key: cord-291790-z5rwznmv
authors: Li, Qianqian; Wu, Jiajing; Nie, Jianhui; Zhang, Li; Hao, Huan; Liu, Shuo; Zhao, Chenyan; Zhang, Qi; Liu, Huan; Nie, Lingling; Qin, Haiyang; Wang, Meng; Lu, Qiong; Li, Xiaoyu; Sun, Qiyu; Liu, Junkai; Zhang, Linqi; Li, Xuguang; Huang, Weijin; Wang, Youchun
title: The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity
date: 2020-07-17
journal: Cell
DOI: 10.1016/j.cell.2020.07.012
sha: 
doc_id: 291790
cord_uid: z5rwznmv

Summary The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious but variants including A475V, L452R, V483A and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious whilst deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is 25 critically important to investigate the biological significance of these mutations. Here we 26 investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to 27 a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several 28 variants containing both D614G and another amino acid change, were significantly more 29 infectious. Most variants with amino acid change at receptor binding domain were less infectious 30 but variants including A475V, L452R, V483A and F490L became resistant to some neutralizing 31 antibodies. Moreover, the majority of glycosylation deletions were less infectious whilst deletion 32 of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of 33 glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing 34 antibodies, whereas N165Q became more sensitive. These findings could be of value in the 35 development of vaccine and therapeutic antibodies. 36

COVID-19 pandemic is a tremendous threat globally. As of July 3, 2020, 216 countries have 38 reported COVID-19 cases, with more than 10 million confirmed cases and approximately 518,000 39 deaths (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). 40

The causative agent of COVID-19, SARS-CoV-2 causes a lower respiratory tract infection that 41 can progress to severe acute respiratory syndrome and even multiple organ failure (Lv et al., 42 2020a; Yang et al., 2020) . 43 SARS-CoV-2 is a single-stranded positive-strand RNA virus whose genome encodes four 44 structural proteins: spike (S), small protein (E), matrix (M) and nucleocapsid (N) (Chan et al., 45 2020) . The S protein is a type I fusion protein that forms trimers on the surface of the virion. It is 46 composed of two subunits, with S1 responsible for receptor binding and S2 for membrane fusion 47 7 single mutants were also constructed to compare with the double mutants with D614G. Group C is 112 comprised of 26 mutants at the putative glycosylation sites (22 sites). This group includes both 113 variants (N74K, N149H and T719A) and investigational mutants that we made for the analyses of 114 the effects of glycosylation. Specifically, all 22 sites (N to Q) were made in the lab to generate 22 115 individual mutants; we also made a combination by deleting the two glycosylation sites in RBD. 116 In total, we have generated 106 pseudotyped viruses, i.e., 80 variants and 26 glycosylation 117 mutants ( Figure 1 ). These viruses were prepared as described previously (Nie et al., 2020 ) (see 118 STAR Methods). 119

To determine the infectivity of these variants and mutants, we first infected 26 cell lines with 121 pseudotyped viruses with either SARS-CoV-2 S protein or VSVG protein (see STAR Methods). 122

As expected, the two types of pseudotyped viruses are different in the infection efficiency in the 123 26 cell lines ( Figure 2) . While almost all cell lines were generally susceptible to infection by VSV 124 G pseudotyped virus, SARS-CoV-2 pseudotyped virus could efficiently infect certain cell lines 125 including three human cell lines (293T-hACE2, 293T and Huh-7) and three non-human primate 126 cell lines (Vero, VeroE6 and LLC-MK2). As such, we selected these four out of the six cell lines 127 in subsequent experiments, including 293T-hACE2, Huh-7, Vero and LLC-MK2. 128

We first tested the infectivity of 106 pseudotyped viruses (80 natural variants and 26 129 glycosylation mutants) in 293T-hACE2 cells, where a difference by 4 -fold in RLU compared 130 with the reference Wuhan-1 strain (GenBank: MN908947) was deemed as being significant 131 ( Figure S1 ). Of all 106 pseudotyped viruses, 22 were determined as low-infectivity (16 natural 132 mutants and 6 glycosylation mutants), with RLU reading decreased by 4 to 100 folds ( Figure 3A) . 133

