key: cord-0926324-0ew4l5x0
authors: Soh, Wai Tuck; Liu, Yafei; Nakayama, Emi E.; Ono, Chikako; Torii, Shiho; Nakagami, Hironori; Matsuura, Yoshiharu; Shioda, Tatsuo; Arase, Hisashi
title: The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN
date: 2020-11-05
journal: bioRxiv
DOI: 10.1101/2020.11.05.369264
sha: 8c13da0040d604d8c107ef40775f89b921818ad7
doc_id: 926324
cord_uid: 0ew4l5x0

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.

to L-SIGN and DC- SIGN. 115 There are seven and eight N-glycosylation sites located on the NTD of SCoV and SCoV2, 116 respectively (Fig. 3a) . The protein sequence alignment of both NTDs revealed that there are two 117 unique glycosylation sites, i.e., N74 and N149, on the SCoV2-NTD (Fig. 3a) . Interestingly, both 118 glycosylation sites are in the most variable region within the NTD. To identify which glycosylation 119 sites interact with DC-SIGN, we substituted SCoV2-NTD glycosylation site Asn (N) with Gln (Q) 120 and analyzed the binding to DC-SIGN (Fig. 3b, c) . The mutation at N149Q diminished the NTD 121 binding to DC-SIGN, while N74Q and N282Q mutation resulted in a substantial reduction of the 122 NTD binding to DC-SIGN. These data suggest that unique glycans on N149 of NTD are 123 responsible for the interaction with SIGNs. We then sought to establish the function of endogenously expressed L-SIGN or DC-SIGN. Since 150 L-SIGN has been reported to be expressed in type II alveolar cells, L-SIGN is likely involved in 151 the SARS-CoV-2 infection of lungs. However, we could not find any cell lines expressing 152 endogenous L-SIGN. On the other hand, DC-SIGN is expressed on monocyte-derived DCs 153 (moDCs). For that reason, we isolated CD14 + cells from human PBMC and differentiated them 154 into DCs in the presence of cytokines, IL-4 and GM-CSF. The moDCs expressed DC-SIGN, low 155 CD14, high MHC-II, and CD74 (Fig. 5a ). As observed with DC-SIGN transfectants (Fig. 2) , 156

SCoV2-NTD-Fc fusion protein bound to moDCs (Fig. 5b) . Anti-DC-SIGN monoclonal antibodies 157 SIGN on moDCs may be infectious to other cells. We incubated moDCs with SCoV2-PV followed 166 by extensive washing. Thereafter, moDCs incubated with SCoV2-PV were cocultured with Vero 167

by anti-DC-SIGN antibodies or mannan ( patient-derived anti-NTD mAbs 10 and C144 is an anti-RBD mAb 18 (Suppl. Fig. 3 ). Both 4A8 and 176 C144 have been reported to neutralize SCoV2 infection. Surprisingly, anti-RBD C144 mAbs as 177 well as anti-NTD 4A8 blocked SCoV2-PV infection of HEK-L-SIGN and HEK-DC-SIGN. In 178 contrast, the 4A2 mAb did not block infection (Fig. 6a, b) . These data indicate that RBD as well SCoV2 is more widespread than its closest human coronavirus, SCoV 3 , and therapeutic 190

interventions are yet to be identified. ACE2 is the primary receptor that interacts with the SCoV2 191 spike glycoprotein (i.e., RBD) and consequently facilitates viral entry 3 . However L-SIGN transfectants were infected with recombinant SCoV2 with the NanoBiT luciferase gene. 586

The luciferase activity was measured 24 h later. Asterisks indicate statistical significance derived 587 from unpaired T-test; *P = 0.001; **P = 0.0006. c, Cell-cell fusion assay of SCoV2 spike 588 transfectants and DC-or L-SIGN transfectants. The effector cells expressing spike and T7 589 polymerase were cocultured with target cells expressing DC-SIGN, L-SIGN, mock, and T7 590 promoter-driven luciferase. Luciferase activities were measured after 24 h. Asterisks indicate 591 statistical significance derived from unpaired T-test ***P<0.0001. d, Cell-cell fusion assay of 592

SCoV2 spike transfectants (red) and DC-SIGN transfectants (green). Representative images are 593

shown. Scale bar represents 50 µm in length. Data are representative of three independent 594 experiments. 595

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Cell surface binding analyses of the SCoV2 spike bound to DC-SIGN and L-SIGN 555 using flow cytometry. a, Cells transfected with ACE2, L-SIGN, DC-SIGN, and CD147 (red line) 556 or mock (shaded gray) were stained with SCoV2-NTD-Fc and RBD-Fc fusion protein

The transfectants were preincubated with mannan (light blue 560 line) or anti-CD209 antibody (dark blue), followed by the staining with NTD-Fc fusion protein. c, 561 Staining of DC-and L-SIGN transfectants (red line) or mock (shaded gray) with SCoV2-NTD-Fc 562 and SCoV-NTD-Fc fusion proteins (10 µg/mL). d, Cells transfected with flag-tagged spike 563 proteins of SCoV2, SCoV, human coronavirus OC43, or HKU1 (red line) or mock (shaded gray) 564 were stained with DC-, L-SIGN-Fc fusion proteins or anti-Flag-tag antibody

5 moDCs facilitate SCoV2 infection in trans through DC-SIGN. a, Phenotype of the 598

CD14 + cells from PBMC cultured in the presence (red line) or absence (purple line) of cytokines 599 (IL-4 and GM-CSF, 500 IU/mL each) for three days. b, Binding of SCoV2-NTD-Fc and RBD-Fc 600 of SCoV2 spike to moDCs. The amount of Fc-fusion proteins bound to moDCs were presented as 601 mean fluorescence intensity (MFI)

Blocking of SCoV2-NTD-Fc binding to moDCs by two specific 603 anti-DC-SIGN mAbs, isotype antibody, and mannan. The percentage of binding is calculated 604 based on the MFI value of non-blocking control. Asterisks indicate statistical significance derived 605 from unpaired T-test

0001. d, SCoV2-PV and VSV-G PV infection on moDCs. Asterisks 606 indicate statistical significance derived from unpaired T-test

SCoV2-PV infection 607 in trans on Vero E6 cells facilitated by moDCs. moDCs was incubated with SCoV2-PV in the 608 presence or absence of anti-DC-SIGN antibody or mannan, and cocultured with Vero E6 cells after 609 extensive washing. Infectivity was calculated based on the luciferase activity of a non-blocking 610 control

Data 611 are representative of three independent experiments

Neutralizing potency of SCoV2 convalescent patients' sera and anti-NTD antibodies 615 to L-SIGN and DC-SIGN mediated infection. Neutralization assay of pseudotyped SCoV2 616 infection on HEK-L-SIGN, HEK-DC-SIGN, and Vero cells by human anti-SCoV2-NTD mAbs (a) 617 and human anti-SCoV2-RBD (C144) specific mAb (b). SCoV2-PV preincubated with mAbs was 618 used to infect the cells. Neutralization assay of SCoV2-PV (c) and VSV-G PV (d) infection of L-619 SIGN or DC-SIGN transfectants by serum from SCoV2

preincubated with sequentially diluted serum was used to infect L-SIGN 621 or DC-SIGN transfectants and luciferase activity was measured. The percentage of infectivity was 622 calculated based on the luminescence value of no serum or mAb control