key: cord-0836786-zpajqwr2
authors: Liu, Yo-Min; Shahed-Al-Mahmud, Md.; Chen, Xiaorui; Chen, Ting-Hua; Liao, Kuo-Shiang; Lo, Jennifer M.; Wu, Yi-Min; Ho, Meng-Chiao; Wu, Chung-Yi; Wong, Chi-Huey; Jan, Jia-Tsrong; Ma, Che
title: A carbohydrate-binding protein from the edible Lablab beans effectively blocks the infections of influenza viruses and SARS-CoV-2
date: 2020-07-24
journal: Cell Rep
DOI: 10.1016/j.celrep.2020.108016
sha: 694f29648f2365b1b762f10f65f8a4271abc0184
doc_id: 836786
cord_uid: zpajqwr2

Summary The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents such as lectins. Here we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex type N-glycans, and neutralizes viruses that possess complex type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest potential application of FRIL for prevention and/or treatment of influenza and COVID-19.

Each year, influenza virus infections cause more than half a million deaths worldwide 50 decrease antibody binding to HA resulting in reduced efficacy (Wu et al., 2014) . 58 Antiviral strategies which aim at components of the virus particle such as 59 post-translational glycosylations that incur a higher fitness cost for mutation can be 60 advantageous for combating influenza. anti-viral lectins, especially those that bind to complex type N-glycans, are of special 85 interest and substantial importance. 86 not been studied. To explore what types of oligosaccharides FRIL binds to, we created 218 differentially-glycosylated egg-based influenza viruses of H1N1 X181 by subjecting 219 them to treatment by the mannosidase I inhibitor kifunensine (KIF) during seeding 220 into allantoic cavity of eggs, followed by a high mannose-cleaving endoglycosidase H 221 (endo H) after harvest (Tseng et al., 2019) . Four types of virus particles were thus 222 created: no treatment (both complex and high mannose-type glycans exist on the virus 223 surface naturally), KIF-treated (high mannose only), endo H-treated (complex glycans 224 remain intact, high mannose glycans digested down to single GlcNAc) and KIF/endo 225 H-treated (single GlcNAc only) (Fig. 3A) . These viruses were then purified by 226 sucrose density gradient centrifugation, and its N-glycan constituency confirmed by 227 glycopeptide analysis with tandem mass spectrometry (supplementary Fig S3A) .

We tested the binding of FRIL to these three types of differentially-glycosylated 230 viruses by FRIL immunoblotting (where lysed whole viruses were blotted onto a 231 PVDF membrane, then probed with FRIL) and live virus ELISA. Immunoblotting 232 showed FRIL binds primarily to hemagglutinin (HA), the glycoprotein that mediates 233 virus attachment and entry (Fig. 3B) . Surprisingly, FRIL bound to non-treated, mostly 234 complex type virus particles at a higher intensity than KIF-treated high mannose 235 viruses, which is different from the oligosaccharide binding affinity of most other 236 known antiviral lectins (Fig. 3B) . In a live virus ELISA, (supplementary Fig S3B) 237 FRIL also showed significantly higher binding to complex type particles, in spite of 238 possible interference by glycolipids and other non-specific components of the 239 glycocalyx in this assay. This preference for complex type glycans is also reflected in 240

MN assays: FRIL only shows neutralization against non-treated and endo H-treated 241 viruses containing complex type sugars, but not against high mannose and single 242

GlcNAc viruses (Fig. 3C ). In contrast, the well-documented high mannose binding 243 lectin ConA had the highest neutralization titers against KIF-treated viruses (Fig. 3D) , 244 and bnAb FI6v3 (an HA stem-specific antibody possibly affected by steric hindrance 245 of N-glycans near its epitope (Magadan et al., 2014)) showed the best neutralization 246 activity against virus particles that contained only a single GlcNAc (Fig. 3E) .

