Contribution of syndecans to the cellular entry of SARS-CoV-2

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is beta-coronavirus initially emerging in China and then rapidly spreading throughout the world, becoming a significant threat to human health^1-8. On 13 March, the WHO declared Europe the epicenter of the SARS-CoV-2 pandemic⁹. Caused by SARS-CoV-2 infection, the coronavirus disease 2019 (COVID-19) poses specific challenges for adequate and effective treatment to avoid the onset of severe clinical manifestations¹⁰. Currently, there is no specific antiviral therapy against SARS-CoV-2 infection¹¹. Several investigational agents have been described in observational studies or being used based on in vitro or extrapolated evidence¹² ^,13. Among the applied anti-COVID-19 therapeutics, remdesivir, an antiviral agent originally developed against Ebola infection, shows one of the most promising clinical efficacy in attenuating the severity of COVID-19¹⁴ ^,15. Given the high mortality despite the use of remdesivir, novel, and more efficient combinatory strategies should be developed to improve patient outcomes in COVID-19¹⁵.

Coronaviruses are endowed with a high tendency to spread from animals to humans, hence enabling cross- species transmission and facilitating severe outbreaks⁵ ^,16-19. The capacity of coronaviruses for cross-species transmission also supports the need to develop highly efficient, yet safe therapeutics and prophylactics to tackle current and future pandemics⁶ ^,20-22.

Development of efficient therapeutics against COVID-19 is hampered by the unknown details of SARS-CoV- 2 cellular biology, which is greatly postulated on previous studies with SARS-CoV, a coronavirus strain responsible for the first SARS outbreak in 2002-2003¹⁰. Exploring the precise molecular events driving SARS-CoV-2 infection is critical for the development of novel and specific medicines against COVID-19.

SARS-CoV-2 is transmitted by multiple means, including liquid droplets, aerosol particles and fomites²³ ^,24. Once SARS-CoV-2 enters the nasal cavity, it binds to epithelial cells and migrates down the respiratory tract while triggering a robust immune response¹⁰ ^,25. The angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for both SARS-CoV-2 and SARS-CoV¹⁰ ^,26-28. However, scientific evidence shows that endocytosis of SARS-CoV also occurs through a novel, clathrin- and caveolae-independent endocytic pathway, mediated by attachment to cell surface heparan sulfate proteoglycans (HSPGs)^29-32. Meanwhile, it has been also revealed that the S1 subunit of the SARS-CoV-2 spike protein, the subunit responsible for cellular attachment, contains the heparin-binding core motif PRRAR^33-36 (Supplementary Fig. S1). According to most recent models for SARS- CoV-2 infection, viral attachment and infection involve the formation of a complex between heparan sulfate (HS) and ACE2³⁷.

HSPGs are glycoproteins containing one or more covalently attached HS chains, a type of glycosaminoglycan (GAG)³⁸ ^,39. The evolutionarily highly conserved syndecans (SDCs) are the only transmembrane HSPG family and possess essential roles in cell interactions, adhesion, migration and signaling^40-42. SDCs share a similar structure: a one-span and highly conserved transmembrane domain (TM) and a relatively short cytoplasmic domain (CD)⁴³ ^,44. The extracellular domain (ectodomain) of SDCs is more diverse, containing HS attachment sites with glycosaminoglycan (GAG) side chains⁴⁵ ^,46. Through their highly sulfated GAG chains, SDCs interact with a myriad of extracellular ligands, hence transmitting extracellular signals intracellularly⁴⁰ ^,45-48. Besides cell signaling, SDCs also mediate the intracellular transport of ligands^45-47. During SDC-mediated endocytosis, ligand-mediated clustering of SDCs induces the redistribution of SDCs to lipid rafts and stimulation of a lipid raft-dependent, but clathrin- and caveolae- independent endocytosis of the SDC-ligand complex⁴⁶ ^,47 ^,49. As ligands internalized through SDC-mediated endocytosis can avoid lysosomal degradation, several parasites, including viruses and bacteria, utilize SDCs as shuttles to enter the cells^50-56.

Members of the SDC family show tissue-specific expression: SDC1 is expressed on epithelial and plasma cells, SDC2 on endothelial cells, SDC3 in the neurons, while SDC4 is more ubiquitous⁴³ ^,44 ^,53 ^,57. The BioGPS gene expression database (http://biogps.org) indicates a high expression of SDC4 in human lung cells⁵³ ^,58. In the lung, SDCs contribute to the balanced progression of inflammation⁵⁹. During SARS-CoV and SARS-CoV-2 infection, certain chemokines such as CXCL10 might be predictive of the subsequent clinical course⁶⁰. CXCL10 and SDC4 display a close interaction during lung inflammation, while SDC1 is essential to limit inflammation and lung injury after influenza infection^61-63. Viruses targeting SDCs in the lung could thus interfere with SDC-dependent signaling, hence influencing the inflammatory response triggered by the infection. The involvement of SDC4 in the regulation of antiviral signaling has also been reported⁵⁶. In their excellent paper, Lin et al. meticulously demonstrated the SDC4 inducing effect of a viral infection, along with SDC4’s influence on attenuating antiviral immunity⁵⁶.

