key: cord-0933265-jhgn25y7
authors: Lu, Qiao; Liu, Jia; Zhao, Shuai; Gomez Castro, Maria Florencia; Laurent-Rolle, Maudry; Dong, Jianbo; Ran, Xiaojuan; Damani-Yokota, Payal; Tang, Hongzhen; Karakousi, Triantafyllia; Son, Juhee; Kaczmarek, Maria E.; Zhang, Ze; Yeung, Stephen T.; McCune, Broc T.; Chen, Rita E.; Tang, Fei; Ren, Xianwen; Chen, Xufeng; Hsu, Jack C.C.; Teplova, Marianna; Huang, Betty; Deng, Haijing; Long, Zhilin; Mudianto, Tenny; Jin, Shumin; Lin, Peng; Du, Jasper; Zang, Ruochen; Su, Tina Tianjiao; Herrera, Alberto; Zhou, Ming; Yan, Renhong; Cui, Jia; Zhu, James; Zhou, Qiang; Wang, Tao; Ma, Jianzhu; Koralov, Sergei B.; Zhang, Zemin; Aifantis, Iannis; Segal, Leopoldo N.; Diamond, Michael S.; Khanna, Kamal M.; Stapleford, Kenneth A.; Cresswell, Peter; Liu, Yue; Ding, Siyuan; Xie, Qi; Wang, Jun
title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2
date: 2021-05-09
journal: Immunity
DOI: 10.1016/j.immuni.2021.05.006
sha: e6fd3d3c23bb732eebe1262ce30beef23b94a1ed
doc_id: 933265
cord_uid: jhgn25y7

Despite mounting evidence for SARS-CoV-2 engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, ACE2. Here, using a myeloid-cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA-sequencing analysis of pulmonary cells from COVID-19 patients indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptors-mediated proinflammatory responses. Our findings suggest SARS-CoV-2-myeloid receptor interactions promote immune hyper-activation, which represents potential targets for COVID-19 therapy.

While proteases such as TMPRSS2 and cathepsins are utilized by both SARS-CoV-2 and SARS-CoV 90 (Hoffmann et al., 2020b) , S cleavage by furin is unique to SARS-CoV-2 due to the presence of a 91 polybasic (RRAR) site at the S1-S2 junction (Xia et al., 2020) . In addition to ACE2, a number of other host 92 molecules reportedly support SARS-CoV-2 binding to cells and act as entry factors, including CD147 93 (Wang et 

To identify myeloid-cell associated receptors for SARS-CoV-2, we built a myeloid cell receptor array that 134 comprised a human cDNA library of ~300 host membrane proteins expressed preferentially in myeloid 135 cells, (i.e., monocyte, macrophage, and dendritic cell (DC) populations) ( Table S1) . We applied a receptor 136 over-expression and detection system that modified from our previous report (Wang et al., 2019) , and 137 tested the binding of human immunoglobulin Fc-tagged SARS-CoV-2 S, S1, and RBD recombinant 138 proteins to HEK293T cells transfected with individual cDNA (Figure 1A ). ACE2 and Fc receptors served 139 as the positive controls. We identified six proteins that interacted with SARS-CoV-2 S protein or its 140 subunits, including five C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety 141 family member 2 (TTYH2) ( Figure 1B) . All six candidate genes were expressed at comparable levels to 142 ACE2 ( Figure S1A) . In contrast to ACE2, which strongly bound to RBD-Fc, S-Fc, and S1-Fc, C-type 143 lectins primarily interacted with S-Fc and S1-Fc, with weak or no binding to RBD-Fc ( Figure 1C) . These 144 data suggest that C-type lectins likely interact with SARS-CoV-2 S protein via non-RBD epitopes (e.g., 145 the N-terminal domain (NTD) and the C-terminal domain (CTD)) within the S1 region. TTYH2 interacted 146 weakly with RBD-Fc but not with S-Fc or S1-Fc ( Figure 1C ). To map the interaction domain, we generated recombinant NTD-Fc, RBD-Fc, CTD-Fc, and S2-Fc   149   proteins. Consistent with the screening results, DC-SIGN, L-SIGN, and LSECtin predominantly   150 associated with CTD-Fc and, to a lesser extent, with NTD-Fc. ASGR1 and CLEC10A mainly interacted 151 with NTD-Fc, whereas ACE2 and TTYH2 primarily bound to RBD-Fc ( Figure 1D and Figure S1B ). We 152 next used an HIV-based lentivirus backbone pseudotyped with SARS-CoV-2 S protein (Crawford et al., 153 2020) to validate these interactions in the context of a viral particle. SARS-CoV-2 pseudovirus bound 154 strongly to HEK293T cells overexpressing ACE2, DC-SIGN, L-SIGN, or ASGR1, and exhibited weaker 155 but significant binding to cells expressing LSECtin, CLEC10A, or TTYH2 ( Figure 1E and Figure S1C ).

