key: cord-0996123-odasjrou
authors: Azad, Taha; Singaravelu, Ragunath; Taha, Zaid; Jamieson, Taylor R.; Boulton, Stephen; Crupi, Mathieu J.F.; Martin, Nikolas T.; Brown, Emily E.F.; Poutou, Joanna; Ghahremani, Mina; Pelin, Adrian; Nouri, Kazem; Rezaei, Reza; Marshall, Christopher Boyd; Enomoto, Masahiro; Arulanandam, Rozanne; Alluqmani, Nouf; Samson, Reuben; Gingras, Anne-Claude; Cameron, D. William; Greer, Peter A.; Ilkow, Carolina S.; Diallo, Jean-Simon; Bell, John C.
title: Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 Spike for viral entry
date: 2021-02-10
journal: Mol Ther
DOI: 10.1016/j.ymthe.2021.02.007
sha: 442baf806e7bf2d6d7566b56d243767492bbbffe
doc_id: 996123
cord_uid: odasjrou

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 lifecycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2)1-3. The bioreporter assay is based on a Nanoluciferase complementation reporter, composed of two subunits, Large BiT and Small BiT, fused to the spike receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to RBD’s antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point towards targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

As of December 22, 2020, there are globally over 75 million confirmed SARS-CoV-2 4 47 infections resulting in nearly 1.7 million deaths 5 and with no signs of the pandemic 48 ebbing in the near future, effective therapeutics and vaccines are desperately needed. 49

Spike (S) protein that binds to the Angiotensin Converting Enzyme 2 (ACE2) cell 51 receptor and initiates fusion of the viral and cell membranes. This critical role in the 52 virus infection cycle has made the S protein the focus of therapeutic development 53

including the identification of neutralizing antibodies 7 , peptide-based S protein binders 6 54 and small molecule inhibitors of proteases involved in S protein maturation 3 . Like many 55 enveloped virus surface proteins, S is heavily glycosylated and it is has been 56 speculated that these post-translational modifications could facilitate immune evasion or 57 perhaps play a fundamental role in the determination of virus tropism 6 . Interestingly, 58 two N-linked glycan modifications occur within the conserved Receptor Binding Domain 59 or RBD of the S protein. The RBD mediates the binding of the S protein to ACE2 7, 8 and 60

while there have been a number of documented polymorphisms in the amino acid 61 sequence of the RBD from clinical isolates around the world 9 , these two glycosylation 62 sites are uniformly conserved. This suggested to us the possibility that glycosylation of 63 the RBD is important for its binding to the cellular ACE2 receptor or as suggested earlier 64 inhibits immune recognition. To test these ideas, we constructed a bioreporter to rapidly 65 assess the interactions between RBD variants and the ACE2 receptor. We took 66 advantage of the recently developed NanoLuc Binary Technology (NanoBiT) 10, 11 12 to 67 J o u r n a l P r e -p r o o f create a surrogate assay for virus:host cell interactions. Our bioreporter provides a 68 simple and rapid system to carry out a structure-function analysis of critical amino acids 69 in the RBD that modulate its interaction with ACE2, as well as screen potential inhibitors 70 of this host-virus interaction. We demonstrate that the two conserved N-glycan 71 modifications in RBD are required for efficient binding to ACE2 and infection with S 72 pseudotyped viruses. 73 74

Several different reporter fragment complementation-based strategies have been 77 employed to interrogate protein-protein interactions 13 , including split-luciferase 78 schemes 14-16 17 . Conventional split-luciferase bioreporters can be limited in their 79 application due to their relatively large sizes, poor stability, and the short half-lives of 80 their catalyzed luminescent reactions. The recently reported Nanoluciferase (or 81 NanoLuc from Oplophorus gracilirostris) 18 does not possess these limitations, and a 82 NanoLuc-based fragment complementation system has been reported 10, 11 12 . Our 83 bioreporter employs NanoLuc fragments linked to RBD and ACE2 creating a bioreporter 84 that can rapidly and sensitively serve as a surrogate for virus:host cell interactions (Fig. 85 1A). Using published sequences and structural homology analysis 7, 8, 19, 20 , we designed 86 a SARS-CoV-2 RBD sequence spanning residues 331 to 524 of the S protein (194 87 amino acids; Fig. S1 ) for one component of the bioreporter. For the other component 88 we used the soluble ectodomain of ACE2 (residues 1 to 740) as this has been shown to 89 be sufficient to interact with RBD 21 . Since RBD is the smaller protein of the two partners 90 J o u r n a l P r e -p r o o f ACE2 residues 82-84 (amino acids NFS) into the human ACE2 (hACE2) sequence was 160 previously shown to strongly impair the hACE2-RBD interaction with SARS-CoV 1 . 161 Introduction of the NFS residues into SmBiT-ACE2 similarly impaired the SARS-CoV-2 162 ACE2-RBD bioreporter ( Fig. 2B-C, Fig. S4B ), suggesting these ACE2 residues 163 contribute to the species tropism of SARS-CoV-2. 164

