key: cord-0838399-gzep793w
authors: Motozono, Chihiro; Toyoda, Mako; Zahradnik, Jiri; Ikeda, Terumasa; Saito, Akatsuki; Tan, Toong Seng; Ngare, Isaac; Nasser, Hesham; Kimura, Izumi; Uriu, Keiya; Kosugi, Yusuke; Torii, Shiho; Yonekawa, Akiko; Shimono, Nobuyuki; Nagasaki, Yoji; Minami, Rumi; Toya, Takashi; Sekiya, Noritaka; Fukuhara, Takasuke; Matsuura, Yoshiharu; Schreiber, Gideon; Nakagawa, So; Ueno, Takamasa; Sato, Kei
title: An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity
date: 2021-04-05
journal: bioRxiv
DOI: 10.1101/2021.04.02.438288
sha: 884162a043b9663c178585990f3dba29d1863477
doc_id: 838399
cord_uid: gzep793w

During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic. Graphical Abstract

The emergence of mutated viruses is mainly due to error-prone viral 126 replication, and the spread of emerged variants is attributed to the escape from 127 immune selective pressures [reviewed in (Duffy et al., 2008) ]. In fact, several SARS-128

CoV-2 mutants can be resistant to the neutralization mediated by the antibodies from 129 COVID-19 patients (Baum et al., 2020; Chen et al., 2021; Liu et al., 2021c; McCarthy 130 et al., 2021; Weisblum et al., 2020) as well as those from vaccinated individuals (Liu 131 et al., 2021b) . Although the B1.1.7 variant is sensitive to convalescent and 132 vaccinated sera (Collier et al., 2021; Garcia-Beltran et al., 2021; Shen et al., 2021; 133 Supasa et al., 2021; Wang et al., 2021) , the B.1.351 and P.1 variants are relatively 134 resistant to anti-SARS-CoV-2 humoral immunity (Garcia-Beltran et al., 2021; 135 Hoffmann et al., 2021a; Wang et al., 2021) . 136

In addition to the humoral immunity mediated by neutralizing antibodies, 137

another protection system against pathogens is the cellular immunity mediated by 138 cytotoxic T lymphocytes (CTLs) [reviewed in (Fryer et al., 2012; Leslie et al., 2004) ]. 139

CTLs recognize the nonself epitopes that are presented on virus-infected cells via 140 human leukocyte antigen (HLA) class I molecules, and therefore, the CTL-mediated 141 antiviral immunity is HLA-restricted [reviewed in (La Gruta et al., 2018) ]. Recent 142 studies have reported the HLA-restricted SARS-CoV-2-derived epitopes that can be 143 recognized by human CTLs (Kared et al., 2021; Kiyotani et al., 2020; Nelde et al., 144 2021; Schulien et al., 2021; Wilson et al., 2021) . More importantly, Bert et al. have 145 recently reported that the functionality of virus-specific cellular immunity is inversely 146 correlated to the COVID-19 severity (Le Bert et al., 2021) . Therefore, it is 147 conceivable to assume that the HLA-restricted CTLs play crucial roles in controlling 148 SARS-CoV-2 infection and COVID-19 disorders. However, comparing to humoral 149 immune responses, it remains unclear whether the SARS-CoV-2 variants can 150 potentially escape from cellular immunity. 151

In this study, we investigate the possibility for the emergence of the SARS-152

CoV-2 mutants that can escape from the HLA-restricted cellular immunity. We 153 demonstrate that at least two naturally occurring substitutions in the receptor binding 154 motif (RBM; residues 438-506) of the SARS-CoV-2 S protein, L452R and Y453F, 155 which were identified in the two major variants, B.1.427/429 (L452R) and B1. 1.298 156 (Y453F), can be resistant to the cellular immunity in the context of HLA-A*24:02, an 157 allele of HLA-I. More intriguingly, the L452R and Y453F mutants increase the binding 158 affinity to ACE2, and the experiments using pseudoviruses show that the L452R 159 substitution increases viral infectivity. Furthermore, we artificially generate the 160 SARS-CoV-2 harboring these point mutations by reverse genetics and demonstrate 161 that the L452R mutants enhance viral replication capacity. 162