Among them, 13 were located in the RBD region. Variant V341I and investigational glycosylation 134 mutant (N331Q +N343Q) were deemed as no-infectivity as demonstrated by over 100-fold 135 8 decrease in RLU values compared with the reference strain. Both of them were located in RBD. It 136 is worth noting that double glycosylation deletions at N331 and N343 resulted in a drastic 137 reduction in viral infectivity (1200-fold), whereas single deletion at each site caused modest 138 reduction in viral infectivity, with the infectivity of N331Q reduced by only 3-fold and N343Q by 139 20-fold. Moreover, the non-natural double glycosylation mutations in RBD (N331Q and N343Q) 140 resulted in significantly reduced infectivity, suggesting that the two glycosylation sites in the RBD 141 region may participate in the binding of the receptor or maintain the conformation of the RBD 142 region. 143

The remaining 63 variants were tested further with other three cell lines for infectivity 144 suggesting that the enhanced infectivity was more likely ascribed to D614G itself. 149

antibodies 151

Having identified the variants with altered infectivity, we next set out to investigate the 152 antigenicity of the infectious mutants using 13 neutralizing monoclonal antibodies (mAbs) (see 153 STAR Methods). It was noted that some changes in RBD region demonstrated altered sensitivity 154 to neutralizing mAbs (Figure 4 and Figure S2 ). Specifically, A475V reduced the sensitivity to 155 mAbs 157, 247, CB6, P2C-1F11, B38 and CA1, while F490L reduced the sensitivity to mAbs 156 X593, 261-262, H4 and P2B-2F6. Moreover, V483A became resistant to mAbs X593 and 157 P2B-2F6, and L452R to mAbs X593 and P2B-2F6. Finally, Y508H reduced the sensitivity to 158 mAbs H014, N439K to mAb H00S022, A831V to mAb B38, D614G+I472V to mAb X593 and 159 D614G+A435S to mAb H014 by more than 4 times. In addition, some changes in the RBD region, 9 including V367F, Q409E, Q414E, I468F, I468T, Y508H and A522V, were observed to be more 161 susceptible to neutralization mediated by mAbs. 162

We next determine how infectious glycosylation mutants reacted to the same panel of mAbs. 163

Mutant N165Q actually became more sensitive to mAb P2B-2F6, whereas N234Q reduced the 164 neutralization sensitivity to different set of mAbs including 157, 247, CB6, P2C-1F11, H00S022, 165 B38, AB35 and H014. These results confirmed that these two glycosylation sites are important for 166 receptor binding. 167

These mAbs have proven to be valuable in our analyses of the amino acid changes. As shown 168

in Figure 4 , five mAbs, i.e., 157, 247, CB6, P2C-1F11 and B38, were unable to effectively 169 neutralize both A475V and N234Q. Neither X593 nor P2B-2F6 was effective in neutralizing 170 L452R, V483A and F490L whilst P2B-2F6 was more effective in neutralizing N165Q. In addition, 171 mAb H014 was incapable of neutralizing N234Q, Y508H and D614G+A435S while mAbs H4 172 and 261-262 were found not to neutralize F490L. Furthermore, Finally, H00S022 was unable to 173 neutralizing N439K and N234Q. 174

Finally, we determined the sensitivity of the strains with amino acid changes to ten 176 COVID-19 convalescent sera (see STAR Methods). None of the variants and mutants 177 demonstrated significantly altered sensitivity to all 10 convalescent sera, i.e., the EC50 values 178

were not altered by more than 4-fold, irrespective of an increase or decrease, when compared with 179 the reference strain ( Figure 5A and Figure S3 ). However, the neutralization sensitivity of both 180 F490L and H519P to three of ten patient sera were found to have decreased by more than 4 times, 181 while six variants and mutants (N149H, N149Q, N165Q, N354D, N709Q and N1173Q) became 182 over 4-fold sensitive to one or two of the ten tested sera. Notably, five out of the six were glycan 183 deletion mutants. 184

