key: cord-0699068-ewf61kj0
authors: Liu, Jiamnin; Lu, Fan; Chen, Yinghua; Plow, Edward; Qin, Jun
title: Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2
date: 2022-02-10
journal: J Biol Chem
DOI: 10.1016/j.jbc.2022.101710
sha: 198bbd2301b00088336f5cdb1e5aaa8a500b294d
doc_id: 699068
cord_uid: ewf61kj0

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.

The COVID-19-induced global pandemic has been ongoing for nearly two years causing > 350 million infections. Although the massive vaccination effort at global scale has reduced the severe threat of the COVID-19 pandemic, the mortality rate remains still relatively high (from ~5% in the beginning of pandemic to ~2% now) with a total of > 5.5 million deaths. The pathogen causing this disastrous disease is SARS-CoV-2 that belongs to the family of coronaviruses including 229E, NL63, OC43, HKU1, MERS-CoV and highly homologous SARS-CoV 1 .

However, SARS-CoV-2 is apparently much more contagious than any other coronaviruses and the mechanism underlying its infectivity is not well understood. Each SARS-CoV-2 is ~50-200 nanometers in diameter containing an RNA segment of 30,000 bases encoding the virus, four structural proteins, the Spike, Envelop, Membrane, and Nucleocapsid proteins. The nucleocapsid protein holds the RNA genome whereas other proteins form the viral envelope 2 . The spike protein contains 1273 amino acids that are divided into S1 domain (1-541), a linker (542-685), S2 domain (686-1213), a single-pass transmembrane segment (1214-1236) and a short cytoplasmic tail (1237-1273) 3, 4 . Importantly, S1 utilizes a fragment (333-529) called its receptor binding domain (RBD) to recognize ACE2 (Angiotensin-converting enzyme 2)a minor variant of ACE (Angiotensinconverting enzyme) 5 to initiate virus entry and infection 2, 4 . Such ACE2-mediated virus entry was first found for the homologous SARS-CoV in 2003 6 , although SARS-CoV only caused ~8000 infections and did not lead to global pandemic. ACE2 contains an N-terminal peptidase domain and a C-terminal collectrin renal amino acid transporter domain on its extracellular region, followed by a single-pass transmembrane domain and a small intracellular domain 4 . ACE2's major physiological role is to lower blood pressure by using the peptidase domain to hydrolyze angiotensin II (a vasoconstrictor hormone peptide) into angiotensin 1-7 (a vasodilator). The SARS-J o u r n a l P r e -p r o o f CoV-2 S1 RBD binding to ACE2 does not affect the activity of ACE2 but rather promotes the attachment of the virus onto the cell surface. The virus attachment is followed by a furin-mediated cleavage at its S1/S2 site to facilitate the entry of SARS-CoV-2 into cells 7 , which ultimately lead to the replication of the virus and its release via exocytosis to infect more cells. While this ACE2mediated SARS-CoV-2 cell entry is widely accepted, the mechanism underlying the progression of the virus infection and the heterogeneous severity (asymptomatic, lightly symptomatic to severe, critical or even lethal conditions) 8 , remains poorly understood. One important finding was that the asymptomatic individuals exhibit similar level of cytokines as the healthy ones, while the symptomatic individuals exhibit elevated level of cytokines 9 . It is well-known now that COVID-19 patients under critical conditions experience hyper-production of cytokines or so-called "cytokine storm" 10 via unknown factors that cannot be purely explained by the ACE2-dependent virus infection in the respiratory system. More importantly, studies have shown that SARS-CoV-2 only causes lung infection without severe disease progression in transgenic mice expressing human ACE2 (hACE2) under endogenous promoter but the SARS-CoV-2 infection can spread to other organs in addition to lung in K18 transgenic mice expressing hACE2 under an ectopic cytokeratin promoter 11 . These observations suggest that although ACE2 is responsible for the initial invasion of SARS-CoV-2, it is highly likely that there exists additional mechanisms for promoting further progression of SARS-CoV-2 infection. Some membrane proteins such as neuropilin-1 and CD147 present in in ACE2-positive cells have been proposed to cooperate with ACE2 to regulate the virus infection 12 , but whether and how SARS-CoV-2 infects non-ACE2 cells that may promote the progression of COVID-19 is unclear. Interestingly, recent studies using mouse models expressing human ACE2 13 and human COVID-19 patient samples [14] [15] [16] revealed that SARS-CoV-2 not only invades ACE2 positive cells but also non-ACE2 cells, suggesting the J o u r n a l P r e -p r o o f existence of unknown ACE2-independent entry mechanism for SARS-CoV-2. Bioinformatics and structure-based analyses revealed that SARS-CoV-2 RBD contains a surface-exposed Arginine-Glycine-Aspartic acid (R 403 GD) motif that is absent in other coronaviruses [17] [18] [19] . Such RGD motif is known to be recognized by several members of a family of cell adhesion receptors, integrins, which are (/) heterodimeric transmembrane proteins containing large extracellular domain, transmembrane domain, and small cytoplasmic tail 20 . Integrins were previously shown to recognize RGD containing viruses such as HIV, adenovirus, foot and mouth virus, and mediate their cell entry via integrin-mediated endocytosis 21 . Increasing computational and cellular studies have therefore been performed to examine whether integrins may engage SARS-CoV-2 and regulate the infectious life cycle of the virus 15, 17, 18, [22] [23] [24] [25] [26] . However, while these studies provided important information of potential role of integrins in SARS-CoV-2 infection, no quantitative analysis has been performed so far to definitively evaluate the SARS-CoV-2 binding to integrin in comparison to ACE2. Moreover, the mechanism of how integrin acts on SARS-CoV-2 in relation to ACE2 remains highly elusive and controversial. For example, a significant number of studies proposed that integrin interacts with ACE2 to cooperatively promote the SARS-CoV-2 infection 15, [17] [18] [19] 22, [24] [25] [26] whereas some indicated that integrin inhibits the SARS-CoV-2 binding to ACE2 23, 27 . Also, one cell-based study suggested the requirement of integrin activation for mediating the SARS-CoV-2 infection 26 but other studies 18, 21-24 did not show such requirement.