Evasion from the HLA-A24-restricted CTL responses by acquiring mutations 164 in the RBM of SARS-CoV-2 S protein 165 We set out to address the possibility of the emergence of the naturally occurring 166 mutants that can potentially confer the resistance to antigen recognition by HLA-167 restricted cellular immunity. A bioinformatic study has suggested that the 9-mer 168 peptide in the RBM, NYNYLYRLF (we designate this peptide "NF9"), which spans 169 448-456 in the S protein, can be the potential epitope presented by HLA-A24 170 (Kiyotani et al., 2020) , an HLA-I allele widely distributed all over the world and 171 particularly predominant in East and Southeast Asian area (Table S1) . Additionally, 172 three immunological analyses using COVID-19 convalescents have shown that the 173 NF9 peptide is an immunodominant epitope presented by HLA-A*24:02 (Gao et al., 174 2021; Hu et al., 2020; Kared et al., 2021) . To verify these observations, we obtained 175 the peripheral blood mononuclear cells (PBMCs) from nine COVID-19 176 convalescents with HLA-A*24:02 and stimulated these cells with the NF9 peptide. 177

As shown in Figure 1A , a fraction of CD8 + T cells upregulated two activation markers, 178 CD25 and CD137, in response to the stimulation with NF9. In the nine samples of 179 COVID-19 convalescents with HLA-A*24:02, the percentage of the CD25 + CD137 + 180 cells in the presence of the NF9 peptide (5.3% in median) was significantly higher 181 than that in the absence of the NF9 peptide (0.49% in median) ( Figure 1B ; P=0.016 182 by Wilcoxon signed-rank test). Additionally, the stimulation with the NF9 peptide did 183 not upregulate CD25 and CD137 in the CD8 + T cells of three seronegative samples 184

with HLA-A*24:02 and the percentage of the CD25 + CD137 + cells in seronegative 185 samples (0.93% in median) was significantly lower than that in COVID-19 186 convalescent samples (Figure 1B ; P=0.011 by Mann-Whitney U test). Consistent 187 with previous reports (Gao et al., 2021; Hu et al., 2020; Kared et al., 2021; Kiyotani 188 et al., 2020) , our data suggest that the NF9 peptide is an immunodominant HLA-189 A*24:02-restricted epitope recognized by the CD8 + T cells of convalescents in our cohort. 191 We next assessed the profile of cytokine production by the NF9 stimulation. 192

As shown in Figure 1C , the stimulation with the NF9 peptide induced the production 193 of IFN-γ, TNF-α and IL-2 in the CD8 + T cells of a COVID-19 convalescent. The 194 analysis using six COVID-19 convalescent samples showed that CD8 + T cells 195 produce multiple cytokines in response to the NF9 stimulation ( Figure 1D) , 196 demonstrating the multifunctional nature of the NF9-specific CD8 + T cells of 19 convalescents. Moreover, the cytotoxic potential of the NF9-specific CD8 + T cells 198

was assessed by staining with surface CD107a, a degranulation marker ( Figure 1E ).

As shown in Figure 1F , the percentage of CD107a + cells in the CD8 + T cells with 200 the NF9 peptide (12.9% in median) was significantly higher than that without the NF9 201 peptide (0.83% in median) (; P=0.031 by Wilcoxon signed-rank test), suggesting the 202 cytotoxic potential of the NF9-specific CD8 + T cells. 203

To assess the presence of naturally occurring variants harboring mutations 204 in this region (residues 448-456 in the S protein), we analyzed the diversity of SARS-205

CoV-2 during the current pandemic. respectively ( Figure 1G and Table S2 ).

To address the possibility that the naturally occurring mutations in the NF9 214 region, L452R and Y453F, evade the NF9-specific CD8 + T cells of HLA-A24-positive 215 COVID-19 convalescents, two NF9 derivatives containing either L452R or Y453F 216 substitution (NF9-L452R and NF9-Y453F) were prepared and used for the 217 stimulation experiments. As shown in Figure S1 , parental NF9 induced IFN-γ 218 expression in a dose-dependent manner. In contrast, the induction level of IFN-γ 219 expression by the NF9-Y453F derivative was significantly lower than that by parental 220 NF9, and more intriguingly, the NF9-L452R derivative did not induce IFN-γ 221 expression even at the highest concentration tested (10 nM) ( Figure S1 ). In the five 222 HLA-A24-positive COVID-19 convalescent samples, parental NF9 peptide 223 significantly induced IFN-γ expression, while the NF9-L452R and NF9-Y453F 224 derivatives did not (Figures 1H and 1I) . Altogether, these results suggest that the 225 NF9 peptide, which is derived from the RBM of SARS-CoV-2 S protein, is an 226 immunodominant epitope of HLA-A24, and two naturally occurring mutants, L452R 227 and Y453F, evade the HLA-A24-restricted cellular immunity. 228 229