Our research group has been focusing on the exploration of SDCs’ drug delivery and therapeutic potential. Our related studies with non-viral drug delivery agents, including cell-penetrating peptides (CPPs) and cationic liposomes, contributed to the understanding of SDCs’ capacity to deliver bioactive macromolecules into the cells⁴³ ^,44 ^,64. Moreover, we also revealed that SDCs contribute to the seeding and prion-like spreading of pathological protein aggregates, the major molecular culprits responsible for the onset of neurodegenerative disorders⁴⁵ ^,46. During these endeavors, we have been developing several SDC specific assays and constructs, enabling thorough analyses of SDCs’ interactions with potential ligands. Considering the scientific evidence on the potential involvement of SDCs in SARS- CoV-2 infection, we progressed to explore the interaction of both SARS-CoV-2 and its spike protein S1 subunit (spikeS1) with SDC isoforms. Our SDC specific transfectants enabled the quantitative analysis of SDCs’ contribution to the cellular entry of SARS-CoV-2, while structural SDC mutants allowed to examine the interaction of the virus and its spikeS1 with various parts of the SDC ectodomain. Besides SDC specific transfectants, our studies also included the lung-specific A549 cell line that, due to its relative resistance against ACE2-mediated SARS-CoV infection, poses a challenge to the current ACE2 paradigm⁶⁵.

Overall, our findings present SDCs as important contributors to SARS-CoV-2 cellular entry and highlight the interplay of SDCs with ACE2 during the cellular uptake of the virus. Exploration of the complex molecular interplay driving SARS-CoV-2 cellular entry also helped us to identify novel inhibitors of virus internalization.

Results

SDCs facilitate cellular uptake of the SARS-CoV-2. SDC isoforms were created in K562 cells, a human myeloid leukemia cell line lacking endogenous HSPGs except for minor amounts of endogenous betaglycan⁵⁵. K562 cells also express no detectable levels of caveolin-1, the main component of caveolae⁶⁶. Due to their limited HSPG expression and inability to form caveolae, the source of caveolar endocytosis, K562 cells offer ideal cellular models to study the contribution of SDCs to cellular uptake of ligands without the interfering effects of other HSPGs or caveolae-mediated endocytosis⁴⁵ ^,46. As HS has already been established as a major binding site for several viruses⁵², including SARS- CoV³², stable SDC transfectants created in K562 cells were standardized according to their HS content⁴⁵ ^,46. (It’s worth noting that SDC transfection did not induce statistically significant changes in ACE2 expression [Supplementary Fig. S2]). Thus, SDC transfectants with an equal amount of HS expression were selected and, along with WT K562 cells, treated with heat-inactivated SARS-CoV-2 (at 1 MOI). After 18 h of incubation, cellular uptake of the virus was detected by incubating the SARS-CoV-2-treated, fixed and permeabilized cells with Alexa Fluor 488 (AF 488) labeled antibodies specific for SARS-CoV-2’s spike glycoprotein. For imaging flow cytometry analyses, surface-attached SARS-CoV-2 was removed with trypsinization (according to the method of Nakase et al.), hence enabling the measurement of the internalized viral particles only⁶⁷. Imaging flow cytometry analyses revealed increased uptake of SARS-CoV-2 into SDC transfectants (Figs. 1a-c). Among SDCs, SDC4 increased SARS-CoV-2 uptake the most (p<0.01). (Incubating the cells with the AF 488-labeled secondary antibodies did not result in any statistically significant difference in cellular fluorescence of applied WT K562 cells and SDC transfectants, showing that no unspecific binding influenced the detected difference in fluorescence intensities of SARS-CoV-2-treated cells [Supplementary Fig. S3]). Colocalization studies revealed a significant degree of colocalization between SARS-CoV- 2 and SDCs, suggesting the same route SDCs and SARS-CoV-2 follow during cellular entry (Figs. 1d and e). Namely, the Mander’s overlap coefficients (MOC) for SDCs and SARS-CoV-2 were around 0.8, an indicator of significant colocalization (Fig. 1e). Colocalization of SARS-CoV-2 with SDCs during virus entry was also confirmed with imaging flow cytometry (Fig. 1d). The Bright Detail Similarity (BDS) score of colocalization between the fluorescent signals of the SDCs and SARS-CoV-2 also showed a high degree of colocalization (generally, a BDS score of 2 or greater represents a high degree of overlap ⁶⁸), especially in SDC3 and 4 transfectants (Fig. 1d).

