Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2

Abstract

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is broadly accepted that SARS-CoV-2 utilizes its spike protein to recognize the extracellular domain of angiotensin-converting enzyme 2 (ACE2) to enter cells for viral infection. However, other mechanisms of SARS-CoV-2 cell entry may occur. We show quantitatively that the SARS-CoV-2 spike protein also binds to the extracellular domain of broadly expressed integrin α5β1 with an affinity comparable to that of SARS-CoV-2 binding to ACE2. More importantly, we provide direct evidence that such binding promotes the internalization of SARS-CoV-2 into non-ACE2 cells in a manner critically dependent upon the activation of the integrin. Our data demonstrate an alternative pathway for the cell entry of SARS-CoV-2, suggesting that upon initial ACE2-mediated invasion of the virus in the respiratory system, which is known to trigger an immune response and secretion of cytokines to activate integrin, the integrin-mediated cell invasion of SARS-CoV-2 into the respiratory system and other organs becomes effective, thereby promoting further infection and progression of COVID-19.

Keywords: COVID-19; SARS-CoV-2; integrin; virus infection.

Figure 1

SARS-CoV-2 spike protein interaction with integrin extracellular domain.A, biotinylated SARS-CoV-2-RBD is able to pull down integrin α5β1 (left) while there is no nonspecific binding of integrin to streptavidin beads (right) (Fig. S2). B, SPR affinity measurements of biotinylated SARS-CoV-2-S1 with integrin α5β1 extracellular domain. Black curves are SPR sensorgrams of various concentrations (7.8 nM, 15.6 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM) of integrin flowed onto the chip surface immobilized with biotinylated SARS-CoV-2-S1. Red curves are 1:1 kinetics fitting, resulting in K_D ∼ 31 nM (n = 2). Low concentration point 3.9 nM had no response and was not used in the data fitting. Note that the fitting especially at high integrin concentration (500 nM) was not ideal likely due to the small degree (5–8%) of self-association (aggregation) and nonspecific binding of integrin despite the addition of BSA. This problem may somewhat affect the accuracy of K_D. Removing the high concentration point (500 nM) made the fitting look better but does not significantly alter the binding kinetics; C, SPR affinity measurements of biotinylated SARS-CoV-2-S1 with hACE2 extracellular domain. Black curves are SPR sensorgrams of various concentrations (3.9 nM, 7.8 nM, 15.6 nM, 31.25 nM, 62.5 nM, 125 nM, and 250 nM) of hACE2 flowed onto the chip surface immobilized with biotinylated SARS-CoV-2-S1. Red curves are 1:1 kinetics binding fitting, resulting in K_D ∼ 26 nM. D, biotinylated SARS-CoV-2-RBD R403 A mutant (RA) is defective in binding to integrin α5β1 compared to its wild type (WT) counterpart (Fig. S3). Integrin α5β1 was detected using antibody specific to flag-tag attached to α5 subunit. ACE2, angiotensin-converting enzyme 2; hACE2, human ACE2; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SPR, surface plasmon resonance.

Figure 2

SARS-CoV-2 RBD association with CHO-K1 cells requires activation of integrin.A, flow cytometry contour plots of CHO-K1 incubated with or without cyanine5 labeled SARS-CoV-2-RBD and with or without MnCl₂. B, biotinylated SARS-CoV-2-RBD binding to integrin α5β1 is inhibited by Cilengitide. Dashed lines indicate spliced borders where data unrelated to the text were removed (see the original full blot in Fig. S4). C, bar graphs generated from flow cytometry data, which show that fixed CHO-K1 cells with activation of integrin by MnCl2 bind to cyanine5-labeled WT RBD, but the binding is substantially reduced upon R403A mutation or addition of integrin inhibitor Cilengitide. RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3