Finally, we assessed the direct protein-protein binding of the S1 subunit from SARS-CoV-2 to the 157 recombinant receptor ectodomains by an enzyme-linked immunosorbent assay (ELISA), except for 158 TTYH2, which has multiple extracellular domains. Analogous to the results with the pseudovirus, there 159 was a clear, albeit weaker, interaction between S1 recombinant protein and Fc-tagged ectodomain of 160 these C-type lectins when compared to ACE2-Fc ( Figure S1D ). Collectively, these data indicate that DC-161 SIGN, L-SIGN, LSECtin, ASGR1, CLEC10A, and TTYH2 can directly associate with SARS-CoV-2 S 162 protein and mediate pseudovirus attachment to cells. 163 164 C-type lectins bind to SARS-CoV-2 S protein via residues distinct from ACE2 and TTYH2 165 Next, we characterized the interaction interfaces of SARS-CoV-2 S protein with these myeloid receptors 166 in comparison with ACE2. Pre-incubation of His-tagged soluble ectodomain of ACE2 recombinant protein 167 (ACE2-His) with S-Fc or RBD-Fc completely blocked their binding to ACE2 over-expressing cells ( Figure   168 2A and Figure S2A ). In addition, soluble ACE2-His blocked the binding of RBD-Fc to TTYH2 ( Figure S2A ). Based on the RBD-ACE2 structure (Shang et al., 2020) , we generated an S1-Fc 175 mutant bearing three point mutations in the RBD (T500A/N501A/G502E), resulting in the loss of two 176 hydrogen bonds between RBD and ACE2 interaction ( Figure 2B ). This S1 protein mutant retained the 177 binding capacity to all five C-type lectins but not to ACE2 (Figure 2C and Figure S2B ), thus confirming 178 that the C-type lectin interface is distinct from that of ACE2. 187 Therefore, we tested whether S protein glycosylation affects interaction with the myeloid cell receptors 188 identified in our screen. We found that the addition of mannan (a mannose polymer) competitively 189 blocked the binding of S protein to DC-SIGN and L-SIGN but not to ACE2, LSECtin, ASGR1, CLEC10A, 190 or TTYH2 (Figure 2D) . Similarly, endoglycosidase H (Endo H), which removes high-mannose 191 oligosaccharides on N-linked glycans (Trimble and Maley, 1984) , only reduced S protein binding to DC-192 SIGN, L-SIGN, and to a weaker extent, to LSECtin ( Figure S2C ). In contrast, treatment of SARS-CoV-2 S 193 protein with peptide:N-glycosidase F (PNGase F), which removes all types of N-linked glycans, 194 significantly reduced S-Fc protein binding to all myeloid cell receptors ( Figure S2C ). PNGase F 195 treatment also reduced S-Fc binding to ACE2 (Figure S2C) , suggesting the involvement of glycans in the 196 interaction with ACE2 and the immune receptors.

To pinpoint the specific glycosylated residues important for receptor binding, we next performed a 199 mutagenesis screen by disrupting the individual 13 N-glycosylation sites and 2 O-glycosylation sites 200 within the S1 subunit. We observed functional mutation sites that could be categorized to an inhibitory 201 group, the single mutation of which led to significant loss of S1-Fc binding to the host receptors (N343 to 202 ACE2, N603 to almost all C-type lectins, and the majority of the mutants to both ASGR1 and CLEC10A).

We also identified an enhancing group, the single mutation of these sites led to significant increase of S1-204 Fc interactions (N74, N149, N282, N603, N616, and N657 to ACE2, N165 to DC-SIGN/L-SIGN/LSECtin, 205 N234, N343 and N657 to DC-SIGN, and N122 to LSECtin) ( Figure 2E -H and Figure S2D ). While the 206 N282Q mutation showed the most enhanced (3-fold) binding to ACE2 among all the glycosylation 207 mutations, we observed that the N165Q mutation greatly enhanced the binding to DC-SIGN, L-SIGN, and 208 LSECtin, but not ACE2 ( Figure 2F and Figure S2D ). Of note, the N343 mutation in the RBD ( Figure 2E ) 209 completely abolished ACE2 binding ( Figure 2G and Figure S2D ), which is consistent with a recent report 210 that the N343 mutation led to reduced infectivity . The N343 mutation also reduced the 211 binding of S1-Fc to ASGR1 and CLEC10A. However, it had no effect on L-SIGN and LSECtin binding but 212 enhanced the S1-Fc binding to DC-SIGN. Another N603 mutant in the CTD ( Figure 2E ) showed 213 decreased binding to most of the C-type lectins, which contrasted with its effects on ACE2 binding 214 ( Figure 2G and Figure S2D ). These data suggest that N603 may be a key glycosylation site supporting 215 SARS-CoV-2 spike binding to C-type lectins but interfering with its ACE2 interaction.