Contribution of various parts of the SDC4 ectodomain to SARS-CoV-2 uptake. Studies on isoform-specific SDC cell lines demonstrated that SDCs increase cellular uptake of SARS-CoV-2. Among SDCs, SDC4 facilitated cellular uptake of SARS-CoV-2 the most. To investigate the molecular mechanisms driving SARS-CoV-2’s interaction with the SDC4 ectodomain, heat-inactivated SARS-CoV-2 (at 1 MOI) was incubated with transfectants expressing various SDC4 structural mutants (Fig. 2a). Deletion mutant Si4 possesses a truncated SDC4 extracellular domain made of only the short signal sequence (Si), while mutant CBD has a mutated ectodomain containing only the cell-binding domain (CBD) and Si, but no HS attachment (HSA) site and HS chains^43-46. We also applied the deletion mutant HSA with an ectodomain comprising the HSA site and HS chains (and also the Si), but no CBD^43-46. To readily detect their expression, all of the SDC4 mutants – along with WT SDC4 – were tagged with GFP and expressed in K562 cells^43-⁴⁶. (As shown in Supplementary S4, expression of the SDC4 mutants did not influence ACE2 expression.) Clones with an equal extent of SDC expression were selected and treated with SARS-CoV-2. After incubation, the cells were trypsinized to remove extracellularly attached viral particles⁶⁷. The cells were then fixed, permeabilized and treated with fluorescently (AF 647) labeled antibodies specific for spike glycoprotein of SARS-CoV-2. Fluorescence was then analyzed with imaging flow cytometry and confocal microscopy. Imaging flow cytometry revealed that both the HSA and CBD of SDC4 has a significant role in interacting with SARS-CoV-2 (Figs. 2b-d). Namely, deleting both the CBD and the HSA with HS chains reduced cellular uptake of SARS-CoV-2, as shown by the markedly reduced intracellular fluorescence detected on Si4 mutants (Figs. 2b-d). However, the insignificant reduction in the cellular fluorescence of SARS-CoV-2-treated CBD mutants showed that the CBD plays an important role in the interaction with the virus. Microscopic colocalization also showed marked colocalization of SARS-CoV-2 with either of the HSA or CBD mutants, with MOC values around 0.8 (Fig. 2e), demonstrating that SARS-CoV-2 could attach to both the HS chains or the CBD of SDC4. Contrary to CBD and HSA mutants, the MOC values measured on Si4 mutants showed significant (i.e. p < 0.001) reduction vs WT SDC4, thus highlighting the importance of CBD and HSA in the interactions with SARS-CoV-2. Overall, our studies with SDC4 deletion mutants revealed that besides the polyanionic HS chains, SARS-CoV-2 also interacts with the CBD of SDC4, highlighting the importance of the HS-independent parts of the SDC4 core protein. (Incubating the cells with the AF 633-labeled secondary antibodies did not induce any difference in fluorescence among the applied SDC4 transfectants and SDC4 mutants, showing that no unspecific binding influenced the difference in the detected fluorescence intensities in SARS-CoV-2-treated cells [Supplementary Fig. S5])

Cellular internalization of SARS-CoV-2 into A549 cells. After assessing the interaction of SARS-CoV-2 with the SDC4 ectodomain, we conducted studies on A549 cells, a human airway epithelia with a reportedly low level of endogenous ACE2 expression⁶⁵. As transfection of ACE2 did not render to A549 cells to support SARS-CoV replication, A549 cells offer an ideal cellular model to study novel pathways for coronavirus entry⁶⁹. Exploration of the SDC expression profile showed modest, yet detectable levels of SDCs in A549 cells (Figs. 3a and b). In terms of ACE2 expression, A549 cells express significantly less ACE2 then WT K562 cells, yet internalize heat-inactivated SARS-CoV-2 more efficiently (Figs. 3e-i), suggesting that ACE2 independent cellular modalities are also involved in the cellular uptake of SARS-CoV-2. Considering A549 cells’ richer expression of SDCs (Figs. 3c and d), along with previous findings of increased SARS-CoV-2 uptake due to SDC overexpression, we also explored the involvement of SDCs in SARS-CoV-2 uptake on A549 cells. Imaging flow cytometry and confocal microscopy analyses demonstrated the high colocalization of SDCs with SARS-CoV-2 (Figs. 4a and b). ACE2, the established receptor for SARS-CoV- 2 also showed high colocalization with the virus in uptake studies on A549 cells (Figs. 4a and b). The next steps showed that ACE2 and SDCs colocalize during SARS-CoV-2 uptake, suggesting that ACE2 and SDCs collaborate in mediating SARS-CoV-2 internalization (Figs. 4c and d). Co-IP studies also confirmed SARS-CoV-2 binding SDC4, but also ACE2 (Supplementary Fig. S6).

