS-CoV-2 variants entry into ACE2-negative cells. (A) HL-mECs (left panel) and A549-ACE2⁺ cells (right panel) were infected with SARS-CoV-2 belonging to B.1, B.1.1, B.1.1.7, B.1.351, B.1.525, B.1.621, and B.1.617.2 lineages at a MOI of 1. The graphs show SARS-CoV-2 genome quantitation in cell supernatants collected at 1, 24, 48, and 120 h p.i. by qRT-PCR. Data are representative of two independent experiments with similar results. (B) Schematic representation of the RBD of SARS-CoV-2 Spike protein. The RBD sequences of the different SARS-CoV-2 lineages were aligned. Numbers refer to the Spike protein sequence. The RGD motif is highlighted in red. (C) HL-mECs were pretreated with 30 µg/mL of RAD or RGD peptides before SARS-CoV-2 B.1 or B.1.617.2 infection. Twenty-four h p.i., the presence of SARS-CoV-2 mRNA at an intracellular level was evaluated by qRT-PCR. The percentage of infectivity inhibition was calculated by comparing SARS-CoV-2 treated cells and SARS-CoV-2 untreated cells. Data are representative of two independent experiments with similar results. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (** p < 0.01) by relating RAD- and RGD-treated cells. (D) Prior to infection, SARS-CoV-2 B.1 or B.1.617.2 were preincubated or not for 1 h, with α_vβ₃ integrin (5 µg/mL). Then, the viruses were used to infect HL-mECs or A549 ACE2⁺ cells for 1 h at a MOI of 1. Twenty-four h p.i., the presence of SARS-CoV-2 mRNA at an intracellular level was evaluated by qRT-PCR. The percentage of infectivity inhibition was calculated by comparing cells infected with α_vβ₃-treated SARS-CoV-2 and cells infected with untreated SARS-CoV-2. Data are representative of two independent experiments with similar results. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (**** p < 0.0001) by relating HL-mECs and A549 ACE2⁺ cells. (E) HL-mECs or A549 ACE2⁺ cells were pretreated or not with mAb against α_vβ₃ integrin (100 µg/mL) for 1 h at 37 °C. Then, mAb pretreated cells were infected, for 1 h, with SARS-CoV-2 B.1 or B.1.617.2 at a MOI of 1. Twenty-four h p.i., the presence of SARS-CoV-2 mRNA at an intracellular level was evaluated by qRT-PCR. The percentage of infectivity inhibition was calculated by comparing SARS-CoV-2-treated cells and SARS-CoV-2-untreated cells. Data are representative of two independent experiments with similar results. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (**** p < 0.0001) by relating HL-mECs and A549 ACE2⁺ cells.

Figure 3

α_vβ₃ counteracts SARS-CoV-2 proangiogenic effects. HL-mECs were mock-infected (mock) or infected with SARS-CoV-2 belonging to the B.1.617.2 lineage (SARS-CoV-2) or with α_vβ₃-treated B.1.617.2 (+α_vβ₃) at MOI 1, for 1 h at 37 °C and then washed and cultured until day 3 p.i. (A) Mock, SARS-CoV-2, and + α_vβ₃ HL-mECs were seeded on reduced growth factor Matrigel-coated wells and then cultured for 12 h at 37 °C. Pictures are representative of one out of three independent experiments with similar results (scale bar, 200 μm). VEGF-A was used as a positive control. Values reported in the graph are the mean ± SD of one representative experiment out of three independent experiments with similar results performed in triplicate. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (*** p < 0.001; **** p < 0.0001). (B) Sprouting of spheroids generated with Mock, SARS-CoV-2, or +α_vβ₃ HL-mECs. Pictures are representative of one out of three independent experiments with similar results (scale bar, 10 μm). VEGF-A was used as a positive control. Values reported in the graph are the mean ± SD of one representative experiment out of three independent experiments with similar results performed in triplicate. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (** p < 0.01). (C) Human proteome array for angiogenic molecules. The results are expressed as mean values ± SD of duplicates given as fold increase, as compared to mock-infected cells. Data are representative of one out of three independent experiments with similar results.

Figure 4

Vaccine elicited antibodies do not inhibit integrin-mediated SARS-CoV-2 entry into HL-mECs. Prior to infection, SARS-CoV-2 belonging to B.1.617.2 lineage was preincubated or not for 1 h, with sera (1:100) collected from volunteers after completing the BNT162b2 vaccine schedule (from 1 to 7). Then, the mixture virus-serum was used to infect HL-mECs or A549 ACE2⁺ cells. Twenty-four h p.i., the presence of SARS-CoV-2 mRNA at intracellular level was evaluated by qRT-PCR. The percentage of infectivity inhibition was calculated by comparing cells infected with serum-treated SARS-CoV-2 and cells infected with untreated SARS-CoV-2. Data are representative of two independent experiments with similar results. Statistical analysis was performed by one-way ANOVA and Bonferroni’s post-test was used to compare data (**** p < 0.0001) by relating HL-mECs and A549 ACE2⁺ cells.

Figure 5

Recombinant SARS-CoV-2 Spike protein binds to α_vβ₃ integrin and then is internalized. (A) Left panel, sensogram overlay showing the binding of increasing amounts of α_vβ₃ integrin (from 1.25 to 20 nM) to immobilized entire RBD of SARS-CoV-2 Spike protein. The response, in resonance units (RU), was recorded as a function of time. Right panel, saturation curve obtained by using the values of RU bound at equilibrium from injection of increasing concentrations of free α_vβ₃ onto sensor chip immobilized entire RBD. (B) HL-mECs were incubated at 4 °C in RPMI (15% FBS) containing 100 ng/mL of recombinant SARS-CoV-2 Spike protein. After 1 h of incubation, cells were washed and then incubated at 37 °C in RPMI (15% FBS) in the absence of Spike protein. After 30 min, cells were washed, fixed with 4% PFA, permeabilized (lower panel) or not (upper panel) with PBS 0.1% Triton X-100, saturated with 0.1% BSA, 0.1% Tween 20 in PBS and probed with a human serum containing IgG to SARS-CoV-2 and with a mouse monoclonal antibody against α_vβ₃ integrin followed by Alexa Fluor 488-conjugated anti-human mouse IgG and by 594 conjugated anti-mouse goat IgG1. Magnified views on the upper panels show α_vβ₃ expression (red) and the recombinant Spike protein (green) on cellular surface (not permeabilized). Magnified views on the lower panels show α_vβ₃ and the recombinant Spike proteins internalized in HL-mECs (permeabilized). Boxes indicate the regions in the merged pictures that are magnified three-fold to show the localization of Spike protein and α_vβ₃ integrin (scale bar, 50 µm).

Figure 6

SARS-CoV-2 enters into HL-mECs through endocytosis. HL-mECs were pretreated for 1 h at 37 °C with Cathepsin L inhibitor III (20 µM), Monensin (2 µM), and Brefeldin A (3 µg/mL). After treatment, cells were infected with SARS-CoV-2 belonging to B.1.617.2 lineage. Twenty-four h p.i., cells were fixed with 4% PFA in PBS and permeabilized with 0.1% Triton X-100 in PBS, and saturated with 0.1% BSA, 0.1% Tween 20 in PBS. For staining, cells were incubated for 1 h with a human serum containing IgG to SARS-CoV-2 (1:1000 dilution) followed by Alexa Fluor 488-conjugated anti-human mouse IgG. Nuclei were counterstained with DAPI (scale bar, 20 µm).