We next utilized previous mutational analyses with SARS-CoV RBD to guide our study 165 of the critical SARS-CoV-2 RBD residues mediating the ACE2 interaction 21 . SARS-CoV 166 RBD cysteine residues 348, 467 and 474 as well as the acidic residues E452 and D454, 167 were shown to be critical to this domain's interaction with ACE2 21 (Fig. 2D ). The 168 homologous residues in SARS-CoV-2 RBD (corresponding to C361, E465, D467, C480, 169 and C488) were mutated to alanine in the RBD-LgBiT construct in order to evaluate 170 their role in ACE2 association. Four of the mutations caused a major loss (>80%) of 171 luminescent signal produced by the bioreporter assay (C361A, D467A, C480A, and 172 C488A) -suggesting these residues in SARS-CoV2 RBD are critical for ACE2 173 interaction ( Fig. 2E-F, Fig. S4C ). On the other hand, the E465A mutation caused a 174 modest drop in signal, suggesting that this residue is less critical for SARS-CoV-2 in 175 mediating an interaction with ACE2 in comparison to 177 Since our bioreporter is sensitive to the initial point mutations we examined, we 178 expanded our mutational analyses to include other potential critical residues, based on 179 analysis of the 3D crystal structure of ACE2-RBD binding interface ( Fig. 3A-E) . We 180 used site directed mutagenesis to create an additional alanine mutations in RBD (Fig.  181 3F) and analyzes their impact on ACE2-RBD interactions ( Fig. 3G-H) . We 182 demonstrated 21 out of the 25 tested SARS-CoV-2 RBD mutations significantly reduced 183 binding to ACE2 (Fig. 3H ). To further illustrate the potential of the assay in a high-184 throughput screen, we analyzed its reproducibility in a 384 well plate assay (Fig. S6 ) 185 and found minimal variability. Collectively, these mutational analyses along with high 186 reproducibility of the assay demonstrate the bioreporter is a useful tool for high-187 throughput structure-function analysis of viral and host determinants of the ACE2-RBD 188 interaction. 189

The SARS-CoV-2 bioreporter is sensitive to neutralizing antibodies 191

Monoclonal antibodies targeting RBD are under consideration as SARS-CoV-2 192 therapeutics 25 . We screened 13 different commercially available monoclonal CoV-2 Spike RBD antibodies with the SARS-CoV-2 bioreporter (Fig. 4A ). Seven of 194 these antibodies 1414, 40592, 9A9C9, 5B7d7, 11D5D3, 6D11F2, 10G6H5) are reported 195 to not only bind RBD but also neutralize infection of cells with an S pseudotyped 196 lentivirus. Interestingly these seven monoclonal antibodies were the most effective at 197 blocking RBD-ACE2 interactions measured with the SARS-CoV-2 bioreporter. We 198 applied the antibody collection to the SARS-CoV-1 bioreporter and found that while 199 most SARS-CoV-2 antibodies did not cross-react, antibodies 5B7d7 and11D5D3 200 showed some ability to disrupt RBD-ACE2 interactions for both virus strains. Non-201 specific mouse IgG and monoclonal antibody 1A9, which binds to the S2 subdomain of 202 the Spike protein, did not disrupt the signal generated by the SARS-CoV-2 bioreporter 203 supporting the specificity of the signals were observed. We tested our bioreporters with 204 serum from two patients recovered from SARS-CoV-2 infections at the Ottawa Hospital 205 and pooled serum from three healthy volunteers (Fig. 4B ). In these experiments, we 206 compared our SARS-CoV-2 bioreporter to a widely used, commercially available ELISA 207 kit that is designed to act as surrogate for virus neutralization (Fig. 4C) 26 . For the 208 bioreporter experiments, SARS-CoV-2 RBD-LgBiT was co-incubated with sera for 25 209 min, followed by the addition of SmBiT-ACE2 for an additional five minutes. At this 210 point, substrate was added and luminescence measured. The bioreporter was able to 211 distinguish seroconverters from healthy donors as both convalescent patients' sera 212 significantly reduce the bioreporter signal ( however, the NanoBiT assay is a more rapid (25 minutes compared to over 1.5 hours 218