The cell-based studies so far mostly relied on cells that contain both integrin and ACE2 (e.g., VERO E6 cells), which might have contributed to the uncertainty of the integrin function.

Furthermore, the use of the integrin antagonists, which have off-target effects and toxicity to cause cell death, may also partially contribute to the uncertainty of the studies.

Here, we use a combination of biochemical, biophysical, and cellular approaches with non-ACE2 cells (CHO-K1 cells) to investigate the role of the integrin-mediated cell entry of SARS-CoV-2. Using pull-down and surface plasmon resonance (SPR), we show that purified SARS-CoV-2 spike protein potently binds to widely distributed integrin 51 ectodomain with an affinity that is comparable to that between SARS-CoV-2 and ACE2. We further demonstrate that such binding leads to the internalization of SARS-CoV-2 into non-ACE2 cells in a manner that is critically dependent on the activation of integrin, its transition to a higher affinity/avidity state for cognate ligands. Our data thus provide proof-of-the-concept evidence for an alternative pathway of SARS-CoV-2 entry into cells. Given that initial SARS-CoV-2 infection via ACE2 in the respiratory system is known to generate cytokines 28 that can activate integrins [29] [30] [31] , our data suggest that more SARS-CoV-2 particles may enter into (via the activated integrin) various human cells and tissues especially those lacking ACE2 thereby contributing to further infection and progression of COVID-19. Our findings may bear important implications in fighting against the global pandemic of COVID-19.

As mentioned above, while numerous studies have indicated the binding of integrin to SARS-CoV-2 15, [17] [18] [19] [22] [23] [24] [25] [26] [27] , no detailed biochemical and biophysical analysis has been performed to elucidate the binding event. To definitively detect the interaction between SARS-CoV-2 and integrin, we first performed the pull down experiments using purified SARS-CoV-2 RBD and J o u r n a l P r e -p r o o f extracellular domain of well-known RGD binding integrin 51, a fibronectin receptor that is broadly distributed, e.g., in fibroblasts, endothelial cells, and blood cells. Fig 1A shows that SARS-CoV-2 RBD exhibits potent binding to 51. We then performed surface plasmon resonance (SPR) experiments, which revealed the binding affinity KD between SARS-CoV-2 RBD and integrin at ~31 nM ( Fig 1B) . This affinity is in the same range as that between SARS-CoV-2 spike protein and ACE2 (KD ~26 nM, Fig 1C) , which was reported before 32 . Fig 1D shows that mutating R403 to alanine within RGD motif on the SARS-CoV-2 RBD abolished its binding to integrin, further demonstrating the specificity of the binding and the importance of R 403 GD motif in SARS-CoV-2 RBD in recognition of integrin.

