key: cord-0333736-vhryv6rj
authors: Escalera, Alba; Gonzalez-Reiche, Ana S.; Aslam, Sadaf; Mena, Ignacio; Pearl, Rebecca L.; Laporte, Manon; Fossati, Andrea; Rathnasinghe, Raveen; Alshammary, Hala; van de Guchte, Adriana; Bouhaddou, Mehdi; Kehrer, Thomas; Zuliani-Alvarez, Lorena; Meekins, David A.; Balaraman, Velmurugan; McDowell, Chester; Richt, Jürgen A.; Bajic, Goran; Sordillo, Emilia Mia; Krogan, Nevan; Simon, Viviana; Albrecht, Randy A.; van Bakel, Harm; Garcia-Sastre, Adolfo; Aydillo, Teresa
title: SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage
date: 2021-08-05
journal: bioRxiv
DOI: 10.1101/2021.08.05.455290
sha: 2fcbc3b88f5b22d65bd9e930f6072d70cf69382a
doc_id: 333736
cord_uid: vhryv6rj

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2’ by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

, indicating a potential role in host replication, 99 transmissibility, and pathogenicity. 100 101

Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to 102 understand their impact on transmissibility, virus pathogenicity and fitness. Using the mink model 103

of COVID-19, we found that the S:H655Y substitution was acquired in vivo after infection with 104 the WA1 isolate (USA-WA1/2020). To investigate the advantage conferred by S:H655Y, we 105 analyzed the kinetics, spike processing by cellular proteases and syncytium formation ability of a 106 panel of SARS-CoV-2 variants harboring 655Y, including human isolates derived from patients 107 seeking care at the Mount Sinai Health System in New York (NY) City which was one of the major 108 early epicenters of COVID-19 pandemic. Our results demonstrated that the 655Y polymorphism 109 enhances spike cleavage and viral growth. Furthermore, the S:655Y substitution was transmitted 110 more efficiently than its ancestor S:655H in the hamster infection model. Finally, and in the context 111 of the current epidemiological situation, we analyzed a set of emerging SARS-CoV-2 variants to 112

investigate how different sets of mutations may impact spike processing. We demonstrated that 113 novel circulating VOCs that became more prevalent have independently acquired mutations 114 associated with a gain in spike cleavage and syncytia formation. Taken together, our study shows 115 a link between an increased spike processing and increased virus transmission due to spike 116 mutations present in SARS-CoV-2 variants that become epidemiologically more prevalent in 117

humans Minks have been suggested to play a role in the initial local spread and evolution of SARS-CoV-126 2 variants in different countries in Europe (3, 4). While minks are susceptible to SARS-CoV-2, 127 they are also capable for zoonotic transmission of SARS-CoV-2 to because of the similarity of the 128 ACE2 receptor between minks and humans. We used the mink model to investigate the replication 129 and pathogenicity of the WA1 (USA-WA1/2020) isolate of SARS-CoV-2, as a representative of 130 the first original human viruses that initiated the SARS-CoV-2 pandemic. This variant corresponds 131 to one of the first USA isolates and does not contain any changes on the S protein when compared 132

to the initial isolates from Wuhan, such as the Wuhan-1 virus. For this purpose, six minks were 133 intranasally infected with 10 6 pfu of WA1 isolate resulting in productive viral replication in the 134 upper respiratory tract with infectious virus recovered from nasal washes at days 1, 3 and 5 post-135 infection (Supplementary Figure 1A-B) . At day 4 post-inoculation, infectious virus was detected 136 by plaque assays from left cranial lung and nasal turbinates but not from any of the other tissues 137 analyzed (Supplementary Figure 1C) . We then selected small and large viral plaques in the Vero-138 E6 cell-based plaque assays from infected mink lung specimens and performed next generation 139 sequencing of the genome from the plaque-isolated viruses. As compared to the Wuhan-1 and 140 WA1 reference sequences, all mink-derived viral isolates encoded the H655Y amino acid 141 substitution within the spike (S) ( Figure 1A) . Additionally, the three viral isolates with the small 142 plaque phenotype encoded the T259K amino acid substitution while the three viral isolates with 143 the large plaque phenotype encoded the R682W amino acid substitution. It is known that 144 S:682W/Q substitution in the furin cleavage site region may emerge after subsequent passages in 145