To further investigate this interesting phenomenon, we utilized glycan array analysis 249 using fluorescent dye Cy3-labeled FRIL (Fig. 3F) . FRIL demonstrated the best 250 binding to complex type N-glycans with α1-3 or α1-4 fucosylated sub-terminal 251

GlcNAc, including Galβ1-4(Fucα1-3)GlcNacβ1-2Man-R (Lewis 252 X/SSEA-1/CD15-carrying N-glycans) and Galβ1-4(Fucα1-4)GlcNacβ1-2Man-R, 253 ranging from 21,085,870 to 6,257,633 relative fluorescent units (RFU). Slightly 254 weaker binding was seen on non-terminally-fucosylated complex and hybrid type were not attached to the N-glycan trimannose core were no higher than other 260 unrelated glycan structures. Terminal desialyation, as would be anticipated from 261 influenza neuraminidase (NA) activity, did not appear to affect FRIL binding 262 (supplementary Fig S3C) . In contrast, Cy3-labeled ConA exhibited the strongest 263 binding to oligomannose structures such as Man3, Man5 and Man9. The result of 264 direct Cy3 labeling was confirmed by using polyclonal anti-FRIL antibodies 265 (supplementary Fig S3D) .

In summary, our glycan array analysis supports our assays with 268 differentially-glycosylated influenza viruses, indicating that FRIL binds preferentially 269 to complex type N-glycans, and only neutralize viruses with complex type glycans on 270 their surface. Compared to other high-mannose-binding anti-viral lectins in previous 271 studies, such as Griffithsin (GRFT), Cyanovirin (CVN) and H84T Banlec, the 272 complex-type-binding FRIL may have an advantage in targeting viral glycoproteins 273 whose complex type glycans are in majority, such as HA of H1N1, Spike of 274 SARS-CoV-2, etc. The molecular mechanism underlying such a binding preference is, 275

however, yet to be revealed. 276

Due to the surprising finding that FRIL's oligosaccharide preference is different from 279 most known antiviral lectins, we were interested in its antiviral mechanism. First, a 280 competitive microneutralization inhibition assay using monosaccharides shows that 281 the two sugars which can inhibit FRIL hemagglutination, α-methylmannopyranoside 282 and D-glucose, were also able to inhibit FRIL MN (Fig. 4A) , suggesting that FRIL's 283 observed neutralization effect is dependent on its carbohydrate binding function. 284

Second, pre-treatment of cells with either FRIL or virus, followed by 18 hours of 285 incubation with the opposite agent (without either coming into direct contact with one 286 another) both failed to neutralize the influenza virus (Fig. 4B) , confirming that FRIL 287 is an inhibitor of virus entry, and must bind directly to the virus particle.

Influenza virus cell entry includes the sequential steps of virus attachment, 290 endocytosis, uncoating, and nuclear import. To test whether FRIL blocks attachment, 291

we employed a FRIL hemagglutination inhibition assay (HAI) against H1N1, H3N2, 292 H5N1 and H7N9 viruses (supplementary Fig. S4A ). In all four strains, no HAI was observed up to 11.7 µg/ml FRIL. An ELISA done on influenza NP in MDCK cells 294

given 1 hour to bind with virus also showed no significant difference in virus 295 attachment whether FRIL was added or not (supplementary Fig. S4B ). We therefore 296 conclude FRIL does not inhibit influenza virus attachment. The next step after virus 297 attachment is cell endocytosis, a process that can be delayed by lowering the 298 temperature to 4 o C (Matlin et al., 1981) . To confirm that FRIL's neutralization effect 299 occurs after virus attachment, virus particles were added to the cell surface at low 300 temperatures so that attachment takes place but endocytosis in inhibited, and FRIL 301 was then added to the virus-attached cells (Fig. 4C ). Under this condition FRIL 302 exhibited a similar neutralization EC 50 compared with when the lectin and virus were 303 mixed and added to cells together (EC 50 4.25 µg/ml and 2.35 µg/ml, respectively). In 304 contrast, giving the virus 1 hour at 37 o C to be endocytosed before FRIL treatment 305 results in a complete loss of neutralization, indicating that FRIL exerts its antiviral 306 effect after virus attachment, only after being in contact with the virus before its 307 endocytosis and subsequent infection (Fig. 4C) . 308 309 Finally, we followed the progression of the influenza virus after its endocytosis into ELISA was used to determine FRIL's binding affinity to recombinant SARS-CoV-2 404 spike protein produced in HEK293T (with native glycosylation) and HEK293S 405 (GnTI-, high mannose N-glycosylation only) cells. We found FRIL was able to bind to 406 recombinant S protein at concentrations as low as 10 ng/ml (89.1 picomole), and the 407 binding to S proteins with predominantly complex type N-glycans (native 408 glycosylation) is 30 times stronger than those with only high mannose glycans (Fig.  409   7A ). This result is consistent with the published glycan profile of SARS-CoV-2 spike 410 ECD that most of its glycosylation sites are complex-type or hybrid, except two sites 411 (N234 and N709) which are >80% high-mannose (Fig. 7B) .