To further examine the association of SARS-CoV-2 RBD with integrin, we next chose to use flow cytometry to detect the association of Cyanine5-labelled RBD (Cy5-RBD) with CHO-K1 cells.

CHO-K1 is a well-established cell line containing RGD binding integrins, notably 51 33,34 . The cell line was derived from Chinese Hamster Ovary and has no detectable ACE2 5 let alone human ACE2. In fact, the entire ovary has no detectable ACE2 5 . Thus, our assay avoids the complication of using cell lines that contain both integrin and ACE2 and is expected to gain definitive cellular evidence of the integrin-RBD interaction. However, first attempt to examine CHO-K1 treated by Cy5-RBD exhibited similar fluorescence intensity as untreated cells (data not shown), indicating that RBD has no significant association with integrins on the cell surface. Since integrins in unstimulated cells exist in predominantly inactive conformation that limits ligand access for binding 20 , we stimulated the cells using MnCl2, a well-known integrin activating reagent 35-38 . The

MnCl2 treatment substantially increased the fluoresce intensity induced by the association of cy5-RBD with CHO-K1 cells (Fig 2A) , demonstrating that activation of integrin leads to robust binding to RBD on cell surface. This finding may explain why genetic screening failed to identify integrin as the SARS-CoV-2 host factor 39-41 since integrin in the screening assay may have been in an inactive state without agonist stimulation. We note that RBD interacted potently with purified integrin 51 ectodomain without Mn2+ treatment in our pull-down and SPR assays (Fig 1) . This is because in the absence of cellular constraints such as plasma membrane and integrin transmembrane-cytoplasmic portion known to control the inactive state of the receptor 20 , the ectodomain can be readily converted from inactive to active state by ligand, resulting in total high affinity binding, e.g., fibronectin repeats 9-10 can bind to 51 ectodomain at KD ~5.2 nM without any activating stimuli 42 . This affinity is comparable to that of the fully activated (extension-open) intact 51 (KD ~1.4 nM) yet is >1730 times lower than inactive (bent-closed) intact 51 (without any cellular stimulation), intact 51 exists in a dynamic equilibrium between inactive and active states, which binds fibronectin repeats 9-10 at KD~1100 nM (stronger than the bentclosed intact receptor at KD ~9000 nM), which may explain why some previous studies 19, 22 detected the SARS-CoV-2 spike binding to integrin without cellular stimulation. Nevertheless, our study is consistent with a recent study showing that activation of integrin is crucial for its binding to UV-inactivated SARS-CoV-2 26 and also with previous studies showing that activation of integrins is required for their binding to some large enveloped viruses such as HIV 37 and foot and mouth virus 43 (SARS-CoV-2 is also enveloped virus).

To further confirm that the association of RBD with CHO-K1 cells is dependent on integrin but not on other receptors such as neuropilin-1, CD147, heparin sulfate, sialic acid, or a number of other molecules that the spike protein has been reported to bind, we examined the RBD binding efficiency using R403A mutation to disrupt the RGD motif and also Cilengitidea RGD analog cyclic pentapeptide that can potently inhibit binding of RGD ligands to v3 (IC50 ~0.61 nM), Shayakhmetov et al previously reported that RGD motif deletion in adenovirus did not significantly affect the virus attachment but reduced the virus internalization 45 . This led us to consider if RBD is rapidly internalized upon binding to the Mn 2+ -activated integrin, which compensated the binding defects from R403A and inhibitor treatment. To circumvent this problem, we modified the cell preparation protocol by fixing cells first before incubating with RBD protein.

In this way, we only look at the protein attachment while excluding the potential internalization of RBD. Fig 2C shows that R403A mutation dramatically reduced association of RBD with CHO-K1 cells, and Cilengitide also significantly inhibited RBD association with CHO cells, demonstrating that RBD specifically binds to active integrin on the cell surface.