VeroE6 cells (29, 30) . Therefore, this mutation may have been selected during the course of the 146

VeroE6 infections and not during the infection in minks. On the other hand, S:655Y appeared 147 dominant in all the mink isolates, indicating that this mutation may confer an advantage in the 148 mink host. 149 150

To understand the magnitude and the spread of the 655Y polymorphism over time, we investigated 151 the frequency of S:655Y over time in sequences sampled worldwide since the initial outbreak to 152 the end of the first wave of SARS-CoV-2 ( Figure 1B ). For this, 7,059 sequences sampled from 153 GISAID up to September 2020 were used. Human variants harboring the 655Y mutation were 154 spread throughout the phylogenetic tree and distributed in all clades with no differences according 155

to temporal distribution, suggesting that the 655Y mutation arose independently multiple times. 156 Remarkably, the S:H655Y polymorphism was also found among the initial variants introduced in 157

New York (NY) City in March 2020. To determine the replication phenotype, we decided to 158 investigate this NY 655Y variant (NY7) together with some of its contemporaneous SARS-CoV-159 2 isolates circulating in New York during the early pandemic outbreak (31). To this end, we 160 isolated 12 viruses based on their genotypes (31), including NY7 which carries the S:655Y 161 mutation for culture directly from nasopharyngeal specimens obtained from COVID-19 infected 162

patients. Of note, the dominant 614G spike polymorphism was present in seven (58%) of the 163 selected human SARS-CoV-2 (hCoV) NY isolates consistent with its early emergence and rapid 164 spread worldwide (17, 18) . Confirmation sequencing of the isolates showed that 682W/Q 165 substitutions appeared in four (33%) viruses after initial isolation and culturing in VeroE6 cells. 166

This is consistent with in vitro adaptative mutations previously described (30). Moreover, a five 167 amino acid sequence (Δ675-679) flanking the furin cleavage site was deleted in five (42%) of the 168

isolates as compared to the sequence from the original specimen. This deletion has been previously 169 reported to be a common in vitro mutation selected in Vero cells (29). Amino acids substitutions 170 of the S protein of these initial human isolates compared to the Wuhan-1 reference are shown 171 Figure 1C . We next studied the replication kinetics of the NY SARS-CoV-2 isolates by comparing 172 their multicycle growth curves at an MOI of 0.01 in VeroE6 and human Caco-2 cells. As expected, 173 NY2 and NY4 containing the 682Q/W showed an advantage in VeroE6, while no differences could 174 be found in Caco-2 cells (Supplementary Figure 2A 2B) when compared to the rest of these early SARS-CoV-2 isolates. These results support our 177 conclusion that the 655Y polymorphism conferred a viral advantage. of TMPRSS2, consistent with enhancement of cell entry ( Figure 2C ). However, differences were 201 found by Western blot and only the isolates bearing 655Y showed enhanced spike cleavage in both 202

VeroE6 and Vero-TMPRSS2 ( Figure 2D ). Finally, and to investigate the ability to induce syncytia, 203