Competitive inhibition of FRIL's S protein binding was done with the 414 monosaccharides α-methylmannopyranoside, glucose, galactose and yeast mannan 415 from Saccharomyces cerevisiae (Fig. 7C) . Results show that as expected, glucose and 416 the α-anomeric configuration of mannose were able to inhibit FRIL binding to S 417 protein, while galactose, which is not a ligand of FRIL, had no such effect. Yeast 418 mannan had only a slight inhibitory effect on FRIL binding, in contrast to single 419 α-anomeric mannose. 420 421

The evolutionary dynamics of sustained influenza virus circulation in a population 423 with pre-existing immunity favors the addition of N-glycans to surface glycoproteins, 424 masking immunogenic epitopes that would otherwise be recognized by the host The discovery that FRIL showed stronger binding to complex type glycans than high 442 mannose was unexpected, as most exogenous lectins that have antiviral properties 443 interact predominantly with high-mannose structures (Mitchell et al., 2017) . FRIL has 444 previously been characterized as a mannose/glucose lectin that bound to both α1-3 445 and α1-6 linked mannose, but did not precipitate yeast mannans (Mo et al., 1999) . Our 446 glycan array results confirm that FRIL binds to single mannose and branched 447 trimannoside as Mo et al reported, but also had equal or stronger affinity to various 448 complex type N-glycans, especially those with α1-3 or α1-4 fucosylated sub-terminal 449

GlcNAc. It is worth noting that, as Figure 7A , 7C and supplementary Fig S3C  450 suggests, FRIL does not entirely abhor attachment to oligomannose glycans. The 451 binding is just considerably weaker.

This affinity for complex type glycans may explain why FRIL was able to 454 demonstrate good neutralization against X181 and SARS-CoV-2, but not HIV. However, this brings up the concern that since complex type N-glycans are commonly 469 expressed on host cell glycoproteins, FRIL given intranasally would bind to host cells, 470

inducing adverse effects. This trepidation can be alleviated by the fact that our in vivo 471 challenge experiment at the highest dosage of 2.9 mg/kg/day FRIL was well-tolerated. 472

In contrast, 2 mg/kg/day of CVN treatment was found to be lethal to mice (Smee et al., ). In our current study we observed a similar phenomenon for both the 490 high-mannose binding ConA and the complex type binding FRIL (Fig. 4E,  491 supplementary Fig. S4D ), hinting that this might be a common mechanism of 492 anti-influenza action for diverse categories of lectins. However, pre-treatment of cells 493

with FRIL 1 hour before virus infection did not neutralize the virus (Fig. 4B) , and the 494 vast majority of FRIL remained extracellular when FRIL was applied to non-infected 495 Fig S4E) , indicating that unlike H84T, FRIL must first bind to 496 the virus particle before being endocytosed. These results, coupled with our finding that FRIL aggregates influenza virions, allows us to put forth a model for FRIL's 498 anti-influenza action (Fig. 5D ): FRIL first binds and cross-links virions extracellularly, 499 which results in either large aggregates rapidly cleared by the host immune system, or 500