SDCs facilitate cellular uptake of the SARS-CoV-2 spike protein S1 subunit. To widen the understanding of SARS-CoV-2’s complex cellular entry, we also explored the cellular interactions of the SARS-CoV-2 spike protein S1 subunit (spikeS1), responsible for mediating attachment to host cells. At first, we explored the potential cellular uptake of spikeS1 into SDC transfectants created in K562 cells. Just like in the case of heat-inactivated SARS-CoV-2, SDC transfectants with an equal amount of HS expression were selected and, along with WT K562 cells, treated with spikeS1. After 18 h of incubation, cellular uptake was detected by incubating the spikeS1-treated, fixed and permeabilized cells with fluorescently (FITC) labeled antibody specific for the N-terminal His-tag of the recombinant spikeS1. For imaging flow cytometry analyses, extracellular fluorescence of surface-attached spikeS1 was removed with trypsinization (according to the method described by Nakase et al.)⁶⁷. Imaging flow cytometry analyses revealed increased uptake of spikeS1 into SDC lines (Figs. 5a-c). Among SDCs, SDC4 significantly increased the uptake of spikeS1 (p<0.01). (Incubating the cells with the fluorescently labeled anti-His tag antibodies without spikeS1 pretreatment did not induce any difference in fluorescence among the applied K562 cells and SDC transfectants, showing that no unspecific binding influenced the detected fluorescence intensities in spikeS1-treated cells [Supplementary Fig. S7]). Colocalization studies revealed a significant degree of colocalization between spikeS1 and SDC4, suggesting the same route SDC4 and spikeS1 follow during cellular entry (Figs. 5d and e). Namely, both the BDS and the MOC for SDC4 and spikeS1 were around 3 and 0.8, respectively, hence indicating significant colocalization (Figs. 5d and e).

Contribution of various parts of the SDC4 ectodomain to spikeS1 uptake. As both SARS-CoV-2 and spikeS1 demonstrated similarly increased internalization into SDC transfectants, suggesting that spikeS1 would be a key modality to facilitate SARS-CoV-2’s interactions with SDCs, we also explored the interaction of spikeS1 with SDC4 structural mutants (Fig. 2a). Transfectants of GFP-tagged Si4, CBD and HSA and SDC4 were incubated with spikeS1 for 18 h. After incubation, the cells were fixed, permeabilized and treated with AF 647-labeled secondary antibodies specific for the N-terminal His-tag of spikeS1. Fluorescence was then analyzed with imaging flow cytometry and confocal microscopy. To remove extracellularly spikeS1, the trypsinization method of Nakase et al. was applied⁶⁷. Just like in the case of SARS-CoV-2, both the HSA and CBD proved to serve a significant role in interacting with spikeS1. Namely, deleting both the CBD and the HSA (with HS chains) significantly reduced cellular uptake of spikeS1, as shown by the markedly reduced intracellular fluorescence detected on Si4 mutants (Figs. 6a-d). However, the insignificant reduction in the cellular fluorescence of spikeS1-treated CBD or HSA mutants showed that deleting either the CBD or the HS chains could not reduce the internalization of spikeS1 significantly (Fig. 6c). Thus, the CBD or the HSA site of SDC4 could compensate for the removal of either the HS chains or the CBD, respectively. In the case of the SDC4 transfectants and the HSA and CBD mutants, the Bright Detail Similarity (BDS) score of colocalization between the fluorescent signals of the SDC4 constructs and spikeS1 also showed a high degree of colocalization (Figs. 6c and d). Compared to SDC4 transfectants, the BDS score of Si4 mutants lacking HS chains and CBD were significantly reduced (p<0.05). Microscopic colocalization also showed marked colocalization of the spikeS1 with either of the HSA or CBD mutants, with MOC values around 0.8, demonstrating that spikeS1 could attach to both the HS chains or the CBD of SDC4 (Fig. 6e). Co-IP studies also confirmed the ability of the CBD or the HS chains of SDC4 to bind spikeS1 (Supplementary Fig. S8). Our studies with the SDC4 deletion mutants thus revealed that besides interacting with the polyanionic HS chains, spikeS1 also interacts with the CBD of SDC4. (Incubating the cells with the fluorescently labeled anti-His tag antibodies without spikeS1 pretreatment did not induce any difference in fluorescence among the applied SDC4 transfectants and SDC4 mutants, showing that no unspecific binding influenced the detected fluorescence intensities in spikeS1-treated cells [Supplementary Fig. S9])

Interaction of spikeS1 with SDC4 in A549 cells. Previous studies showed modest, yet detectable levels of SDC4 expression in A549 cells (Figs. 3a-d). As SDC4 demonstrated highest uptake efficacy of spikeS1, we created an SDC4 transfectant exhibiting elevated SDC4 expression (Figs. 7a and b). It’s worth noting that SDC4 overexpression did not affect the modest expression of ACE2 in A549 cells (Supplementary Fig. S10). Increased SDC4 expression, with unaffected ACE2 levels, resulted in increased cellular uptake of spikeS1 (Figs. 7c-g). Namely, overexpression of SDC4 increased spikeS1 entry from a low level of WT A549 cells by almost twofold (Fig. 7c-e). Colocalization studies revealed that spikeS1 colocalizes with SDC4 during increased spikeS1 entry (as shown by the high BDS and MOC scores obtained with imaging flow cytometry and confocal microscopy, see details in Figs. 7f and g, while co- immunoprecipitation showed increased binding of spikeS1 to SDC4 due to SDC4 overexpression [Fig. 7h]). (Incubating the cells with the fluorescently labeled anti-His tag antibodies without spikeS1 pretreatment did not induce any difference in fluorescence among the applied A549 cell line and SDC4 transfectants, showing that no unspecific binding influenced the detected fluorescence intensities in spikeS1-treated cells [Supplementary Fig. S11]).