As shown in Figure 5B , when the data of individual convalescent sera were pooled together 185 to analyze the sensitivity of all variants, no marked difference was observed (>4 fold). However, 186 modest differences between some variants and reference strain (within 4-fold) were observed in 187 their reactivity to grouped convalescent sera. These differences were statistically significant 188 (P<0.05). It is worth mentioning that some variants including F338L, V367F, I468F, I468T and 189 V615L ( Figure 5B ) were even more sensitive to the convalescent sera compared with reference 190 strain, whereas more variants were found to be resistant to the convalescent sera. These variants 191 include single amino acid change such as Y145del, Q414E, N439K, G446V, K458N, I472V, 192 A475V, T478I, V483I, F490L and A831V, as well as the double amino acid changes including 193 D614G + Q321L, D614G +I472V, D614G +A831V, D614G +A879S and D614G +M1237I. 194 Similar to natural variants, although the magnitude of some glycosylation deletions in 195 sensitivity to the sera is less than 4-fold, the differences between mutants and the reference strain 196 (Wuhan-1) were found to be still several-folds and statistically significant, i.e., glycosylation 197 mutants N331Q and N709Q significantly increased the sensitivity to convalescent sera ( and ambiguous sequences, we narrowed down to 80 variants. Moreover, as glycosylation of viral 206 protein is well documented to affect viral replication and immune response and SARS-CoV-2 S 207 protein is heavily glycosylated, we also made 26 substitutional mutations at all 22 putative 208 glycosylation sites. In total, we made 106 pseudotyped viruses, allowing us to characterize them 209 using the established method (Nie et al., 2020) (see STAR Methods). 210 Table 1 summarize the characteristics of variants and investigational mutants. Of all variants, 211 D614G is of particular note. This variant has been shown to rapidly accumulating since its 212 emergence and linked to more clinical presentations (Korber et al., 2020) . At the beginning of this 213 study (May 6, 2020), it accounted for 62.8% of all circulating strains, but by July 3, it had reached 214 75.7%. This dominant strain could effectively infect the four cell lines tested, being 10-fold more 215 infectious than the original Wuhan-1 strain ( Figure 3) . 216

Another important finding is that natural variants capable of affecting the reactivity to 217 neutralizing mAbs were almost all located in the RBD region (except A831V and 218 D614G+A831V), as all antibodies used in this study were targeting the RBD ( with decreased sensitivity to neutralization by P2B-2F6 mAb; as both L452R and F490L remain 225 sensitive to P2C-1F11, suggesting this mAb is not derived from the same clone for P2B-2F6. 226

Moreover, both mutants displayed decreased sensitivity to another neutralizing mAb X593 by 227 10-fold compared with the reference strain (Figure4). 228

While we identified multiple variants with decreased sensitivity to neutralizing mAbs, we 229 need to look at how frequent these variants are in the field. V483A in RBD is one of the two 230 variants with a mutation frequency of over 0.1%. It showed decreased reactivity to the two mAbs 231 (P2B-2F6 and X593) ( Figure 6A and 6B) (Ju et al., 2020) . Another RBD variant A475V sits in the 232 binding epitope of RBD. It is significantly resistant to several neutralizing mAbs including 233 P2C-1F11, CA1, 247 and CB6. It is noteworthy that CB6 mAb targets the receptor binding 12 epitope ( Figure 6C and 6D) (Shi et al., 2020) . Specifically, Y508 was buried in the epitope 235 targeted by mAb H014 (Figure 6E and 6F) (Lv et al., 2020b) . Indeed, the Y508H was found to be 236 resistant to this mAb. 237

It is worth mentioning that D614G+I472V has shown increased infectivity and more 238 resistance to neutralizing antibodies (Table 1 ), but only one sequence (originated from Canada) 239 was reported in GISAID. Moreover, some variants, including N439K, L452R, A475V, V483A, 240 F490L and Y508H, do have decreased sensitivity to neutralizing mAbs. However, only V483A 241 exceeded 0.1% in frequency at the beginning of the study, all of which were found in US, with 28 242 sequences reported as of May 6, 2020, and 36 up to July 3, 2020. Variants containing N439K 243 showed a significant increase in circulation, i.e., with 5 case reported as of May 6, 2020 (all in UK) 244 to 47 by July 3, 2020 (45 in UK, 2 in Romania). In addition, only one sequence from France 245 containing Y508H was deposited in GIRSAID as of May 6, while four sequences reported as of 246

July 3, 2020, of which two originated from Netherlands, one from Sweden, and one from France. 247