Augmentation of the binding affinity to ACE2 by the L452 and Y453 mutations 230 We next addressed whether the mutations of interest affect the efficacy of virus 231 infection. Structural analyses have shown that the Y453 and N501 residues in the 232 RBM are located on the interface between the SARS-CoV-2 RBM and human ACE2 233

and directly contribute to the binding to human ACE2, while the L452 residue is not 234 on the RBM-ACE2 interface (Lan et al., 2020; Wang et al., 2020; Zhao et al., 2020) 235 ( Figure 2A) . To directly assess the effect of these mutations in the RBM on the 236 binding affinity to ACE2, we prepared the yeasts expressing parental SARS-CoV-2 237 receptor binding domain RBD (residues 336-528) and its derivatives (L452R, Y453F 238 and N501Y) and performed in vitro binding assay using the yeast surface display of 239 the RBD and soluble ACE2 protein. Consistent with recent studies including ours 240 (Supasa et al., 2021; Zahradník et al., 2021b) , the N501Y mutation, which is a 241 common mutation in the B1. 1.7, B1.351 and P.1 variants [reviewed in (Plante et al., 242 2021)] as well as the Y453F mutation (Bayarri-Olmos et al., 2021; Zahradník et al., 243 2021b) significantly increased the binding affinity to human ACE2 (Figures 2B and  244 2C; RBD parental KD = 2.05 ± 0.26 nM; RBD N501Y KD = 0.59 ± 0.03 nM; and RBD 245 Y453F KD = 0.51 ± 0.06 nM). We also found that the L452R mutant significantly 246

increased the binding affinity to human ACE2 (Figures 2B and 2C ; RBD L452R KD 247 = 1.20 ± 0.06 nM). Intriguingly, the L452R mutations increased the surface 248 expression, which reflects protein stability (Traxlmayr and Obinger, 2012) , while the 249 Y453F and N501Y mutations decreased ( Figure 2D ).

Increase of pseudovirus infectivity by the L452R mutation 252

To directly analyze the effect of the mutations of interest on viral infectivity, we 253 prepared the HIV-1-based reporter virus pseudotyped with the SARS-CoV-2 S 254 protein and its mutants and the 293 cells transiently expressing human ACE2 and 255

TMPRSS2. As shown in Figure 2E , although the N501Y mutation faintly affected 256 viral infectivity in this assay, the L452R mutations significantly increased viral 257 infectivity compared to parental S protein. In contrast to the yeast display assay 258

( Figures 2B and 2C) , the infectivity of the Y453F mutant was significantly lower than 259 that of parental S protein ( Figure 2E ). Altogether, these findings suggest that the 260 L452R substitution increases the binding affinity of the SARS-CoV-2 RBD to human 261 ACE2, protein stability, and viral infectivity. Although the L452 residue is not directly 262 located at the binding interface (Figure 2A) , structural analysis and in silico 263 mutagenesis suggested that the L452R substitution can cause a gain of 264 electrostatics complementarity (Selzer et al., 2000) ( Figure 2F ). Because the 265 residue 452 is located close proximity to the negatively charged patch of ACE2 266 residues (E35, E37, D38), the increase of viral infectivity by the L452R substitution 267

can be attributed to the increase in the electrostatic interaction with ACE2. 268 269