subsequently retained in the late endosome/lysosome and prevented from nuclear 502 entry, until its ultimate degradation 24 hours post-infection. However, there is the 503 possibility that the mechanism for FRIL inhibition of SARS-CoV-2 may be different 504 from influenza virus, given that we did not observe strong N protein signal retained 505 inside cell punctae in FRIL-treated samples, and further investigations will be needed 506 for a more complete mechanistic understanding. In conclusion, we found that FRIL is a tetrameric lectin with potent anti-influenza and 519

anti-SARS-CoV-2 activity. It preferentially binds to complex type N-glycans to halt 520 influenza virus entry at the late endosomal stage, and we have demonstrated that FRIL 521 is effective both in vitro and in vivo. Furthermore, FRIL's neutralizing ability is at 522 least on par with most known antiviral neutralizing monoclonal antibodies. We 523 believe its utility as a preventive or therapeutic agent in influenza and the current 524 COVID-19 pandemic warrants further investigation: for example, to coat it on masks 525 or be included in aerosol mists in a closed space such as an airplane cabin for 526 reducing transmission, or to be used in an inhaler (like Relenza for influenza), which 527 will require vigilant clinical trials to evaluate its safety and efficacy.

We thank Sue-Jane Chen and Yung-Chieh Tseng for insightful discussions. We thank 531 Academia Sinica Biological Electron Microscopy Core Facility for EM technical 532 support. The core facility is funded by the Academia Sinica Core Facility and 533

Innovative Instrument Project (AS-CFII-108-119). We thank Academia Sinica 534 

Resource availability 557

Further information and requests for resources and reagents should be directed to the 559 Lead Contact, Che Ma (cma@gate.sinica.edu.tw). 560 561

All unique/stable reagents generated in this study are available from the Lead Contact 563 with a completed Materials Transfer Agreement. Waltham, Massachusetts). 620

For plaque reduction assay, MDCK or Vero E6 cells were plated onto a 6-well plate at 621 2x10 5 cells/well overnight for 90% confluence. FRIL and viruses were co-incubated 622 at 37 o C for 1 hour, before the mixture is added onto the monolayer for another hour. Glycan array analysis was performed as described previously (Shivatare et al., 2016) . For Western blotting, purified X181 viruses were loaded onto a 4~15% SDS-PAGE (Bio-Rad) with a non-reducing loading dye, the transferred onto PVDF membrane 670 with semi-dry method (Trans-Blot SD, Bio-Rad). The membrane is blocked with 5% 671 BSA in PBST for 1 hour at room temperature, then 1.2 µg/ml FRIL protein is added. 672

The membrane was then sequentially treated with polyclonal anti-FRIL primary 673 antibody and HRP-conjugated secondary antibody. Finally, Clarity ECL substrate 674 (Bio-Rad) was added for chemiluminescence, and visualized with ImageQuant 675 LAS4000 (GE). 676 677

Our mouse challenge experiments were performed by following an intranasal 679 administration method described previously (Sidwell et al., 1998) the pH back up to 7. Finally, TPCK-trypsin was added to the mixture at a 1:50 molar 787 ratio and the mixture incubated at 37 o C for 30 minutes before the digestion was 788 stopped by the addition of SDS-PAGE loading dye (non-reducing), and denatured at 789 100 o C for 10 mins. Samples were run on a 4~15% SDS-PAGE (Bio-Rad) to assess 790 trypsin susceptibility.

Quantification and statistical analysis 793

All data are presented as mean ± SEM except Fig 1B and 

Notes coronavirus infection is inhibited by griffithsin

Antiviral lectins: Selective 922 inhibitors of viral entry

Purification and 924 characterization of Dolichos lablab lectin

Broad-spectrum in vitro 928 activity and in vivo efficacy of the antiviral protein griffithsin against emerging 929 viruses of the family Coronaviridae

Potent anti-influenza 932 activity of cyanovirin-N and interactions with viral hemagglutinin

937 Modular synthesis of N-glycans and arrays for the hetero-ligand binding 938 analysis of HIV antibodies

Inhibition of influenza 941 virus infections in mice by GS4104, an orally effective influenza virus 942 neuraminidase inhibitor

Treatment of influenza A (H1N1) virus infections in 945 mice and ferrets with cyanovirin-N

Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a 950 Soluble Trimeric Immunogen

956 Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral 957 activity

Playing hide and seek: how glycosylation of the influenza virus 960 hemagglutinin can modulate the immune response to infection

Egg-based influenza split virus vaccine with 964 monoglycosylation induces cross-strain protection against influenza virus 965 infections

Immunomodulatory glc/man-directed Dolichos 968 lablab lectin (DLL) evokes anti-tumour response in vivo by counteracting 969 angiogenic gene expressions

Site-specific glycan analysis of the SARS-CoV-2 spike

Manual for the laboratory diagnosis and 973 virological surveillance of influenza

Influenza A surface glycosylation and vaccine design

High-throughput profiling of influenza A virus 981 hemagglutinin gene at single-nucleotide resolution

Lewis X-carrying 983 N-glycans regulate the proliferation of mouse embryonic neural stem cells via 984 the Notch signaling pathway

Reassortment and mutations associated with 987 emergence and spread of oseltamivir-resistant seasonal influenza A/H1N1 988 viruses in

Legume lectin FRIL preserves neural progenitor cells in suspension 991 culture in vitro

Lablab purpureus. (B) Microneutralization of Lablab purpureus seed crude aqueous extract against 998

IVR-165 (H3N2), and RG32A (H7N9) viruses. A single experiment was 999 performed in this screening. (C) Purified anti-viral reagent exhibits five bands on SDS-PAGE (left) 1000 which are confirmed as different truncations of the α-and β-subunits of FRIL by mass spectrometry

The same sample exhibits only one single band of higher molecular weight on native PAGE (right)

Black arrows indicate molecular weights (kD) of the protein ladder for SDS-PAGE, no marker was 1003 used on native PAGE. Data representative of 3 independent experiments. (D) SEC-MALS of purified 1004 FRIL in solution (phosphate buffer) shows a single narrow peak. The MALS trace (black line) indicates 1005 a molecular mass of 112.1±0.8 kDa. Data representative of 2 independent experiments

Negative-stain EM density of purified FRIL (grey) fitted with its previously solved crystal structure 1007 (PDB code 1qmo) confirms its tetrameric state in solution, with different shades of blue for each 1008 monomer

Figure 2. FRIL exhibits potent broad-spectrum anti-influenza activity in vitro and in vivo. (A) FRIL plaque reduction assay with H1N1 X181 (A/California/07/2009-like) virus. Data representative of 3 independent experiments performed in triplicate (mean±SEM). See also Figure S2A. (B) FRIL (blue) and bnAb FI6v3 (FI6, orange) MN of

Data representative of 2 independent experiments performed in triplicate (mean±SEM) for each strain. (C) HA phylogenetic tree created with MEGA-X shows microneutralization EC 50 values (nM) of FRIL and bnAb FI6v3 (FI6) against 11 representative vaccine and laboratory strains of group 1, group 2 and influenza B viruses. Each block is colored by EC 50 values (nM): the darker the color, the higher the neutralizing activity. Data representative of 2 independent experiments performed in triplicate (mean±SEM) for each strain. See also Figure S2B. (D) Treatment schedule of X181 challenge and FRIL administration. 29 or 2.9 µg of FRIL protein was given intranasally to BALB/c mice (n = 10) 4 hours before challenge. Influenza virus intranasal challenge was conducted using 5LD 50 of X181 virus, and 29 or 2.9 µg of FRIL protein was then given intranasally twice per day for 8 days following challenge. Survival (E) and body weight (F) were tracked for 21 days following influenza virus challenge

Schematic diagram showing the generation of non-treated (KIF (-) Endo H (-), complex and high mannose type glycans), KIF-treated (KIF (+) Endo H (-), high mannose type glycans only), endo H-treated (KIF (-) Endo H (+)

SDS PAGE (left) and FRIL immunoblotting (right) of non-treated, KIF-treated, and KIF and endo H-treated lysed virus particles. FRIL immunoblotting was done by incubating FRIL with viral proteins transferred onto membrane, followed by detection with anti-FRIL antibodies. Data representative of 3 individual experiments. (C, D and E) Microneutralization assay of non-treated (blue square), endo H-treated (light blue square) KIF-treated (green circle) and KIF and endo H-treated (grey triangle) viruses with FRIL (C)