Only one or two isolates were reported for other variants, which have not been observed to have 248 increased during the time frame we have been monitoring. Nevertheless, as RNA viruses mutate 249 all the time and some variants may only appears during certain period of time, while others could 250 emerge in an unpredictable fashion, continued analyses of the circulating strains in terms of the 251 mutation frequency and temporal pattern are warranted. 252

Our results suggest that the 13 mAbs used in this study could be divided into seven groups as 253 they appear to be different in the inhibitory effects on the variants. As such, it would be interesting 254 to formulate a therapeutic regimen comprised of at least two mAbs. For example, a combination 255 of P2C-1F11 and X593 should be effective to inhibit all variants in this study. It would be of 256 interest to test more neutralizing antibodies which could be targeting epitopes outside RBD. 257

With regard to the glycosylation mutants analyzed in this study, N165Q increased the 258 sensitivity to mAb P2b-2F6 whilst N234Q displayed resistance to neutralizing mAbs such CA1, 259 CB6, 157 and others. Although neither of them is found in circulation, the reactivity of these two 260 13 mutants to neutralizing mAb is still worth noting. As N165 and N234 are located near the RBD 261 region (Watanabe et al., 2020) , these mutants may affect some epitopes targeted by neutralizing 262 mAbs. Specifically, N165 glycosylation site is involved in the binding of mAb to the RBD region 263 of S protein (Cao et al., 2020) . It is likely that the sugar chain can mask the epitope targeted by the 264 antibody. This type of glycan shield has been observed in other virus such as HIV-1. Specifically The use of sera from 10 convalescent patients in neutralizing assay largely confirmed the 277 results obtained with the well characterized neutralizing mAbs. It is understood that the magnitude 278 of altered reactivity is slightly smaller with human sera than that with mAbs, given that polyclonal 279 antibodies from convalescent patents are directed against multi-epitopes on the full-length S 280 protein; as a result, these polyclonal antibodies could complement one another. However, the 281 differences in their reactivity to the human antibodies were found to be by several folds in most 282 cases and all determined as statistically significant. Notably, some RBD variants such as A475V 283 and F490L have been confirmed to have decreased sensitivity to both human sera and multiple 284 neutralizing mAbs. A475V reduced the sensitivity to 6 mAbs out of the 13 mAb used in this study, 285 while F490L reduced the sensitivity to neutralization by 3 mAbs. It is possible that antibodies in 286 14 convalescent sera are able to neutralize these critical epitopes targeted by these mAbs that are 287 known to disrupt the binding of the S protein to hACE2 receptor (Ju et Serial dilutions of mAb preparations were pre-incubated with the pseudotyped viruses at 37°C for 355 one hour before they were added to Huh-7 cells. Luciferase activity was measured 24 hours later 356 to calculate EC50 of each antibody. The ratio of EC50 between the variant or mutant strains and 357 the reference strain (Wuhan-1) was calculated and analyzed to generate heatmap using Hem I 17 (Deng et al., 2014) . The data were the results from 3-5 replicates. The red and blue boxes indicate 359 the increase or decrease of the neutralization activity as shown in the scale bar. See also Figure S2 . Serial dilutions of mAb preparations were pre-incubated with the virus at 37°C for one hour 393 before they were added to Huh-7 cells. Luciferase activity was measured 24 hours later to 394 calculate EC50 of each antibody. The y-axis represents the ratio of EC50 between the 395 variant/mutant strain and the reference strain (Wuhan-1). The data were the results from 3-5 396

replicates. The horizontal dashed lines indicate the threshold of 4-fold difference. The significant 397 changes were marked with colored symbols, blue for decreased, red for increased. Related to 398 

Further information and requests for resources and reagents should be directed to and will be 409 fulfilled by the Lead Contact, Dr. Youchun Wang (wangyc@nifdc.org.cn). 410

All the unique reagents generated in this study are available from the Lead Contact with a 412 completed Materials Transfer Agreement. 413