Promotion of SARS-CoV-2 replication in cell cultures by the L452 mutation 270

To investigate the effect of the mutations in the RBM on viral replication, we artificially 271 generated the recombinant SARS-CoV-2 viruses that harbor the mutations of 272 interest as well as parental recombinant virus by a reverse genetics system (Torii et  273 al., 2021). The nucleotide similarity of SARS-CoV-2 strain WK-521 (GISAID ID: 274 EPI_ISL_408667) , the backbone of the artificially 275 generated recombinant SARS-CoV-2, to strain Wuhan-Hu-1 (GenBank: 276 NC_045512.2) (Wu et al., 2020) is 99.91% (27 nucleotides difference) and the 277 sequences encoding the S protein between these two strains are identical, indicating 278 that the strain WK-521 is a SARS-CoV-2 prototype. We verified the insertions of the 279 targeted mutations in the generated viruses by direct sequencing ( Figure 3A ) and 280 performed virus replication assay using these recombinant viruses. As shown in 281 Figure 3B , we revealed that the growth of the L452R mutant in VeroE6/TMPRSS2 282 cells was significantly higher than that of parental virus. Together with the findings in 283 the binding assay (Figures 2B-2D ) and the assay using pseudoviruses (Figure 2E) , 284 our results suggest that the L452R mutation potentially increase viral replication. 285 286

Dynamics of the spread of the RBM mutants during the current pandemic 287

We finally assessed the epidemic dynamics of the naturally occurring variants 288

containing the substitutions in L452 and Y453. As shown in Figure 4A and Table  289 S3, the L452R mutants were mainly found (3,967 sequences) in the B. Figure  299 4B, top). In 2021, this lineage has expanded throughout the USA, and currently, is 300 one of the most predominant lineages in the country ( Figure 4B , bottom and Table  301 S4).

For the Y453F mutation, 1,274 out of the 1,380 mutated sequences belong 303 to the B.1.1.298 lineage, which has been exclusively detected in Denmark (Table  304 S3). The oldest sequence that contains the Y453F mutation in the B.1.1.298 lineage 305 was isolated from a human in Denmark on April 20, 2020 (GISAID ID: 306 EPI_ISL_714253) ( Figure 4C) . Intriguingly, the B.1.1.298 variants containing either 307 Y453 or F453 are detected not only in humans but also in minks ( Figure 4D ). 308

The phylogenetic analysis of the whole genome sequences of the B.1.1.298 lineage 309 SARS-CoV-2 suggested multiple SARS-CoV-2 transmissions between humans to 310 minks ( Figure S2) . Additionally, the three sequences that contain the Y453F 311 mutation were isolated from cats in Denmark: the two sequences out of them 312 (GISAID ID: EPI_ISL_683164 and EPI_ISL_683166) made a single clade, while the 313 other one (GISAID ID: EPI_ISL_683165) had a distinct origin ( Figure S2 ). These 314 results suggest that this SARS-CoV-2 variant has transmitted from humans to cats 315 multiple times and some of them may spread among Danish cat population. However, 316 the epidemic of a fraction of the B.1.1.298 lineage containing the Y453F mutation in 317

Denmark peaked during October to November, 2020, and subsequently, gradually 318 reduced ( Figure 4D ). The variant containing the Y453F mutation was last collected 319

in Denmark on January 18, 2021 (GISAID ID: EPI_ISL_925998) and it has not been 320

reported worldwide since then ( Figure 4D ). 321

In the present study, we demonstrated that at least two naturally occurring mutations 323

in the SARS-CoV-2 RBM, L452R and Y453F, escape from an HLA-restricted cellular 324

immunity and further reinforce the affinity to viral receptor ACE2. We further 325 demonstrate that the L452R mutation increase the stability of S protein, viral 326 infectivity and thereby enhances viral replication. Our data suggests that the L452R 327 mutant escapes from the HLA-A24-restricted cellular immunity and further 328 strengthens its infectivity. 329

Lines of recent studies have shown the emergence of the SARS-CoV-2 330

variants that evade the anti-SARS-CoV-2 neutralizing humoral immunity ( Although it remains unclear whether the emergence of the Y453F mutant potentially 356

associates with the evasion from the acquired immunity in mink, here we showed 357 that this mutation can be resistant to the HLA-A24-resticted human cellular immunity. 358

Because the Y453F mutation did not increase the infection efficacy using mink ACE2, 359 our results suggest that the emergence of this mutant is not due to improving viral 360 fitness to mink. Nevertheless, the host range of SARS-CoV-2, in terms of the use of 361 ACE2 molecule for infection receptor, is broad in a variety of mammals (Liu et al.,  362 2021a; OIE, 2021). More importantly, although murine ACE2 cannot be used for the 363 infection of prototype SARS-CoV-2, recent studies have revealed that some SARS-364