Inhibitor studies support the complexity of SARS-CoV-2 uptake. Utilizing SDC transfectants and A549 cells, we managed to reveal an interplay of ACE2 and SDCs in mediating the cellular uptake of SARS-CoV-2. Developing efficient SARS-CoV-2 therapeutics requires the consideration of the complexity of SARS-CoV-2’s cellular interplay. This complexity was also demonstrated in our studies with representative inhibitors of various cellular pathways. The following inhibitors were applied: amiloride hydrochloride (amiloride) as the well-established inhibitor of macropinocytosis⁷⁰; DX600 as a selective ACE2 blocker⁷¹; Gö 6983 as a selective PKC antagonist^72-74; heparin as the inhibitor of electrostatic interactions of GAGs⁷⁵; a heparin-binding peptide (WQPPRARI, abbreviated as HBP) derived from fibronectin^76-78; and a small peptide (SPRRAR) derived from the heparin binding motif of SARS-CoV-2. Among them, amiloride and heparin is considered as more general inhibitors, DX600 and Gö 6983 are selective. As PKC activation is required for triggering SDC-mediated uptake, the application of Gö 6983 served the exploration of SDCs in SARS-CoV-2 internalization. The HBP (WQPPRARI) from fibronectin competes to the attachment of HS chains of SDCs, while SPRRAR, derived from spikeS1 contains a very efficient heparin-binding motif. As shown in. Figs. 8a-c, uptake studies demonstrated that while all of the applied inhibitors efficiently reduced SARS-CoV-2 uptake, SPRRAR, a peptide derived from the spikeS1 of SARS-CoV-2 emerged as the most potent one, demonstrating that molecularly detailed understanding the of SARS-CoV-2 internalization could indeed lead to the rational development of potent SARS-CoV-2 therapeutic leads. (Preincubating the cells with the inhibitors did not influence cell viability, demonstrating that the reduced SARS-CoV-2 uptake due to inhibitor treatment did not arose from disturbed cellular viability [Supplementary Fig. S12]).

Discussion

The current outbreak of SARS-CoV-2, a novel coronavirus with a yet undetermined origin, causes an unprecedented threat to modern societies⁵ ^,79 ^,80. Due to the WHO declared SARS-CoV-2 pandemic, intense research is being manifested to deliver specific medicines halting virus spread and infection¹². Pharmaceutical efforts in delivering efficient, yet safe medicines against SARS-CoV-2 are being hampered by the unknown details of SARS-CoV-2 cellular biology¹⁰ ^,81. While most studies on SARS-CoV-2 emphasize the difference between SARS-CoV-2 and SARS-CoV, several findings on SARS-CoV are also being regarded as applicable for SARS-CoV-2⁸². One such fundamental finding on SARS-CoV that has been widely accepted for SARS-CoV-2 is ACE2 serving as the primary, yet sole cellular entry receptor, facilitating virus internalization after transmembrane protease serine type 2 (TMPRSS2) mediated cleavage of the spike protein²⁷ ^,28 ^,83 ^,84. However, the failure of highly specific ACE2 pharmaceuticals to stop SARS-CoV-2 infection and related disease, COVID-19, clearly highlights the complexity of SARS-CoV-2 internalization⁸⁵. Emerging evidence show the collaboration of ACE2 and HSPGs during SARS-CoV-2 uptake³⁷.

It has been widely accepted that cell surface HSPGs provide efficient cellular entry for a whole range of pathogens^50-52. SDCs are the only transmembrane family of HSPGs³⁸ ^,39. Due to their versatile and polyanionic HS chains, SDCs bind a myriad of extracellular ligands, including several viruses. Due to their evolutionary conserved intracellular PDZ domains, SDCs also interact with a whole range of intracellular signaling molecules, thus providing a transmembrane link between extracellular HS-mediated processes and intracellular signaling cascades⁴⁰ ^,42 ^,47 ^,86. Attachment to HS chains of HSPGs and interaction with PDZ domains has been already explored for SARS-CoV, but not yet investigated for SARS-CoV-2²⁹ ^,30 ^,32. However, recent studies explored the interaction of the SARS-CoV-2 with heparin⁸⁷. According to these studies, the conformation change induced by the attachment of spikeS1 to heparin is required for efficient SARS-CoV-2 entry into the cells⁸⁷. Another very recent findings of also showed heparin effectively blocking SARS-CoV-2 invasion into Vero cells⁸⁸. It has to be noted that the primary sequence of spikeS1 (namely Pro681 - Arg685) contains the heparin-binding core motif PRRAR^33-35 (Supplementary Fig. S1). According to current reports, this heparin-binding motif may facilitate SARS-CoV-2 host cell entry³⁶.