for the ELISA-based assay) and more accessible, in terms of cost and technical 219 feasibility. 220 SARS-CoV-2 genome sequencing has revealed the emergence of RBD 221 mutations in global strains. We investigated the influence of six emerging RBD 222 mutations found in SARS-CoV-2 genome sequences worldwide on the ACE2-RBD 223 interaction: V367F (France and Hong Kong/China), N354D (China), A435S (Finland), 224 F342L (England), K458R and V483R (United States) 27 (Fig. 4D ). The bioreporter assay 225 revealed that these SARS-CoV-2 variants displayed variable binding to ACE2 (Fig. 4E -226 F). Interestingly, the V367F mutant displayed over 3-fold enhanced interaction with 227 ACE2, while the F342L mutation decreased reporter activity 2-fold. The enhanced 228 J o u r n a l P r e -p r o o f affinity of V367F RBD mutation to ACE2 is consistent with a recent study describing 229 enhanced viral entry in HEK293T-ACE2/TMPRSS2 cells with lentivirus pseudotyped 230 with V367F Spike compared to wildtype Spike 9 . Similarly, these mutations also have 231 the potential to impact the efficacy of RBD-targeted monoclonal antibodies and 232 vaccination strategies. We analyzed the cross-reactivity of two SARS-CoV-2 RBD-233 targeted monoclonal antibodies towards the different RBD variants using the bioreporter 234 assay ( Fig. 4G-H) . Our results demonstrated that both monoclonal antibodies tested 235 could effectively block all the mutants' interactions with ACE2 -highlighting that specific 236 monoclonal RBD antibodies have the potential to work effectively against multiple 237 circulating SARS-CoV-2 strains encoding the different RBD variants. 238

We found that bacterially produced recombinant SARS-CoV-2 RBD was not able to 241 block SmBiT-ACE2's interaction with SARS-CoV-2 RBD-LgBiT (Fig. S5A ), in contrast to 242 our results with recombinant RBD produced in mammalian cells (Fig. 1G ). An important 243 distinction with bacterial expression systems is their inability to produce mammalian-244 type glycosylation, suggesting a potential role for protein glycosylation in the ACE2-RBD 245 interaction. As discussed earlier, a recent study demonstrated that the spike protein We then used site-directed mutagenesis to create full-length Spike mutants (N331A and 281 N343A) and used these to create S pseudotyped lentiviruses. Consistent with our 282 SARS-CoV-2-NanoBiT data (Fig. 5E ), both mutations produced significant decreases in 283 S pseudotyped lentivirus infectivity (Fig. 5G, Fig. S5F -G). Overall, these data provide 284 direct evidence that SARS-CoV-2 S depends on N-linked glycosylation of RBD to 285 mediate its interaction with the ACE2 ectodomain. 286

SARS-CoV-2 S is glycosylated with oligomannose-and complex-type glycans 6 . We 289 sought to examine the therapeutic potential of targeting these N-linked glycans by 290 testing mannose-binding plant lectins for anti-viral effects. We screened lectins from 291

Canavalia ensiformis (jack bean), Pisum sativum (pea), Galanthus nivalis (snow drop), 292

Datura stramonium (jimson weed/thorn apple), Musa acuminata (banana), and Lens 293 culinaris (lentil) for their ability to disrupt the SARS-CoV-2 RBD-ACE2 interaction using 294 our bioreporter ( Fig. 5H; Fig. S5H ). Our results demonstrate a diverse range of antiviral 295 effects. While the lentil lectin displayed no significant inhibition of the interaction across 296 the tested concentration range (8-1000 ng/µL), other lectins showed some efficacy, with 