CoV-2 variants including the B.1.351 and P.1 variants gained the ability to use 365 murine ACE2 for infection and expanded their host range to mice (Li et al., 2021; 366 Montagutelli et al., 2021) . In addition to the evasion from human acquired immunity, 367

zoonotic and zooanthroponosis SARS-CoV-2 transmissions can contribute to the 368 accumulation of mutations in the spreading viruses and further impact viral 369 phenotypes including infectivity, replication efficacy, pathogenicity, transmissibility 370 and even host range. Therefore, the surveillance on the emergence of novel variants 371 even in nonhuman mammals and assessing their potentials to adapt to use 372 nonhuman ACE2 for infection receptor will be critical. 373

In contrast to the B. All authors reviewed and proofread the manuscript. 452

The Genotype to Phenotype Japan (G2P-Japan) consortium contributed to the 453 project administration. 454 455

Consortia 456

The Genotype to Phenotype Japan (G2P-Japan See also Figure S1 and Tables S1 and S2. 848 849 surface structure of the SARS-CoV-2 S and ACE2 (PDB: 6M17) . 870

The residue See also Figure S2 and Tables S3 and S4 . 917 

Establishment of a reverse genetics 732 system for SARS-CoV-2 using circular polymerase extension reaction. Cell Rep Pre-733 proof

Directed evolution of proteins for 735 increased stability and expression using yeast display

Evaluating the Effects of 739 SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Antibody Resistance of SARS-CoV-2 Variants B.1.351 743 and B

Structural and Functional Basis of SARS-CoV-2 Entry by 746 Using Human ACE2

Escape 749 from neutralizing antibodies by SARS-CoV-2 spike protein variants. Elife 9

SARS-CoV-2 mink-associated variant strain -Denmark

Total predicted 756 MHC-I epitope load is inversely associated with population mortality from SARS-757 CoV-2

Activation-induced expression of CD137 permits detection, 760 isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen 761 without requiring knowledge of epitope specificities

A new coronavirus associated with human respiratory 764 disease in China

Structural basis for 766 the recognition of SARS-CoV-2 by full-length human ACE2

DNA vaccine protection 769 against SARS-CoV-2 in rhesus macaques

Structural and 772 Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

An enhanced yeast 775 display platform demonstrates the binding plasticity under various selection 776 pressures

SARS-CoV-2 RBD in vitro 779 evolution follows contagious mutation spread 780 yet generates an able infection inhibitor

Emergence of a novel SARS-CoV-2 variant in Southern California

Broad and Differential Animal Angiotensin-Converting Enzyme 786 2 Receptor Usage by SARS-CoV-2

SARS-CoV-2 spike 789 D614G change enhances replication and transmission

A pneumonia outbreak associated with a new 792 coronavirus of probable bat origin

Robust SARS-CoV-2-specific T 795 cell immunity is maintained at 6 months following primary infection

Briefly, human PBMCs were pulsed with 1008

μg/ml of the SARS-CoV-2 PepTivator peptide pools

130-126-700) and maintained in RPMI 1640 medium

Scientific, cat# 11875101) containing 10% FCS and 30 U/ml recombinant human IL-1011 2 (Peprotec, cat# 200-02) for 10-14 days. The in vitro expanded CD8 + T cells (i.e., 1012 the CTL lines) were stimulated with or without the NF9 peptide (NYNYLYRLF, 1013 residues 448-456 of the SARS-CoV-2 S protein

After the incubation for 20 min on ice, the cells were fixed 1018 with 1% paraformaldehyde (Nacalai Tesque, cat# 09154-85) and the levels of protein 1019 surface expression were analyzed by flow cytometry using a FACS Canto II

The data obtained by flow cytometry were analyzed by FlowJo 1021 software

L5R in NF9) and the NF9-Y453F peptide 1026 (NYNYLFRLF, Y6F in NF9); synthesized by Scrum Inc.] at concentrations from 0.1 1027 to 10 nM at 37°C for 1 h. The cells were washed twice with PBS, mixed with the CTL 1028 lines generated from COVID-19 convalescents (see above) and incubated with 1029 RPMI 1640 medium (Thermo Fisher Scientific, cat# 11875101) containing 10% FCS, 1030 5 μg/ml brefeldin A (Sigma-Aldrich, cat# B7651), 2 μM monensin (Biolegend, cat# 1031 420701) and BV421-anti-CD107a antibody (Biolegend, cat# 420404) in a 96-well U 1032 plate at 37°C