Considering scientific evidence supporting the involvement of HSPGs in SARS-CoV-2 infection, we set up a study exploring the contribution of SDCs to the cellular uptake of SARS-CoV-2 and spikeS1, the subunit responsible for cell attachment of the virus. Up-till-now, this is the first study exploring the specific involvement of the whole SDC family in the cellular uptake of SARS-CoV-2. According to our results, the overexpression of SDCs, including SDC4, the isoform most abundant in the lung, significantly increases cellular uptake of SARS-CoV-2. Thus, SDCs have a key involvement in facilitating the cellular entry of SARS-CoV-2, while the spikeS1 plays a major role in the interactions with SDCs. Binding of SARS-CoV-2 to the SDC4 ectodomain is not exclusively driven by the HS chains, but also influenced by other parts of the SDC ectodomain, including SDC4’s CBD mediating cell-to-cell attachment. In our studies with SDC4 deletion mutants, the CBD mutants lacking HS chains exhibited internalization characteristics comparable to WT SDC4 transfectants, thus supporting the involvement of the SDC core protein in the interaction with the virus. SDC4’s CBD also played a dominant role in spikeS1-SDC4 interactions, hence emphasizing the need to go beyond standard HS-virus interactions in understanding the molecular interplay between SARS-CoV-2 and SDCs. In our studies, heparin, a polyanionic agent effectively inhibiting the attachment of several viruses to polyanionic HS on cell surface proteoglycans⁵², even at very high doses, did not emerged as the most potent blocker of virus uptake, while SPRRAR, a heparin-binding peptide derived from spikeS1 showed superior efficacy to block virus uptake.

It’s worth noting that the sulfation pattern of proteoglycans, contributing significantly to the structural diversity of proteoglycan HS chains, defines the binding of ligands³⁸ ^,45 ^,48 ^,89-91. Although most cells express more than one HSPG at their cell surface, several lines of evidence indicate that HS chains attached to different core proteins on the same cell surface have the same sulfation patterns³⁹ ^,92-94. The detected difference in the cellular uptake of SARS- CoV-2 (and also spikeS1) between various SDC transfectants expressing similar level of HS also indicates that interaction of SARS-CoV-2 with SDCs is also influenced by HS independent parts of the SDC core protein. Investigating the cellular uptake of SARS-CoV-2 in stable SDC transfectants with an equal amount of HS expression was therefore critical in understanding the influence of the core protein (i.e. the non-GAG parts) on interactions with the virus. As the fine structure and ligand binding of SDCs’ HS chains show cell type-specific differences^94-96, the involvement of different cell lines in the analyses of SDCs interaction with SARS-CoV-2s was also crucial in the exploration of factors influencing the virus’ cellular interactions.

Exploration of SARS-CoV-2 internalization in A549 cells also revealed the interplay of SDCs and ACE2, where both modalities (i.e. ACE2 and SDCs) interact with SARS-CoV-2 to facilitate its cellular entry. Therefore, our results do not abolish the ACE2 hypothesis, but depicts a molecularly more diverse scenario involving the collaboration of SDCs and ACE2 in mediating SARS-CoV-2 internalization. The finding that SDC overexpression, with unaffected ACE2 expression levels, results in increased SARS-CoV-2 cellular entry highlights the importance of SDCs in the complex molecular interplay of SARS-CoV-2 internalization.

SDCs have been already regarded as a favorite binding site for several viruses and bacteria⁵¹ ^,52. In line with these findings, our results suggest that SDCs play a key role in facilitating SARS-CoV-2’s cellular entry. Although most abundant in the lungs, the widespread distribution of SDC4 and other SDCs in the human body can explain the ability of SARS-CoV-2 the spread to various organs. Also, the increased uptake of SARS-CoV-2 into cells expressing other SDCs, including neuronal SDC3 might imply the virus’s ability to infect the cells of the CNS. Considering the ability of HIV-1 to exploit SDCs to enter the brain⁹⁷, our finding on the increased uptake of SARS-CoV-2 into SDC3 transfectants might explain the molecular determinants of SARS-CoV-2’s potential CNS entry⁹⁸. Moreover, altered expression of SDCs is associated with cardiovascular disorders, diabetes, obesity and Alzheimer’s disease, just some of the most common comorbidities correlating with poorer clinical outcomes in COVID-19^99-107.

Overall, our results obtained in highly specific SDC assays presents SDCs as important mediators of SARS- CoV-2 cellular entry and highlight SDCs, including the lung abundant SDC4, as a novel therapeutic target against SARS-CoV-2 infection. Our findings are in line with recent reports suggesting the potential role of HSPGs in the cellular entry of SARS-CoV-2 and explore a molecularly more detailed interplay of ACE2 and SDCs in facilitating SARS-CoV-2 uptake. Inhibitor studies show the efficacy of inhibitors of both pathways (i.e. ACE2 or SDC-dependent) in blocking SARS-CoV-2 internalization, while highlighting the efficacy of peptide (SPRRAR) derived from the heparin-binding motif of SARS-CoV-2. Pharmaceutical development of efficient medicines against SARS-CoV-2 infection therefore should consider a more complex interplay of potential receptors in order to develop efficient mono- or combinatorial therapeutic strategies against SARS-CoV-2 infection.