we infected Vero-TMPRSS2 at an MOI of 0.01 with the mink (MiA1) and human isolates (NY7, 204 NY13 and NY6) and used specific antibodies to detect the S and N protein, as well as nuclei 205 staining with DAPI after 24 p.i. by immunofluorescence microscopy. As shown in Figure 2E , experiments. For this, one hamster of each pair was infected intranasally with 10 5 pfu of a mix of 213 WA1 and WA1-655Y viruses at a one-to-one ratio ( Figure 3A ). Direct infected (DI) and direct 214 contact (DC) hamsters were euthanized after day 5 and 7 post-infection, respectively, and lungs 215 and nasal turbinates were harvested for subsequent viral titer quantification. In addition, nasal 216 washes were collected on day 2 and 4 in both DI and DC, and on day 6 p.i. of DC animals. One of 217 the DI hamsters died after nasal wash collection at day 2, leaving 4 animals subjected to follow up 218 in the DI group. Hamsters were also monitored daily for body weight loss. After 2 days p.i. DC 219 hamsters exhibited a decrease in weight indicating early viral transmission from infected animals 220 ( Figure 3B ). This observation was further supported by detection of infectious virus in nasal 221 washes at 2 days p.i in both DI and DC hamsters. At day 4 p.i., viral titers were detected in 3 out 222 of 4 animals and at day 6 p.i., viral replication was not detected in two of the DC nasal wash 223 samples ( Figure 3C ). In general, we observed a decrease over time in the infectious virus present 224 in nasal washes from DI and DC animals. We then determined the relative abundance of S:655Y 225

on the viral RNA present in the nasal washes by next-generation sequencing ( Figure 3D ). when compared to the rest of the animals. Next, we analyzed the viral growth in lungs and nasal 232 turbinates from DI (collected at day 5 p.i.) and DC (collected at day 7p.i.) hamsters ( Figure 3E ). 233

No differences were found in viral titers in the tissues from both animal groups. However, we 234 observed that three DC hamsters had lower lung titers compared to the rest of the animals. These 235 same hamsters also exhibited low viral loads in the nasal turbinates. We then sequenced the viral 236 RNA present in these tissues ( Figure 3F -G). The RNA from one lung and nasal turbinate of one 237 DI and DC hamster could not be amplified by specific PCR for downstream sequencing. Figure  238 3F-G shows that all lungs and nasal turbinate tissues from DI animals analyzed had the S:655Y 239 mutation. In addition, S:655Y was present in 75% of the nasal turbinates and lungs from DC 240 animals. also found to be a highly variable position and present in widely circulating variants (36). We then 259 spatially and structurally mapped these amino acid changes within and surrounding the furin 260 cleavage sequence of the S protein ( Figure 4A ). The 655 position was located in close proximity 261

to the furin cleavage site. Next, we performed a phylogenetic analysis of sequences sampled 262 worldwide from February 2020 to June 2021 to illustrate the temporal distribution and 263 phylogenetic relationship of the high prevalent S mutations ( Figure 4B ). For this, a sample set of 264 13,847 sequences deposited in GISAID up to June 2021 were analyzed. While the H665Y 265 frequency was higher in the Gamma lineage (P.1), it could be found also in 19B clade ( Figure 4C ), 266

in line with our identified NY7 isolate. The P681H substitution located in the furin cleavage site 267 of the spike protein was identified in the Alpha variant that emerged in September 2020. 268

Interesting, this mutation was also found in the Theta variant, first detected in February 2021 269 ( Figure 4D ). In contrast, Kappa and Delta variants harbor polymorphism P681R ( Figure 4D ). 270

Finally, the frequency of A701V mutation was higher in Beta and Iota variants which emerged in 271

October and November 2020, respectively ( Figure 4E ). 272 273

Next, we analyzed the in vitro phenotype of some of the most prevalent SARS-CoV-2 VOCs. 274

Multiple protein sequence alignment of the VOCs used are shown in Figure 5A . ( Figure 5B ). Additionally, viral supernatants were titrated on both VeroE6 and Vero-TMRPSS2 280 cells ( Figure 5C ). Substantial differences in plaque phenotypes were observed, especially for the 281 Kappa (B. Figure 5D ). N protein was used as a control for viral replication and loading. Similarly, WA1 287 was included as a reference since no selective mutations are found in the S protein. Figure 5D  288 shows similar cleaved and uncleaved S protein levels for all the SARS-CoV-2 VOCs in the 289 presence of TMRPSS2 expressed in Vero-TMPRSS2 cells. In contrast, only Beta (B.1.351) and 290