CD14 and CD19) were stained with the antibodies listed in Key Resources 1034

After the incubation at 37°C for 30 min, the cells were fixed and 1036 permeabilized with Cytofix/Cytoperm Fixation/Permeabilization solution kit

After the incubation 1039 at room temperature for 30 min, the cells were washed and the levels of protein 1040 expression were analyzed by flow cytometry using a FACS Canto II

The data obtained by flow cytometry were analyzed by FlowJo 1042 software

Plasmid Construction

-1047 N501Y) were generated by site-directed mutagenesis PCR using pC-SARS2-S 1048 (kindly provided by Kenzo Tokunaga) (Ozono et al., 2021) as the template and the 1049 following primers: S forward

The resultant PCR fragment was digested with 1055

KpnI and NotI and inserted into the KpnI-NotI site of pCAGGS vector

To construct the expression plasmid for human ACE2 (GenBank: 1058 NM_021804.3) (pLV-EF1a-human ACE2-IRES-Puro), the MluI-SmaI fragment 1059 pTargeT-human ACE2 (kindly provided by Shuetsu Fukushi) (Fukushi et al., 2007) 1060 was inserted into the MluI-HpaI site of pLV-EF1a-IRES-Puro

Nucleotide sequences were determined by a DNA sequencing service (Fasmac), 1062 and the sequence data were analyzed by Sequencher v5.1 software (Gene Codes 1063 Corporation). 1064 1065 Preparation of Soluble Human ACE2 1066 To prepare soluble human ACE2, the expression plasmid for the extracellular 1067 domain of human ACE2 (residues 18-740) based on pHL-sec (Addgene, cat# 99845) 1068 (Zahradník et al., 2021a) was transfected into Expi293F cells using ExpiFectamine 1069 293 transfection kit (Thermo Fisher Scientific, cat# A14525) according to the 1070 manufacturer's protocol. Three days posttransfection, the culture medium was 1071 harvested

cat# 09-740-114). The filtered medium was applied on a 5-ml of 1073

The 1075 column was washed with PBS and the pure human ACE2 protein (residues 18-740) 1076 was eluted using the PBS supplemented with 300 mM imidazole

Ultracel-3 regenerated cellulose membrane (Merck, cat# UFC900324), the buffer 1078 was exchanged to PBS and the purified protein was concentrated. The purity of 1079 prepared protein was analyzed by a Tycho NT

To prepare the plasmids with the 1086 mutated RBD, megaprimers were amplified by PCR using KAPA HiFi HotStart 1087 ReadyMix kit (Roche, cat# KK2601) and the following primers: RBD L452R forward 1088 5

TTG-3'; pCT_seq Reverse: 5'-CAT GGG AAA ACA TGT TGT 1093 TTA CGG AG-3'; and pCTCON_seq Forward: 5'-GCA GCC CCA TAA ACA CAC 1094 AGT AT-3', according to the manufacturer's protocol. The PCR products were 1095 integrated into pJYDC1 by integration PCR as previously described

Analysis of the Binding Affinity of the SARS-CoV-2 S RBD Variants to Human 1099 ACE2 by Yeast Surface Display 1100 The pJYDC1-based yeast display plasmids expressing SARS-CoV-2 RBD and its 1101 mutants were transformed into yeast

) overnight at 30°C (220 rpm) and used to inoculate the expression cultures in 1105 the 1/9 medium (Zahradník et al., 2021a) with 1 nM bilirubin (Sigma-Aldrich, cat# 1106 14370-1G). The cells were washed with PBS-B buffer [PBS supplemented with 1107 bovine serum albumin (1 g/l)] and aliquoted in analysis solutions. The analysis 1108 solutions consist of the PBS-B buffer with the 14 different concentrations (covering 1109 the range from 100 nM to 1 pM) of the human ACE2 protein (residues 18-740) that 1110 is labeled with CF640R succinimidyl ester (Biotium, cat# 92108). The volume of the 1111 analysis solution was adjusted (1-100 ml) in order to reduce the effect of ligand 1112 depletion