We sought to examine the possibility that deglycosylation of Spike/RBD was disrupting 311 protein conformation. To investigate this, we also analyzed cell surface RBD 312 expression of the Spike glycosite mutant (N331A/N343A). In our models, we observed 313 no major differences in cell surface expression of the Spike N331A/N343A double 314 mutant relative to the wildtype protein, using either immunofluorescence or flow 315 cytometric analyses ( Fig. 6A-B) . Further, Native-PAGE analyses revealed that the 316 Spike mutant retained its ability to trimerize (Fig.6C ). Taken together, these data 317 suggest the glycosite mutations do not cause major structural changes. Our data 318 cannot exclude, however, that minor conformation changes in the proximity of the RBD 319 glycosites result in a reduced ACE2 binding capacity. Similar to the wildtype construct, 320

we demonstrated that the N331A/N343A RBD-TMD construct maintained its ability to 321 trimerize and localize to the cell surface as demonstrated by immunofluorescence ( Fig 7F) . However, the glycosidase-treated lysate was only recognized by the non-364 neutralizing antibody. This pointed towards the importance of N-linked glycosylation to 365 RBD antigenicity. Similar results were observed using sera from convalescent COVID-366 19 patients to perform dot blot analysis on N331A and N343A Spike mutants. While the 367 N331A mutation had minimal impact on sera binding, the N343A cause a major drop in 368 patient sera binding -suggesting an important role for N-linked glycosylation of Spike 369 N343 in the protein's antigenicity (Fig. S7E) . This was further supported by the fact that 370 recombinant RBD from bacteria, which are incapable of matching mammalian 371 glycosylation profile, was poorly recognized by 1414 and the mouse sera (Fig. 7G ). An Previous studies using split-luciferase reporters have examined viral protein 380 interactions 36, 37 , however we believe the data presented here is the first report of a 381 NanoLuc complementation reporter-based assay to probe virus binding to host receptor 382 ACE2. While the RBD in our bioreporter may not capture trimerization-related and full 383 length-Spike epitopes, we have validated the system's ability to successfully test 384 potential therapeutics, including monoclonal antibodies and receptor decoys. The 385 bioreporter also enabled the evaluation of emerging RBD mutations on SARS-COV-2 386 infectivity and monoclonal antibody efficacy. This represents a valuable application as 387 we begin to identify the novel emerging SARS-CoV-2 spike mutants in the global 388

Our observation that monoclonal RBD antibodies have conserved efficacy against 391 various RBD variants suggests that vaccines capable of inducing a strong neutralizing 392 antibody response against SARS-COV-2 RBD should display strong cross-reactive 393 efficacy in the global population. Although it has been speculated that glycan clusters on 394 the Spike protein could impede immune recognition or antibody activity, our bioreporter 395 data suggests that, for SARS-CoV-2 Spike RBD, this may not be the primary role of 396 glycosylation. Indeed, given the strong conservation of these glycosylation sites in 397 clinical isolates around the world we believe that appropriate glycosylation at N331 and 398 N343 could provide a conserved target for vaccine development. 399

We believe that our data provides direct evidence demonstrating that N-linked 400 glycosylation of SARS-CoV-2 RBD is an important mediator of ACE2 binding. This is Lectins or another carbohydrate binding agent may similarly act as a lead candidate to 412 enable the development of a SARS-CoV-2 spike glycan-targeted lectin. Alternatively, 413 our finding that glycosylation is essential for RBD binding to ACE2 suggests that it may 414 be possible to use specific glycosylation inhibitors as an antiviral approach to blunt 415 SARS-CoV-2 infections especially if given acutely in a locoregional fashion. For 416 example, iminosugars that disrupt appropriate processing of N-linked glycan groups 417

have been shown to act as broad-spectrum antivirals against viruses which dependent 418 on one N-linked glycan on a glycoprotein for infectivity 43 . Our results are consistent 419 with a recent study describing an important role for the N331 and N343 glycosites in 420 viral entry 39 . 421

Previous studies have highlighted the importance of glycosylation on viral 422 antigenicity. In fact, several HIV neutralizing antibodies have been shown to be glycan-423 dependent, with viral escape associated with deletion of a glycan 44, 45 . Our study 424 establishes a similar role for N-linked glycosylation in the antigenicity of RBD (Fig. 7E-425 G). Specifically, our data suggests that the N343 glycan regulates epitope recognition 426 of several RBD-targeted neutralizing antibodies. This is also consistent with a recent 427 reports describing potent neutralizing antibodies forming directed interactions with the 428 N343 glycan 46 . 429