Next, we wanted to experimentally examine if SARS-CoV-2 RBD can indeed internalize into cells by binding to integrin 51 as we speculated. If so, this would suggest that SARS-CoV-2 can J o u r n a l P r e -p r o o f enter non-ACE2 cells through the route of integrin endocytosis. Integrin is well known to quickly internalize soluble ligands into endosomes via endocytosis 21, 46 . Integrin bound ligand can be also internalized but may dissociate from integrin and gradually degrade in lysosomes while free integrin can recycle back to the cell membrane 47 . Although the membrane fusion is deemed to mediate the cell entry of Sars coronavirus via ACE2 route, recent evidence showed that SARS-CoV can also be internalized via an endocytosis route 48 . It is thus possible that viruses take advantage of routes of membrane fusion and/or endocytosis, depending on which route is available.

In support of our speculation, Fig 3 shows that RBD can indeed be internalized into Mn 2+ treated cells but not in cells without the Mn 2+ treatment. Thus activation of integrin is required for effective internalization of RBD into cells, which is consistent with the above data of RBD binding to activated integrin 51 (Fig 2A) . We also stained integrin 1 to mark the cell boundary without disruption of the cell membrane. Importantly, R403A mutation substantially reduced the internalization, indicating that RBD is specifically internalized through the association with the integrin (Fig 3) . We also treated cells with Cilengitide, and our results show that Cilengitide can significantly inhibit RBD internalization. Our results thus provide strong evidence that SARS-CoV-2 RBD can internalize into cells through binding to activated integrin 51 in ACE2indepenent manner. As mentioned earlier, several viruses such as Human adenovirus, Foot and mouth disease virus, Human herpes viruses, etc. were shown to internalize into cells via integrin endocytosis 21 and thus integrin 51 highly likely uses the same endocytosis route to internalize SARS-CoV-2. upon Mn 2+ treatment to activate integrin, the SARS-CoV-2 entry was dramatically impaired in the hACE2 expressing CHO-K1 cells, whereas the inhibition of integrin by cilengitide can gradually recover the cell entry of the virus apparently through the hACE2 route (the integrin route is suppressed by cilengitide) (Fig 5) . These results indicate that the two entry receptors do not cooperate with each other. Rather, they are independent and mutually exclusive from each other when both are expressed on the same cell surface with integrin being also activated. Pull down experiments reveal that integrin could still bind to SARS-CoV-2 spike protein or RBD in the presence of ACE2, albeit with reduced capacity (Fig S1A and S1B) probably due to the steric clash between integrin and ACE2 when binding to the same SARS-CoV-2 since RGD site is spatially close to the ACE2 site on RBD 17, 27 . There is no detectable interaction between integrin and ACE2 remains to be further investigated. Nevertheless, our studies using non-ACE2 cells demonstrate clearly that integrin 51 functions independently of ACE2 to mediate the SARS-CoV-2 internalization.

Although SARS-CoV-2 has been intensively studied ever since the global pandemic started, its high contagiousness and high hospitalization/mortality rate compared to seasonal influenza still remain poorly understood. The infection clearly started locally in upper respiratory system but how it progresses further to severe, critical or even lethal medical conditions remains enigmatic.

Using multiple independent methods, we provide strong evidence that in addition to binding to well-known receptor ACE2, SARS-CoV-2 spike protein also associates with integrin with an affinity that is comparable to that between SARS-CoV-2 and ACE2. More importantly, we demonstrate that SARS-CoV-2 invades cells upon binding to activated integrin 51 and such infection is ACE2-independent. The infection appears to be also independent from other molecules that the spike protein has been reported to bind because we used the integrin-specific RGD mutation, inhibitor, and Mn 2+ treatment. The requirement of integrin activation to bind to SARS-CoV-2 explains why previous genetic screening failed to identify integrin as the SARS-CoV-2 host factor 39-41 since integrin in the screening assay is apparently in inactive state without agonist stimulation. Our findings suggest that while ACE2-mediated SARS-CoV-2 invasion plays a crucial role in initiating the infection in the respiratory system, activation of RGD binding integrins J o u r n a l P r e -p r o o f such as 51 may further contribute to deeper infection by binding and internalizing virus into more cells and tissues especially those non-ACE2 containing ones. The latter is consistent with the detections of SARS-CoV-2 in non-ACE2 cells of mouse model 13 , lung 52 , brain 14 , and tissue samples of COVID-19 patients 15, 16 . A key question is: when and how are integrins activated to mediate the SARS-CoV-2 infection in connection with the ACE2 pathway? As we mentioned earlier, although ubiquitously expressed, integrins are highly controlled receptors that are typically in the inactivated conformation with a very low ligand binding affinity. The control of integrin inactivation is likely even tighter for epithelial cell integrins that are exposed to the external environment such as in the upper respiratory system. Even if there exist a small population of active integrins, they might be tightly bound to natural ligands and unavailable for virus binding.