This study did not generate any unique datasets or code. primers. Following site-directed mutagenesis PCR, the template chain was digested using DpnI 452 restriction endonuclease (NEB, USA). Afterwards, the PCR product was directly used to 453 transform E. coli DH5α competent cells; single clones were selected and then sequenced. The 454 primers designed for the specific mutation sites are listed in Table S2 , and the frequency of 455 different variants in the epidemic population is listed in Table S1 . 456 Highlights Over 100 mutations were selected for analyses on their infectivity and antigenicity The dominant D614G itself and combined with other mutations are more infectious Ablation of both N331 and N343 glycosylation at RBD drastically reduced infectivity Ten mutations such as N234Q, L452R, A475V, V483A was markedly resistant to some mAbs

Eighty natural variants and twenty-six glycosylation spike mutants of SARS-CoV-2 were analyzed in terms of infectivity and antigenicity using high throughput pseudovirus assay in conjunction with neutralizing antibodies. Reference  L5F  L8V  L8W  H49Y  Y145del  F338L  P384L  N354D  N354K  S359N  V367F  K378R  P384L  R408I  Q409E  Q414E  A435S  N439K  G446V  L452R  K458R  K458N  I468F  I468T  I472V  A475V  G476S  T478I  V483A  V483I  F490L  Y508H  H519P  H519Q  A520S  A522S  A522V  V615L  A831V  D839E  D936Y  S943T  S943R  G1124V  Y145del+R408I  D614G+Q239K  D614G+Q321L  D614G+V341I  D614G+A435S  D614G+K458R  D614G+I472V  D614G+H519P  D614G+A831V  D614G+A845S  D614G+A879S  D614G+D936Y  D614G+S939F  D614G+S943T  D614G+M1229I  D614G  D614G+M1237I  D614G+P1263L  D614G+L5F  N17Q  N61Q  N74Q  N149Q  N165Q  N234Q  N282Q  N331Q  N603Q  N616Q  N657Q  N709Q  N1098Q  N1134Q  N1158Q  N1173Q  N1194Q  N74K  N149H  A   B   Reference   CS1   CS2   CS10   CS3   CS86   CS7   CS4   CS87   CS6   CS8   L5F  L8V  L8W  H49Y  Y145del  F338L  A348T  N354D  N354K  S359N  V367F  K378R  P384L  R408I  Q409E  Q414E  A435S  N439K  G446V  L452R  K458R  K458N  I468F  I468T  I472V  A475V  G476S  T478I  V483A  V483I  F490L  Y508H  H519P  H519Q  A520S  A522S  A522V  D614G  V615L  A831V  D839E  D936Y  S943T  S943R  G1124V  Y145del+R408I  D614G+L5F  D614G+Q239K  D614G+Q321L  D614G+V341I  D614G+A435S  D614G+K458R  D614G+I472V  D614G+H519P  D614G+A831V  D614G+A845S  D614G+A879S  D614G+D936Y  D614G+S939F  D614G+S943T  D614G+M1229I  D614G+M1237I  D614G+P1263L  N17Q  N61Q  N74Q N149Q Reference  L5F  L8V  L8W  H49Y  Y145del  F338L  A348T  N354D  N354K  S359N  V367F  K378R  P384L  R408I  Q409E  Q414E  A435S  N439K  G446V  L452R  K458R  K458N  I468F  I468T  I472V  A475V  G476S  T478I  V483A  V483I  F490L  Y508H  H519P  H519Q  A520S  A522S  A522V  D614G  V615L  A831V  D839E  D936Y  S943T  S943R  G1124V  Y145del+R408I  D614G+L5F  D614G+Q239K  D614G+Q321L  D614G+V341I  D614G+A435S  D614G+K458R  D614G+I472V  D614G+H519P  D614G+A831V  D614G+A845S  D614G+A879S  D614G+D936Y  D614G+S939F  D614G+S943T  D614G+M1229I  D614G+M1237I  D614G+P1263L  N17Q  N61Q  N74Q  N149Q  N165Q  N234Q  N282Q  N331Q  N603Q  N616Q  N657Q  N709Q  N1098Q  N1134Q  N1158Q  N1173Q  N1194Q  N74K 