Gamma (P.1), exhibited an increased spike cleavage when the infections were performed in wild 291 type VeroE6 cells. Interestingly, the spike and nucleocapsid expression of Kappa (B.1.617.1) and 292

Delta (B.1.617.2) variants was not detectable by Western blot analysis of VeroE6 supernatants. 293

The canonical cleavage at the S1/S2 site occurs at the last arginine (R) of the multibasic PRRAR 294 motif and is performed by furin proteases at this specific residue. Thus, we next quantified the 295 abundance of furin-cleaved peptide of VOCs in Vero-TMPRSS2 cell supernatants by targeted 296 mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs 297 and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cell extracts 298

were collected after 24 hours post-infection and samples were prepared. The abundance of the C-299

terminal peptide resulting from endogenous furin cleavage at the terminal arginine (PRRAR \ 300 SVASQSIIAYTMSLGAE) was quantified as a proxy of cleavage efficiency since this peptide is 301 common for all SARS-CoV-2 VOCs, except for the Beta that contained a V instead of A at the 302 end of the peptide (SVASQSIIAYTMSLGVE). Fold change peptide-level abundance for each 303 variant compared to WA1 control was calculated and plotted in Figure 5E . Isolates WA1-655Y, 304

NY7 and Gamma, all of them harboring the 655Y mutation, and Beta harboring 701V, showed the 305 higher abundance of furin-cleaved peptide. Conversely, lower levels of C-terminal cleaved peptide 306

were found for the VOCs harboring the 681H/R amino acid change suggesting that an introduction 307 of an amino acid change at this position might modify the canonical cleavage residue at the last R 308 of the furin cleavage site. Nonetheless, when we assessed the fusogenic capacity of the S protein 309

of VOCs by immunofluorescence microscopy of infected Vero-TMPRSS2 cells, we found strong 310 syncytium formation induced by all the variants ( Figure 5F ). Interestingly, extensive fusogenic 311 capacity was also exhibited by Delta and Kappa variants ( Figure 5F -G) consistent with abundant 312 cleaved S form found by Western blot ( Figure 5D ). Because cleavage at the multibasic furin motif 313 is believed to be required for optimal syncytium formation (10), we finally compared the ability 314 to induce cell fusion by the Kappa variant and a mutated form lacking amino acids at the furin 315 cleavage site (S: Δ678-682). This Δ678-682 Kappa was obtained after consecutive passage and 316 culturing in VeroE6 cells. As shown in Figure 5G , a loss of fusogenic activity was observed when 317 compared to the intact Kappa VOC. Altogether, our results are consistent with the notion that 318 current highly transmissible circulating VOCs have evolved independently to acquire mutations 319 associated with increased spike protein processing and transmission. 320 321

Discussion 322 323

Emerging SARS-CoV-2 VOCs contain novel spike polymorphisms with unclear functional 324 consequences on epidemiology, viral fitness, and antigenicity. In this study, we evaluated the 325 impact of different spike mutations on viral infection, pathogenicity, and in vivo transmission. We 326

found that in the mink animal model the 655Y spike substitution is selected after infection with 327 the WA1 isolate. Phylogenetic analysis of genome sequences collected worldwide showed an early 328 sporadic appearance of S:655Y during the first pandemic wave in New York in March 2020, and 329 the presence of this mutation in several posterior lineages, including SARS-CoV-2 Gamma 330 variant, pointing to a potential role in adaptation and evolution. To better understand the impact of 331 this polymorphism, we isolated and in vitro characterized a panel of SARS-CoV-2 viruses bearing 332 the 655Y spike mutation. Our results demonstrated that S:655Y enhances the viral growth and the 333 spike protein processing required for optimal cell entry and viral-host membrane fusion. In 334 addition, we performed viral competition and transmission experiments in the hamster animal 335 model and showed that S:655Y became predominant in both direct infected and direct contact 336

animals. Finally, we showed that VOCs converge to gain spike cleavage efficiency and fusogenic 337 potential. 338 339