SARS-CoV-2-RBD is able to internalize into CHO-K1 cells upon integrin is activated, and the internalization is dependent on RGD motif. The first row, cyanine5-labeled SARS-CoV-2-RBD WT shown in magenta is able to internalize into CHO-K1 cells in the presence of MnCl₂. The second row, cyanine5-labeled SARS-CoV-2-RBD WT is unable to internalize into CHO-K1 cells in the absence of MnCl₂. The third row, cyanine5-labeled SARS-CoV-2-RBD R403A mutant (RA) is unable to internalize into CHO-K1 cells in the presence of MnCl₂. The fourth row, cyanine5-labeled SARS-CoV-2-RBD WT internalization into CHO-K1 cells in the presence of MnCl₂ is impaired by Cilengitide. RBD, receptor binding domain; R⁴⁰³GD, arginine–glycine–aspartic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 4

SARS-CoV-2 pseudovirus infection to CHO-K1 cells.A, representative images of SARS-CoV-2 pseudovirus particle (PP) infection on CHO-K1 cells upon integrin activation by MnCl₂ and the inhibition by integrin inhibitor Cilengitide. The first row, SARS-CoV-2 PP barely infects CHO-K1 cells in the absence of MnCl₂. The second row, SARS-CoV-2 PP massively infects CHO-K1 cells in the presence of MnCl₂. The third and fourth rows, SARS-CoV-2 PP infection on CHO-K1 cells in the presence of MnCl₂ is impaired by Cilengitide. B, quantification of the relative infection of three randomly selected regions from each of three independent experiments. Data are averages ± SEM for nine images (three randomly selected images from each three independent experiments). ∗∗∗p < 0.001. ns, not significant, p > 0.05. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 5

Integrin and ACE2 are independent receptors for SARS-CoV-2 pseudovirus infection on hACE2 transiently transfected CHO-K1 cells.A, representative images of SARS-CoV-2 pseudovirus particle (PP) infection on hACE2 transiently transfected CHO-K1 cells upon integrin activation by MnCl₂ and the inhibition of integrin by inhibitor Cilengitide. The first row, SARS-CoV-2 PP massively infects hACE2 transiently transfected CHO-K1 cells in the absence of MnCl₂. The second row, SARS-CoV-2 PP barely infects hACE2 transiently transfected CHO-K1 cells upon integrin activation in the presence of MnCl₂. The third and fourth rows, SARS-CoV-2 PP infection on hACE2 transiently transfected CHO-K1 cells upon integrin activation in the presence of MnCl₂ is gradually recovered by Cilengitide. B, quantification of the relative infection of three randomly selected regions from each of three independent experiments. Data are averages ± SEM for nine images (three randomly selected images from each three independent experiments). ∗∗∗p < 0.001. Note: the infect rate here is lower than Figure 4, which is because hACE2 here is transiently transfected while integrin is endogenously expressed. ACE2, angiotensin-converting enzyme 2; hACE2, human ACE2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Supplementary Figure S1

SARS-CoV-2 relationship with integrin and ACE2. A, biotinylated SARS-CoV-2-S1S1 or (B) biotinylated SARS-CoV-2-RBD (SINO BIOLOGICAL #40592-V08B-B) is able to pull down integrin α5β1 in the presence or absence of hACE2 (Figs. S5S6). The hACE2 slightly reduced the binding of SARS-CoV-2 RBD to integrin probably because the binding sites of hACE2 and integrin on RBD are spatially close and thus the two proteins sterically clash when both bind to RBD. C, SPR experiment showing no significant interaction between immobilized biotinylated ACE2 (SINO BIOLOGICAL #10108-H27B-B) and integrin α5β1.

Supplementary Figure S2

Unprocessed blots for Figure 1A.

Supplementary Figure S3

Unprocessed blots for Figure 1D.

Supplementary Figure S4

Unprocessed blots for Figure 2B.

Supplementary Figure S5

Unprocessed blots for Fig. S1A.

Supplementary Figure S6

Unprocessed blots for Fig. S1B.