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Pseudotyped viruses incorporated with spike protein from either SARS-CoV-2, variants or 458 mutants were constructed using a procedure described by us recently (Nie et al., 2020) . On day 459 before transfection, 293T cells were prepared and adjusted to the concentration of 5 -7 × 10 5 460 cell/ml, 15 ml of which were transferred into a T75 cell culture flask and incubated overnight at 461 37 0 C in an incubator conditioned with 5% CO 2 . The cells generally reach 70-90% confluence after 462 overnight incubation. Thirty microgram of DNA plasmid expressing the spike protein was 463 transfected according to the user's instruction manual. The transfected cells were subsequently 464 infected with G*∆G-VSV (VSV G pseudotyped virus) at concentration of 7.0 × 10 4 TCID50/ml. 465These cells were incubated at 37°C for 6-8 hours in the presence of in 5% CO 2 . Afterwards, cell 466 supernatant was discarded, followed by rinsing the cells gently with PBS +1% FBS. Next, 15ml 467 fresh complete DMEM was added to the flask and cultured for 24 h. Twenty-four hours post 468 infection, SARS-CoV-2 pseudotyped viruses containing culture supernatants were harvested, 469 filtered (0.45-µm pore size, Millipore, Cat#SLHP033RB) and stored at −70°C in 2-ml aliquots 470 until use. 471The 50% tissue culture infectious dose (TCID50) of SARS-CoV-2 pseudovirus was 472 determined using a single-use aliquot from the pseudovirus bank to avoid inconsistencies resulted 473 from repeated freezing-thawing cycles. 474For titration of the pseudotyped virus, a 2-fold initial dilution with six replicates was made in 475 96-well culture plates followed by serial 3-fold dilutions. The last column was employed as the 476 cells control without pseudotyped virus. Subsequently, the 96-well plates were seeded with HuH-7 477 cells adjusted to 2×10 5 cells/ml. After 24 h incubation at 37°C in a humidified atmosphere with 5% 478 CO 2 , the supernatant was aspirated and discarded gently to leave 100 µl in each well; next, 100 µl 479 of luciferase substrate (Perkinelmer, Cat#6066769) was added to each well. After 2-min 480 incubation at room temperature in the dark, 150 µl of lysate was transferred to white 96-well 481 plates for the detection of luminescence using a luminometer (PerkinElmer, Ensight). Positive was 482 23 determined to be ten-fold higher than the negative (cells only) in terms of relative luminescence 483 unit (RLU) values. The 50% tissue culture infectious dose (TCID50) was calculated using the 484 Reed-Muench method (Nie et al., 2020) . 485

Before quantification, all the pseudotyped viruses were purified through a 25% sucrose cushion by 487 ultra-centrifugation at 100,000× g for 3 h (Nie et al., 2020) Resources Table. 495

Using the quantitative RT-PCR, we normalized the pseudotyped virus particles to the same 497 amount. After normalization, 100 µl of the pseudotyped virus with 10-fold dilution was added to 498 wells in 96-well cell culture plate. After the cells were trypsin-digested, 2×10 4 /100 µl cells were 499 added to each well in the 96-well plates. The plates were then incubated at 37°C in a humidified 500 atmosphere with 5% CO 2 . After incubation for 24 hours, chemiluminescence detection was 501 performed as described in the titration of pseudotyped viruses. Each group contained 3-5 502replicates. 503

The virus neutralization assay was conducted as described previously (Nie et al., 2020) . Briefly, 505 100 µl serial dilutions of human sera or monoclonal antibody preparations were added into 506 96-well plates. After that, 50 µl pseudoviruses with concentration of 1300 TCID50/ml were added 507 into the plates, followed by incubation at 37°C for 1 hour. Afterwards, HuH-7 cells were added 508 24 into the plates (2×10 4 cells/100 µl cells per well), followed by incubation at 37°C in a humidified 509 atmosphere with 5% CO 2 . Chemiluminescence detection was performed after 24 hours incubation. 510The Reed-Muench method was used to calculate the virus neutralization titer. The results are 511 based on 3-5 replicates unless specified. In order to validate the test operation process, the 512 Coefficient of Variance (CV) control of replicates is set within 30% of six wells, so is the CV for 513 the duplicate sample wells. 514

GraphPad Prism 8 was used for plotting and statistical analysis; the values were expressed as 517 mean ±SEM. One-way ANOVA and Holm-Sidak's multiple comparisons test was used to analyze 518 the differences between groups. A P-value of less than 0.05 was considered to be significant. * 519 P<0.05, ** P<0.01, *** P<0.005, **** P<0.001, ns represents no significant difference. 520