While N-glycosylation greatly affect S1 interaction with ACE2 and myeloid receptors, O-glycosylation may 218 not be required for the binding of S1-Fc to most of the host receptors, except for CLEC10A ( Figure S2D ).

In parallel, we analyzed over 5,000 genomes of SARS-CoV-2 natural variants documented as of May 

HIV-GFP virus pseudotyped with SARS-CoV-2 S protein were generated by co-transfecting pcDNA3

CD-511B-1) (encoding the GFP reporter) at 1:2:3 762 mass ratio into Expi293F cells using PEI (Polysciences) at 3:1 (PEI:DNA) mass ratio. Four days post-763 transfection, the supernatant was harvested and filtered through a 0.45 µm sterile syringe filter, followed 764 by concentration using Amicon Ultra-15 Centrifugal Filter Unit (Millipore, UFC910024)

SARS-CoV-2 virus experiments

2020) (MOI=0.1 -10) with 774 or without centrifugation at 1200 x g for 2 hr at RT. Media were then changed and cells were incubated at 775 37°C until analysis

Total RNA was extracted from cells using Trizol or lysis buffer 778 from commercially available RNA extraction Kit (Thermo Fisher Scientific or Qiagen) at 24 hr after 779 incubation at 37°C. Reverse transcription was performed with High-Capacity RT kit

RT-PCR was performed using the CFX96 Touch™ Real-Time PCR Detection System

Applied Biosystems) with a 20 µL reaction, composed of 50 ng of 782 cDNA, 10 µL Power SYBR Green master mix (Applied Biosystems) for IL1A, IL1B, IL6, IL8, IL10

master mix (Applied Biosystems) for SARS-CoV-2 N protein, and 200 nM of each forward and reverse 785 primer. All SYBR Green primers and Taqman probes used

Total RNA was extracted from human PBMC-derived myeloid cells in culture with 788 conditioned media (mock) or a clinical isolate of SARS-CoV-2 using Trizol. RNA sample quality was 789 examined by the NanoDrop spectrophotometer (Thermo Fisher) and Bioanalyzer 2100 (Agilent)

Langmead and Salzberg, 2012) to map clean reads to reference gene and using HISAT2 to 792 reference genome with the following parameters: --phred64 --sensitive -I 1 -X 1000. Reads were counted 793 using Subread and differential gene expression analysis was performed using DESeq2BGI

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Binding of DC-SIGN to the 1074 hemagglutinin of influenza A viruses supports virus replication in DC-SIGN expressing cells

A Multibasic Cleavage Site in the 1077

Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

SARS-CoV-2 Cell Entry Depends on 1081 ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

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Clec10a regulates mite-1091 induced dermatitis

Human Dendritic Cell DC-SIGN and TLR-2 Mediate Complementary Immune 1094 Regulatory Activities in Response to Lactobacillus rhamnosus JB-1

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the 1097 ACE2 receptor

Fast gapped-read alignment with Bowtie 2

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

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BiNGO: a Cytoscape plugin to assess 1118 overrepresentation of gene ontology categories in biological networks

DC-SIGN and DC-SIGNR interact with the 1122 glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome 1123 coronavirus

Yeast surface display platform for rapid 1126 discovery of conformationally selective nanobodies

Therapeutic blockade of granulocyte macrophage colony-1129 stimulating factor in COVID-19-associated hyperinflammation: challenges and opportunities

Pathological inflammation in patients with COVID-19: a key 1132 role for monocytes and macrophages

Inhibition of HIV-1 replication by a 1135 nonnucleoside reverse transcriptase inhibitor

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Human Sialome 1139 and Coronavirus Disease-2019 (COVID-19) Pandemic: An Understated Correlation? Front 1140 Immunol 11

Inhalable Nanobody (PiN-21) prevents and 1143 treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses

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DC-SIGN (CD209) 1185 mediates dengue virus infection of human dendritic cells

Optimizing hydrolysis of N-linked high-mannose 1187 oligosaccharides by endo-beta-N-acetylglucosaminidase H

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Escape from neutralizing 1204 antibodies by SARS-CoV-2 spike protein variants

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CLEC5A is critical for dengue virus-induced inflammasome activation in human 1218 macrophages

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to 1221 its receptor ACE2

The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion 1224 in the presence or absence of trypsin

An Infectious cDNA Clone of SARS-CoV-2

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Flow Cytometric Analysis of Myeloid 1233 Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues

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Asialoglycoprotein receptor interacts with the preS1 domain of hepatitis B 1243 virus in vivo and in vitro

The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for

Inflammatory Response Induced by Ebola Virus Glycoprotein

Active replication of Middle East respiratory syndrome coronavirus and 1249 aberrant induction of inflammatory cytokines and chemokines in human macrophages: 1250 implications for pathogenesis

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