Subsequently, the yeasts were washed with the PBS-B buffer, passed through a 40-1115 µm cell strainer (SPL Life Sciences, cat# 93040), and the binding affinity to the 1116 CF640R-labeled human ACE2 protein (residues 18-740) was analyzed using an

The fluorescent signal was processed 1118 as previously described (Zahradník et al., 2021b) and the standard non

Hill equation was fitted by nonlinear least-squares regression using Python v3

HIV)-based, luciferase-expressing 1124 reporter viruses that are pseudotyped with the SARS-CoV-2 S protein and its 1125 derivatives, HEK293T cells (1 × 10 6 cells) were cotransfected with 1 μg

2020), and 1 μg of pWPI-Luc2

Two days posttransfection, the culture supernatants 1130 were harvested, centrifuged, and treated with 37.5 U/ml DNase I (Roche, cat# 1131 Sigma-Aldrich, cat# 11284932001) at 37°C for 30 min. The amount of the 1132 pseudoviruses prepared was quantified by HiBiT assay and the measured value was 1133 normalized to the level of HIV p24 antigen as previously described

2021) and 1138 500 ng of pC-ACE2 (a human ACE2 expression plasmid) (Ozono et al., 2021) using 1139 Lipofectamine 2000 (Thermo Fisher Scientific, cat# 11668019) according to the 1140 manufacturer's protocol. Two days posttransfection, the transfected cells (22,000 1141 cells/100 μl) were seeded into 96-well plates and infected with 100 μl of the 1142 pseudoviruses prepared at 4 different doses (1, 3, 5 and 10 ng of p24 antigen). Two 1143 days postinfection, the infected cells were lysed with One-Glo luciferase assay 1144 system (Promega

Lentiviral Transduction

Lentiviral transduction was performed as described previously

Briefly, the VSV-G-pseudotyped lenvirus vector expressing 1150 human ACE2 was generated by transfecting 2.5 μg of pLV-EF1a-human

IRES-Puro plasmid with 1.67 μg of pΔ-NRF (expressing HIV-1 gag, pol, rev, and tat 1152 genes

Two days posttransfection, the 1155 culture supernatants were harvested, centrifuged, and the supernatants were filtered 1156 with 0.45 µm pore size filter (Millipore, cat# SLGVR33RB) and collected as the 1157 lentiviral vector. The lenvirus vectors were concentrated by centrifugation (at 22,000 1158 × g for 2 h at 4°C) and the concentrated lentiviral vectors were inoculated into the 1159 target cells and incubated at 37°C. Two days posttransduction, the transduced cells 1160 were placed under the drug selection using the culture medium containing 1 µg/ml 1161 puromycin (Invivogen, cat# ant-pr-1). The puromycin-selected cells with relatively 1162 higher ACE2 expression were sorted by a FACS Aria II (BD Biosciences) and 1163 expanded

The L452R substitution (Figure 2F) was prepared using UCSF Chimera 1174 v1

Reverse Genetics 1177 Recombinant SARS-CoV-2 was generated by circular polymerase extension 1178 reaction (CPER) as previously described (Torii et al., 2021). In brief, the 9 DNA 1179 fragments encoding the partial genome of SARS-CoV-2 (strain

Additionally, a linker 1182 fragment encoding hepatitis delta virus ribozyme (HDVr), bovine growth hormone 1183 (BGH) polyA signal and cytomegalovirus (CMV) promoter was prepared by PCR. 1184 The corresponding SARS-CoV-2 genomic region and the templates and the primers 1185 of this PCR are summarized in Table S6. The obtained 10 DNA fragments were 1186 mixed and used for the CPER

One day posttransfection, the culture 1190 medium was replaced with Dulbecco's modified Eagle's medium (high glucose) 1191 (Sigma-Aldrich, cat# R8758-500ML) containing 2% FCS, 1% PS and doxycycline (1 1192 μg/ml; Takara, cat# 1311N)

Two days postinfection, the culture medium 1203 was harvested, centrifuged, and the supernatants were collected as the working 1204 virus. 1205 To generate the recombinant SARS-CoV-2 mutants, mutations were 1206 inserted into the fragment 8 (Table S6) using GENEART Site-Directed mutagenesis 1207 system (Thermo Fisher Scientific, cat# A13312) and the following primers: Fragment 1208 8_S L452R forward, 5'