FRIL halts influenza virus entry in the late endosome. (A) (upper) Hemagglutination inhibition of 59 µg/ml FRIL (4 HAU) by the monosaccharides α-methylmannopyranoside

Mean±SEM of three replicates. (B) FRIL microneutralization where FRIL and virus were added, either together (circle) or sequentially (square), in a 1 hour pre-treatment step after which they are removed or an ensuing 18 hour incubation step. Mean±SEM of three replicates. (C) FRIL microneutralization with a 1 hour low-temperature arrest of viral endocytosis, separating viral entry steps into attachment (black circle), internalization (blue square), and infection (grey triangle), with FRIL added during the different steps. Data representative of 3 independent experiments performed in triplicate (mean±SEM). (D) Progression of influenza RNP after virion endocytosis with or without FRIL inhibition, visualized by immunofluorescence tracking of viral NP protein (anti-NP, green) and nuclei (DAPI, blue)

Dynamic light scattering (DLS) analysis of virus particle aggregation under increasing concentrations of FRIL (from 1.5 µg/ml in purple line to 490 µg/ml in dark red line) (B) Negative stain EM images of purified X181 virions alone (upper panels) and aggregated X181 virus particles after mixing with 150 µg/ml FRIL (lower panels). Data representative of 3 independent experiments. (C) Quantitation of aggregated virions calculated from 20 images for each FRIL concentration. Virions that directly contact each other are considered aggregated. Concentrations above 32 µg/ml proved difficult to ascertain due to formation of large overlapping aggregates. (D) A proposed model for FRIL's anti-influenza mechanism: large FRIL/virus aggregations may occur outside the cell to prevent virus entry

Black triangles represent areas of focal CPE, while FRIL concentrations below 0.78 µg/ml (6.96 nM) were unable to prevent diffuse and widespread CPE. Data representative of 2 experiments. (B) FRIL plaque reduction neutralization assay with hCoV-19/Taiwan/NTU04/2020 virus

Data representative of 2 experiments performed in quadruplicate (mean±SEM). (D) Immunofluorescence tracking of SARS-CoV-2 (hCoV-19/Taiwan/NTU04/2020) N (green) and S protein (cyan) production 4 to 24 hpi, with or without 33 µg/ml (0.29 µM) FRIL inhibition. Nuclei are counterstained with DAPI (blue)

ELISA of FRIL binding to SARS-CoV-2 S protein, with either native N-glycosylation (containing complex type, hybrid and high mannose glycans, blue square) or high mannose only (green circle). The unglycosylated bovine serum albumin (BSA, grey triangle) was used as control. Mean±SEM of three replicates. (B) Cryo-EM structure of SARS-CoV-2 Spike protein modeled with glycans according to either the published glycan profile (left, native) or the high-mannose (Man5) type produced by HEK293S cell line (right)

The hemagglutination titer of FRIL was determined beforehand. 709Methyl-α-D-mannopyranoside, D-galactose, D-glucose and L-arabinose were serially 710 diluted in V-bottom 96-well plates with PBS, 25 µl/well. 25 µl of 59 µg/ml FRIL (4.1 711 HAU) were then added to each well, followed by 50 µl of 0.5% turkey red blood cells 712 

• FRIL is a plant lectin with potent anti-influenza and anti-SARS-CoV-2 activity.• FRIL preferentially binds to complex type N-glycans on viral glycoproteins.• FRIL inhibits influenza virus entry by sequestering virions in late endosomes.• Intranasal administration of FRIL protects against lethal H1N1 challenge in mice.

Liu et al. demonstrate that FRIL, a plant lectin isolated from the hyacinth bean, has potent antiviral activity against SARS-CoV2 and diverse influenza virus strains. FRIL is effective in vivo against H1N1. FRIL's antiviral activity is mediated by binding to complex type N-glycans on viral glycoproteins, interfering with viral entry.