Here, we demonstrate that viruses containing the H655Y polymorphism confer a growth advantage 340

in both VeroE6 and human-like Vero-TMPRSS2 cells. Interestingly, the early human isolate NY7 341

harboring the 655Y mutation also showed higher replication in human Caco-2 cells. However, it 342 is known that other mutations outside of the S gene could be impacting viral replication and 343 infection (37, 38). Therefore, we confirmed the S:655Y mutation alone was responsible for the 344 enhanced growth and spike cleavage phenotype when comparing WA1 wild type and WA1-655Y 345

isolates. These variants have the same viral protein amino acid sequence except for the amino acid 346 present at position 655 of the spike. Since most of the isolates used in this study contain a 347 constellation of mutations across the genome that could increase viral fitness, comparison of both 348 viruses in parallel allowed to detect differences in growth and spike cleavage that can be attributed 349 only to 655Y polymorphism. S:655Y is present in the S1 spike domain outside of the RBD and 350

has been associated with a decrease of the neutralizing activity when targeted by some monoclonal 351 antibodies(26). However, H655Y has been also naturally selected in cats and mice suggesting a 352 beneficial impact of this substitution in widen viral host range and susceptibility (27, 28). Our data 353 further supports this argument because we also found that S:655Y is selected after replication in 354 minks, a natural host for SARS-CoV-2. Besides, when we assessed the viral transmission 355 efficiency of 655Y versus the ancestor 655H in competition experiments in the hamster model, we 356 also found that 655Y becomes more prevalent, as the bulk of infectious viruses recovered from the 357 infected animals harbored this mutation, except for one hamster. This indicates that S:655Y can 358 overcome S:655H in vivo. 359 360

Intense worldwide surveillance has established that SARS-CoV-2 variants are constantly 361 emerging. In particular, the spike protein has shown high plasticity (6). Most of the spike mutations 362 associated with a decrease in neutralization by antibodies against earlier viruses are located in the 363 RBD or N-terminal domain (NTD), which are critical for binding and interacting with the ACE2 364 cellular receptor. While mutations at these domains may impact SARS-CoV-2 vaccine efficacy, it 365

is also vital to characterize other mutations that might explain the gain in transmissibility observed 366 for the VOCs. Since the Gamma variant that emerged in November 2020 also harbors the 655Y 367 polymorphism ( Figure 5A ), we decided to investigate its phenotype in vitro. Similar to the earlier 368 S:655Y isolates, this variant also exhibited an increase in spike processing efficiency. More 369 importantly, this phenotype was also confirmed in all emerging VOCs analyzed when infections 370

were performed in the Vero-TMPRSS2 cells indicating that additional mutations within S confer 371 this advantage. Most likely, the spike mutations P681H in Alpha variant -first identified in United 372

Kingdom-and P681R harbored by Kappa and Delta variants -first emerged in India-allowed this 373 enhanced S cleavage. Interestingly, for these variants, optimal cleavage appeared to be dependent 374 on TMPRSS2 protease activity ( Figure 5D ). 375 376

To confirm the cleavage at the putative furin cleavage site, we determined the relative abundance 377 of the furin cleaved peptide produced after the 685-terminal arginine. We observed higher amount 378 of cleavage at this position as compared to the previous circulating viruses, although lower 379 amounts were detected in Alpha, Kappa and Delta variants as compared to the viruses harboring 380 the 655Y mutation. This suggests that a change in residue 681 may introduce an additional 381 cleavage site, perhaps recognized by TMPRSS2 protease that enhances spike cleavage of these 382 variants and produces an additional cleavage peptide different in size and amino acid sequences. 383