To verify the inserted mutation in the working viruses, 1224 viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen, cat# 52906)

cat# 18080085) according to manufacturers' protocols. The DNA 1227 fragments including the mutations inserted were obtained by RT-PCR using 1228

Takara, cat# R050A) and the following primers: 1229 WK-521

Nucleotide sequences were determined as described above, and the sequence 1232 chromatograms (Figure 3A) were visualized using a web application Tracy 1233

Plaque Assay 1236

After removing the culture media, Vero cells were infected with 500 µl of 1241 the diluted virus at 37 °C. Two hours postinfection, 1 ml of mounting solution [1 × 1242 minimum essential medium containing 3% FCS and 1.5% carboxymethyl cellulose 1243 (Sigma, cat# C9481-500G)] was overlaid and incubated at 37 °C. Three days 1244 postinfection, the culture media were removed, and the cells were washed with PBS 1245 three times and fixed with 10% formaldehyde (Nacalai Tesque, cat# 37152-51) or 1246 4% paraformaldehyde (Nacalai Tesque, cat# 09154-85). The fixed cells were 1247 washed with city water, dried up, and stained with staining solution

000 cells of VeroE6/TMPRSS2 and 293-ACE2 cells 1254 were seeded into the 96-well plate. Recombinant SARS-CoV-2 (100 pfu) was 1255 inoculated and incubated at 37 °C for 1 h. The infected cells were washed and 1256 replaced with 180 µl of culture media. The culture supernatant (10 µl) was harvested 1257 at 0, 6, 24, 48 and 72 hours postinfection and used for real-time PCR to quantify the 1258 copy number of viral RNA

The amount of viral RNA in the culture supernatant was quantified by real-time RT-1262 PCR as previously described (Shema Mugisha et al., 2020) with some modifications. 1263 In brief, 5 μl of culture supernatants was mixed with 5 μl of 2 × RNA lysis buffer

40% glycerol, 0.8 U/μl 1265 recombinant RNase inhibitor (Takara, cat# 2313B)] and incubated at room 1266 temperature for 10 min. RNase-free water (90 μl) was added and the diluted sample 1267 (2.5 μl) was used as the template of real-time RT-PCR

PrimeScript PLUS RT-PCR kit (Takara, cat# RR096A) and the following primers

The copy number of viral RNA was standardized by 1272 SARS-CoV-2 direct detection RT-qPCR kit (Takara, cat# RC300A). The fluorescent 1273 signal was acquired on a QuantStudio 3 Real-Time PCR systems (Thermo Fisher 1274 Scientific), a CFX Connect Real-Time PCR Detection system (Bio-Rad) or a 7500 1275 Real Time PCR System (Applied Biosystems) was used. 1276 1277 QUANTIFICATION AND STATISTICAL ANALYSIS 1278 Data analyses were performed using Prism 7 (GraphPad Software)

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies FITC-conjugated anti-human CD3 antibody Biolegend Cat# 300440

BV510-conjugated anti-human CD3 antibody Biolegend Cat# 317331

APC-Cy7-conjugated anti-human CD8 antibody Biolegend Cat# 301016

5-conjugated anti-human CD14 antibody Biolegend Cat# 325622

5-conjugated anti-human CD19 antibody Biolegend Cat# 302230

PE-Cy7-conjugated anti-human CD25 antibody Biolegend Cat# 356107

APC-conjugated anti-human CD137 antibody Biolegend Cat# 309809; RRID: AB_830671 BV421-conjugated anti-human CD107a antibody Biolegend Cat# 328625

PE-conjugated anti-human IFN-γ antibody BD

PE-Cy7-conjugated anti-human TNF-α antibody Biolegend Cat# 502930

APC-conjugated anti-human IL-2 antibody Biolegend Cat# 500310; RRID: AB_315097 Anti-ACE2 antibody R&D systems Cat# AF933

APC-conjugated anti-goat IgG R&D systems Cat# F0108; RRID: AB_573124 Goat normal IgG R&D systems Cat# AB-108-C

RRID: AB_354267 Bacterial and Virus Strains SARS-CoV-2 (strain WK-521

MAFFT suite (v7.467) (Katoh and Standley

uk/software/figtree trimAl