Materials And Methods

Heat-inactivated SARS-CoV-2, recombinant proteins and peptides. Heat-inactivated SARS-CoV-2 (strain: 2019- nCoV/USA-WA1/2020) was purchased from ATCC® (cat. no. ATCC® VR-1986HK™), the N-terminally His-tagged recombinant SARS-CoV-2 Spike Protein S1 Subunit (region Val16 - Gln690) from RayBiotech, recombinant human syndecan-4 from R&D Systems (cat. no. 2918-SD) and recombinant human ACE2 from Abcam (ab151852). Peptide inhibitors utilized for the studies were purchased from either BACHEM (DX600) or Genscript (SPRRAR and WQPPRARI).

SDC constructs, cell culture and transfection. Full-length SDC1-4 and SDC4 deletion mutants and transfectants, established in K562 cells (ATCC® CCL-243™), were created as described previously ⁴³ ^,45 ^,46. No His-tags were applied for the SDC constructs. SDC4 overexpressing A549 clones, along with WT A549 cells (ATCC® CCL-185™) were then grown in Advanced MEM medium (Thermo Fischer Scientific) supplemented with 10% FCS (Gibco) at 37 °C in a humified 5% CO2 containing air environment.

Flow cytometry analysis of HS, ACE2 and SDC expression. HS and SDC expression of applied cell lines (WT K562 and SDC transfectants) were measured with flow cytometry by using anti-human HS antibody (10E4 epitope [Amsbio]), AF 488-labeled anti-mouse IgM and APC-labeled antibody, as described previously⁴⁵ ^,46. SDC transfectants with almost equal amount of HS expression were selected for further uptake studies. ACE2 expression of WT K562 and A549 cells, along with SDC transfectants was measured with human ACE-2 AF 647-conjugated antibody (R&D Systems, cat. no. FAB9332R) according to the manufacturer’s protocol.

Flow cytometry analysis of SARS-CoV-2 and spikeS1 uptake. WT K562 and A549 cells, SDC transfectants, along with SDC4 structural mutants were utilized to quantify internalization of spikeS1. Briefly, 6 x 10⁵ cells/ml in DMEM/F12 medium were incubated with either SARS-CoV-2 (at 1 MOI) or spikeS1 (at a concentration of 2 μg/ml) for 18 h at 37 ºC. After 18 h of incubation, the cells were trypsinized (with the method described by Nakase et al ⁶⁷) to remove the extracellularly attached virus particles or spikeS1 from the cell surface. Then the cells were washed, fixed, permeabilized and blocked with the appropriate serum for 1h at room temperature. In case of spikeS1, the cells were then treated with fluorescently (either FITC or in case of SDC4 mutants, AF 647) labeled anti-His-tag antibodies (rabbit poly- and monoclonal, Abcam, cat. no. ab1206 and ab237337) for 1 h. In case of SARS-CoV-2, the cells were then treated with mouse monoclonal (1A9) to SARS spike glycoprotein (Abcam, cat.no: 273433), followed by treatment with either AF 488- (SDC transfectants and A549 cells) or AF 633-labeled (SDC mutants) goat anti-mouse IgG (both Invitrogen, cat.no.: A-11001 and A-21052, respectively). For the colocalization studies, SDCs made visible by incubating the cells with either APC-labeled SDC or AF-labeled antibodies (1:100), while ACE2 was detected by using either AF 647-labeled human ACE2 antibody (R&D systems, cat.no: FAB9332R) or rabbit polyclonal Ace2 antibody (Abcam, cat.no. ab272690) and fluorescently (FITC) labeled anti-rabbit antibody (Sigma). The samples were then rinsed three times with PBS containing 1% BSA and 0.1% Triton X-100, and progressed towards flow cytometry. Cellular uptake was then measured by flow cytometry using an AMNIS® FlowSight imaging flow cytometer (AMNIS Corporation). A minimum of 10,000 events per sample was analyzed. Appropriate gating in a forward-scatter-against- side-scatter plot was utilized to exclude cellular debris and aggregates. Fluorescence analysis was conducted with the IDEAS™ analysis software. To examine the influence of the fluorescently labeled anti-His-tag antibodies or secondary IgGs, some of the cells were also treated with either anti-His-tag or secondary antibodies without preincubation with spikeS1 or SARS-CoV-2 and cellular fluorescence was measured with imaging flow cytometry.

Inhibitor studies. To reveal the involvement of various endocytosis pathways, A549 cells were preincubated with the following inhibitors 30 min before SARS-CoV-2 admission: amiloride hydrochloride (at a concentration of 100 µM) as the well-established inhibitor of micropinocytosis (Sigma); DX600 as a selective ACE2 blocker (10 µM; BACHEM); Gö 6983 as a selective PKC antagonist (10 µM; Sigma); heparin as the inhibitor of electrostatic interactions of GAGs (200 ug/ml; Sigma); a heparin-binding peptide (WQPPRARI, 100 µM; Genscript) derived from fibronectin; SPRRAR derived from spikeS1 (100 µM; Genscript). After incubation with these inhibitors, the cells were treated with SARS-CoV-2 and processed for the flow cytometric analyses as described above.