Further research is needed to confirm the existence of a recognition site for additional proteases 384 different than furin in the amino acid motif SH/RRRAR when the P681S/H mutation is present. In 385 any case, all the VOCs analyzed proved to be strong syncytia inducers which could potentially 386 indicate a role in pathogenesis and lung damage mediated by TMPRSS2 activity after infection in 387

humans (39). On the other hand, the Beta variant, which was first identified in South Africa in 388

October 2020, does not contain a change in the furin cleavage site or in the spike position 655, but 389 instead a change in the residue found at position 701. Although this residue is found around 20 390 amino acids away from the furin cleavage motif, we found similar results when the extent of the 391 spike processing was investigated ( Figure 4A -E; 5D-G). It is important to note that the VOCs 392 investigated in here independently acquired S mutations around the furin cleavage site that became 393 epidemiologically more prevalent in humans. When we investigated the spatial distribution by 394 superimposition of the crystal structure of the S protein, we found that these highly prevalent 395 polymorphisms were all located in close proximity to the furin site loop ( Figure 4A ). CoV-2 S and N protein localization in Vero-TMPRSS2 infected cells at an MOI of 0.01 and 24 527 hours p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N 528 protein was detected using a polyclonal antiserum (red) and 4',6-diamidino-2-phenylindole 529 (DAPI) was used to stain the nucleus. 530 531 Figure 3 . The 655Y polymorphism prevails over the 655H in the transmission in vivo model. 532 A) Ten 3-weeks-old female Syrian hamsters were placed in pairs. Only one hamster per cage was 533 infected intranasally with a total of 10 5 pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a 534

one-to-one ratio. Nasal washes were collected at day 2, 4 and VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein 583 was detected using a polyclonal antiserum (red) and 4',6-diamidino-2-phenylindole (DAPI) was 584 used to stain the nucleus. 585 586

Cell lines: VeroE6 and Caco-2 cell lines were originally purchased from the American Type 589

Culture Collection (ATCC FBS, non-essential amino acids, HEPES, penicillin (100 UI/mL) and streptomycin (100 UI/mL)) 623 at 37°C in 5% CO2. Infected cells were monitored by microscopy and cell-infected supernatants 624

were collected at day 2 post-infection when cytopathic effect was observed. Viral supernatants 625

were clarified of cell debris by spin down followed by centrifugation at 2000 x g for 20 min in 626

Amicon Ultra-15 centrifugal filters (Sigma, 100 kDa cutoff) to concentrate the viral stocks. 627

Aliquots were stored at -80°C until titration by plaque assay. All SARS-CoV-2 variants were 628 sequence-confirmed before performing the experiments. 629 630

Infection of cell cultures: Approximately 3.2 x 10 5 VeroE6 or VeroE6-TMPRSS2 or Caco-2 were 631 seeded in a 12 well-plate and cultured at 37°C in 5% CO2 for 16 hours. Cells were infected with 632 the corresponding SARS-CoV-2 isolate at an MOI of 0.01. Cells were incubated with the virus for 633 1 hour and then, cells were washed with PBS to ensure removal of non-attached virus. After 634 infection, cells were maintained in infection media. Supernatants were collected at and the 635 indicated time points and stored at -80°C for plaque assay analysis and virus quantification. 636 637

Western blotting: VeroE6 or VeroE6-TMPRSS2 cells were infected with the indicated SARS-638

CoV-2 isolates, similar to the description above. Viral supernatants were collected at 24-and 48-639

hours post-infection. Supernatants were clarified by low-speed spin. Viral supernatants and cell 640 extracts were mixed with RIPA buffer (Sigma Aldrich) containing EDTA-free protease inhibitor 641 cocktail (Roche) and 10% SDS (Invitrogen) to a final concentration of 1%. Then, samples were 642 boiled for 10 minutes at 100°C and centrifuged for 10 minutes at 4°C and maximum speed. Viral 643