Whereas several other SARS-CoV-2 regions are less conserved, the RBD 431 glycopeptide is highly conserved and represents a prime immunogen to drive 432 neutralizing antibody responses. Our study illustrates that the immunogenicity of RBD 433 is strongly influenced by N-linked glycosylation (Fig. 7A-D) . Mutational analyses 434 revealed that the N343 glycan was critical for RBD's recognition by neutralizing 435 antibodies and, in the context of an RBD-targeted vaccine, the N343A mutation 436 significantly decreased the immunogenicity as measured by blunted induction of anti-437 RBD IgG and neutralizing antibodies (Fig. 7B-D) . These results suggest that a crucial 438 consideration for vaccines using RBD as an immunogen is the utilization of a production 439 system that will generate the proper glycosylation of RBD as this influences the efficacy 440 of the neutralizing antibody response. 

Inserts outlined in Table S1 were ordered from GenScript. Bioreporter subunits were 453 Center, Seattle, WA) 29 . For glycosite mutants, HDm-IDTSpike-fixK was mutated using 508

QuikChange SDM kit (Stratagene) using the primers listed in Table S1 , as per 509 manufacturer's protocols. Briefly, HEK293 cells were co-transfected with HDM-510

IDTSpike-fixK, pHAGE-CMV-Luc2-IRES-ZsGreen-W, and pSPAX2. 48 hours post-511 transfection, cell supernatants containing virus were collected and treated with either 512 PNGase F or EndoH for 1 hours. HEK293T-ACE2 cells were subsequently transduced 513 and transduction efficiency was assessed by luciferase assay using the Bright-Glo 514

Luciferase Assay system (Promega) or fluorescence microscopy (EVOS Cell Imaging 515 System, Thermo Fisher). Where indicated, lentivirus titers were measured using Lenti-516 X GoStix Plus (Takara) as per manufacturer's protocols. For lectin inhibition assays, 517

Spike pseudotyped lentivirus was co-incubated with lectins for 1 hour, and then 518 virus/lectin mixture was applied to HEK293-ACE2 cells as described above. 519 520

Receptor-ligand binding ELISA was performed as per manufacturer's protocols 522 (GenScript L00847). 523 524

All graphs and statistical analyses were generated using Excel or GraphPad Prism v.8. 526

Means of two groups were compared using two-tailed unpaired Student's t-test. Means 527 of more than two groups were compared by one-way ANOVA with Dunnett's or Tukey's 528 multiple comparisons correction. Alpha levels for all tests were 0.05, with a 95% 529 confidence interval. Error was calculated as the standard deviation (SD). Measurements 

Inserts (See Table S1 for detailed sequences) were ordered from GenScript 541 (Piscataway, NJ, USA). SARS-CoV-2 RBD-TMD constructs were cloned into the 542

VSV∆51 viruses expressing SARS-CoV-2 wildtype or mutant RBD-TMD were rescued 544 as previously described 48 . 545 546

Female 6 week-old BALB/C mice (Charles River Laboratories, Malvern, PA) were 548 vaccinated intravenously with 1E7 PFU of VSV∆51 expressing RBD-TMD wildtype or 549 mutants (N331A or N343A). Mice sera was collected using saphenous vein bleeds at 550 days 7 and 14 post-inoculation using sera collection tubes. Blood was incubated on ice 551 for 30 min and then centrifuged to separate sera. LgBiT-YAP15 and 14-3-3-SmBiT plasmids were gifts from Dr. Yang (Queen's 623 Editing. Jean-Simon Diallo: Writing -Reviewing and Editing. John C. Bell -Supervision, 644 WT  C361A  K417A  V445A  G446D  Y449A  Y453A  L455A  F456A  E465A  D467A  Y473A  A475D  C480A  F486A  N487A  C488A  Y489A  Q493A  G496D  Q498A  P499A  T500D  N501A  G502D C361A  E417A  V445A  G446D  Y449A  Y453A  L455A  F456A  E466A  D467A  A475A  C480A  F886A  WT  N487A  C488A  Y489A  Q493A  G496D  Q498A  P499A  T500D  G502D  N501A  G502D WT Y473A 

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