Cell viability measurements. The effect of the applied inhibitors were assessed by incubating A549 cells were with either of the following inhibitors: amiloride hydrochloride (amiloride, at a concentration of 100 µM), DX600 (10 µM); Gö 6983 (10 µM, Sigma); heparin (200 ug/ml); a heparin-binding peptide (WQPPRARI) and SPRRAR (100 µM). Eighteen hours after incubation with these inhibitors, viability of inhibitor-treated WT A549 cells, along with untreated controls, were assessed with the EZ4U Cell Proliferation Assay (Biomedica Gmbh, cat. no. BI-5000) according to the instructions of the manufacturer. Absorbance was measured with a BioTek Cytation 3 multimode microplate-reader.

Microscopic visualization of uptake. The internalization of SARS-CoV-2 or spikeS1 was visualized by confocal microscopy. WT A549 and WT K562 cells, along with SDC transfectants and SDC4 mutants were grown on poly-D- lysine-coated glass-bottom 35-mm culture dishes (MatTek Corp.). After 24 h, the cells were preincubated in DMEM/F12 medium at 37 °C for 30 min before incubation with the spikeS1 at a concentration of 2 μg/ml. After incubation with either SARS-CoV-2 (1MOI) or spikeS1, the cells were rinsed two times with ice-cold PBS, fixed in 4% paraformaldehyde (Sigma), the cell membranes were permeabilized (0,1% Triton X-100), and the cells were blocked with the appropriate serum for 1h at room temperature, followed by the specific antibody treatments as described for the flow cytometry analyses. The samples were then rinsed three times with PBS containing 0.1% Triton X-100, then stained with DAPI (1:5000) for 5 min, washed three times with PBS and embedded in Fluoromount G (SouthernBiotech)⁴⁵ ^,46. The distribution of fluorescence was then analyzed on a Leica DMi8 microscope equipped with Aurox Clarity Laser Free Confocal Unit. Sections presented were taken approximately at the mid-height level of the cells. Photomultiplier gain and illumination power were identical within each experiment. The Aurox Visionarysoftware was used for image acquisition by confocal microscopy. For colocalization analyses, the images were analyzed in the ImageJ software (NIH, Bethesda, Maryland, US) with the plug-in JACoP as described by Wesén et al.¹⁰⁸ 12 images (18 images per sample, experiments performed in triplicate) were analyzed, and the data is presented as mean ± SEM.

Co-immunoprecipitation experiments. SDC4 transfectants or WT K562 or A549 cells, incubated with or without SARS-CoV2 or spikeS1 (as mentioned above), were processed for co-immunoprecipitation experiments as described previously⁴⁵ ^,46. Briefly, after incubation, the cells were washed twice with ice-cold PBS and treated with cold Pierce IP lysis buffer. Then the cells were scraped off to clean Eppendorf tubes, put on a low-speed rotating shaker for 15 min and centrifuged at 14,000 g for 15 min at 4°C. The supernatant was then transferred to new tubes and combined with 5 µg of either of mouse monoclonal (1A9) SARS-Cov2 spike glycoprotein S1 antibody (Abcam, cat. no. ab273433) or, in the case of the GFP-tagged SDC4 mutants, GFP antibody (Abcam, cat. no. ab6662). The antigen sample/SDC/SARS-CoV-2 or GFP antibody mixture was then incubated for overnight at 4°C with mixing. The antigen sample/antibody mixture was then added to a 1.5 ml microcentrifuge tube containing pre-washed Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific) and after incubation at room temperature for 1 hour with mixing, the beads were then collected with a MagJET Separation Rack magnetic stand (Thermo Fisher Scientific) and supernatants were discarded. To eluate the antigen, 100µl of SDS-PAGE reducing sample buffer were then added to the tubes and samples were heated at 96°C for 10 minutes in 1% SDS and the samples were proceeded to SDS-PAGE⁴⁵ ^,46. The samples were then immunoblotted onto PVDF membranes and the proteins were detected with specific antibodies as described above. Image acquisition was conducted with Uvitec’s Alliance Q9 Advanced Imaging Platform.

Statistical analysis. Results are expressed as means ± standard error of the mean (SEM). Differences between experimental groups were evaluated by using one-way analysis of variance (ANOVA). Values of p<0.05 were accepted as significant⁴⁵ ^,46. During imaging flow cytometry, the IDEAS™ feature Bright Detail Similarity (BDS) was used to measure colocalization between two signals⁶⁸. A BDS score of 2 or greater represents a high degree of overlap. For microscopic colocalization analyses, the Mander’s overlap coefficient (MOC) was calculated as described by Wesen et al.¹⁰⁸

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Contribution of syndecans to the cellular entry of SARS-CoV-2

Abstract

Introduction

Results

Discussion

Materials And Methods

Declarations

Acknowledgements

Author contributions statement

Additional information

References