supernatants were subjected to SDS-PAGE protein electrophoresis using precast 10% TGX gels 644 (Bio-Rad). Gels were run at 120 V and subsequently transferred to polyvinylidene fluoride (PVDF) 645 membranes (BioRad) using BIO-RAD semi-dry transfer system. Then, membranes were fixed 646 with 100% methanol for 1 minute and blocked with 5 % non-fat dry milk-containing Tris-buffered 647 saline with Tween-20 (TBST) with 0.1% Tween-20 for 1 hour in shaking and room temperature 648 (RT). Next, membranes were incubated with primary antibodies overnight at 4°C followed by 649 incubation with secondary antibodies in a 3% milk diluted in TBST for 1 hour at RT. Primary 650 antibodies against SARS-CoV-2 Spike S2 protein (Abcam; ab6823) and nucleocapsid (Novus 651

Biologicals; NB100-56576) were purchased from the indicated suppliers and used at a dilution of 652 1:3000 and 1:2000 respectively. Anti-mouse secondary IgG-HRP antibody (abcam, 6823) was 653 used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP 654 antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid. 655 656

Plaque assay: To determine viral titers, 3.2 x 10 5 VeroE6 or VeroE6-TMPRSS2 were seeded in a 657 12 well-plate the day before plaque assay was performed. Briefly, ten-fold serial dilutions were 658 performed in infection media for SARS-CoV-2 and inoculated onto confluent VeroE6 or VeroE6-659 TMPRSS2 cell monolayer. After one-hour adsorption, supernatants were removed, and cells 660 monolayers were overlaid with minimum essential media (MEM) containing 2% FBS and purified 661 agar (OXOID) at a final concentration of 0.7%. Cells were then incubated 3 days at 37°C. Cells 662

were fixed overnight with 10% formaldehyde for inactivation of potential SARS-CoV-2 virus. 663

Overlay was removed and cells were washed once with PBS. Plaques were visualized by 664

immunostaining For targeted analysis, the samples were separated in 62 minutes to concentrate the analytes in 763 narrower peaks and increase signal. The gradient employed was from 3% B to 34% in 40 minutes 764 then B was increased to 42% in 10 minutes and then finally to 95% in 5 minutes. As for the DDA 765 the column was washed for 5 minutes at 95% B before the next run. included as controls: 2 from the C-term spike fragment to be used as proxy for total spike quantity 841 and 2 from Orf3a and N protein to be used as internal standard to normalize across variants. 842

Following acquisition, the PRM data was imported into the Skyline document with the following 843 transition settings: MS1 filtering was enabled, and MS/MS filtering was changed to targeted using 844

Orbitrap as mass analyzer (35000 resolution) and high selectivity extraction. A minimum of 6 845 transitions and a maximum of 18 having m/z > precursors were selected for data analysis. After 846 manual peak boundaries selection and elimination of interferences the transition results were 847 exported. Transitions where the signal/background ratio was less than 5 were removed to ensure 848 robust quantitative accuracy. C   1  1,273  1,200  1,150  1,100  1,050  1 K  950  900  850  800  750  700  650  600  550  500  450  400  350  300  250  200  150  100 Figure 5 .

000 genome copies per reaction) and nuclease-free 679 water were included as controls. Reactions were performed in duplicate using the following 680 cycling conditions on the Roche LightCycler 480 Instrument II (Roche Molecular Systems; 681 05015243001): 50 °C for 20 min, 95 °C for 1 s, 95 °C for 5 min, followed by 40 cycles of 95 °C 682 for 15 s and 60 °C for 45 s. To determine the limit of detection for SARS-CoV-2, we used a 683 commercially available plasmid control (Integrated DNA Technologies;10006625)

After 24 post-infection, cells were fixed with 10% methanol-free formaldehyde 691 and incubated with primary antibodies against spike KL-S-3A7 (43) and nucleoprotein polyclonal 692 anti-serum (44) diluted in 3% bovine serum albumin (BSA) for 1 hour at RT. Then, cells were 693 washed and stained with secondary antibodies

A21202) and anti-Rabbit Alexa Fluor 568 (ThermoFisher; A11011) in 5% BSA for 1h at RT