To activate integrin, a cascade of cellular signals triggered by cytokines [29] [30] [31] or other stimuli may impinge on integrin cytoplasmic tails causing inside-out conformational change of the receptor 20 .

Such feature makes integrin an inferior entry receptor for virus. On the other hand, although the expression of ACE2 is low and restricted in certain organs, especially in the respiratory system exposed to the external environment, ACE2 is constantly available to bind the viral ligand. Based on this scenario and our results, we speculate two sequential events leading to the progression of 

We thank technical assistance by Dr. Gauravi Deshpande for cell imaging and Dr. Eric Roush from Cytiva for useful discussion on SPR data analysis, respectively. We also thank Dr. Jae Jung for valuable discussion. The work was supported by NIH grants R01 HL58758 to JQ, P01s HL73311, P01HL154811 to EFP and JQ, and AHA Career Development grant #18CDA34110364 to JL.

SARS-CoV-2 RBD purification and labelling. SARS-CoV-2 RBD cDNA (T333-K529) was cloned into PHL-mMBP-10 vector (addgene #72348). MBP tagged RBD protein was produced from HEK293T cells. The cells were seeded in DMEM supplemented with 10% FBS for overnight.

When cells grew to 70-80% confluency, plasmid DNA was transfected using JetOPTIMUS J o u r n a l P r e -p r o o f transfection reagent (Polyplus). After 4 hours, medium was replaced by fresh DMEM without FBS.

Conditioned medium containing secreted mMBP-RBD was harvested three days after transfection.

Conditioned medium was adjusted to 10mM imidazole, 150 mM NaCl and 20 mM HEPES PH 8.0 (binding buffer). 1 ml pre-equilibrated NI-NTA agarose slurry (thermofisher) was added and allowed to incubate overnight at 4 °C using a rotary shaker. Beads were collected and washed with binding buffer plus extra 10 mM imidazole. Proteins were eluted with binding buffer plus 350 mM imidazole. Elution fractions were concentrated and subjected to superdex 200 size-exclusion chromatography pre-equilibrated with 100 mM NaCl, 20 mM HEPES pH 8.0 for purification. R403A mutation was made by in-fusion cloning kit (Takara Bio). Biotinylated RBD and RA RBD were generated using EZ-link Sulfo-NHS-Biotin kit (thermofisher). Cy5-labelled RBD was made with Cyanine5 NHS ester (lumiprobe).

Pull-down and western blot. Biotinylated SARS-CoV-2 S1, biotinylated SARS-CoV-2 S1S2, 

All the relevant data are within the manuscript and its Supporting information files. 

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Low incidence rate of diarrhoea in COVID-19 patients is due to integrin

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Biotinylated SARS-CoV-2-RBD binding to integrin 51 is inhibited by Cilengitide. Dashed lines indicate spliced borders where data unrelated to the text were removed (see the original full gel in Fig S4). (C) Bar graphs generated from flow cytometry data

The first row: cyanine5 labeled SARS-CoV-2-RBD wild type (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 wild type (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 wild type (WT)

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 pseudovirus particle (PP) barely infects CHO-K1 cells in the absence of MnCl2. The second row: SARS-CoV-2 pseudovirus particle (PP) massively infects CHO-K1 cells in the presence of MnCl2. The third and fourth rows: SARS-CoV-2 pseudovirus particle (PP) infection on CHO-K1 cells in the presence of MnCl2 is impaired by cilengitide. (B) Quantification of the relative infection of