DAPI (4′,6-diamidino-2-phenylindole) was used to visualize the nucleus

On days 2, 4, 701 6 post-infection, animals were anesthetized with 100 mg/kg Ketamine and 20 mg/kg Xylazine and 702 nasal washes were collected in 200 ul PBS. Directly infected (DI) and direct contact (DC) 703 hamsters were humanely euthanized for collection of lungs and nasal turbinates on day 5 and 7 704 post-infection, respectively. Anesthetized hamsters were euthanized by intracardiac injection of 705 sodium pentobarbital (Sleepaway -Zoetis) euthanasia solution. Samples were collected for viral 706 quantification by plaque assay and next-generation sequencing

All mink were individually housed, given ad libitum 712 access to food and water, and maintained on a 12-hour light/dark cycle. Six minks were infected 713 with an infectious dose of 10 6 pfu of WA-1 isolate

Minks were anesthetized by 715 intramuscular administration of 30 mg/kg Ketamine and 2 mg/kg Xylazine prior to intranasal 716 infection, collection of specimens, or euthanasia. Nasal washes, rectal swabs, and oropharyngeal 717 swabs were collected on days 1, 3, and 5 post-infection. On days 4 and 7 post-infection, three mink 718 per day were humanely euthanized for collection of tissue specimens for viral quantification by 719 plaque assay and sequencing. Body weights of mink were collected days 0, 1, 3, 4, 5 and 7 post-720 infection. Anesthetized minks were euthanized by intracardiac injection of sodium pentobarbital 721 (Sleepaway -Zoetis) euthanasia solution. The Institutional Animal Care and Use Committee

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1.617, show enhanced spike cleavage by furin. bioRxiv

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational 919 escape seen with individual antibodies

Transmission of SARS-CoV-2 in domestic cats imposes a narrow 921 bottleneck

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in 923 obese and aged mice and is neutralized by convalescent and post-vaccination human sera

Identification of Common Deletions in the Spike Protein of Severe Acute 926 Respiratory Syndrome Coronavirus 2

Human airway cells prevent SARS-CoV-2 multibasic cleavage site 928 cell culture adaptation

Introductions and early spread of SARS-CoV-2 in the New 930

SARS-CoV-2 infection, disease and transmission in domestic cats

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters

Defining the Syrian hamster as a highly susceptible preclinical model 936 for SARS-CoV-2 infection

SARS-CoV-2 variants, spike mutations and immune escape

Spatiotemporal dissemination pattern of SARS-CoV-2 B1.1.28-derived 940 lineages introduced into Uruguay across its southeastern border with Brazil. medRxiv

Mutational spectra of SARS-943

CoV-2 orf1ab polyprotein and signature mutations in the United States of America

Specific mutations in SARS-CoV2 RNA dependent RNA polymerase and 946 helicase alter protein structure, dynamics and thus function: Effect on viral RNA 947 replication

Syncytia formation by SARS-CoV-2-infected cells

A neutralizing human antibody binds to the N-terminal domain of the Spike 951 protein of SARS-CoV-2

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the 953 ACE2 receptor

Shedding of Viable SARS-CoV-2 after Immunosuppressive Therapy for 955 Cancer

Murine Monoclonal Antibodies against the Receptor Binding Domain of 957 SARS-CoV-2 Neutralize Authentic Wild-Type SARS-CoV-2 as Well as B.1.1.7 and 958 B.1.351 Viruses and Protect In Vivo in a Mouse Model in a Neutralization-Dependent 959

Inhibition of Beta interferon induction by severe acute respiratory 961 syndrome coronavirus suggests a two-step model for activation of interferon regulatory 962 factor 3

Multiplex PCR method for MinION and Illumina sequencing of Zika and 964 other virus genomes directly from clinical samples

COBALT: constraint-based alignment tool for multiple 966 protein sequences

Nextstrain: real-time tracking of pathogen evolution

ggplot2: Elegant Graphics for Data Analysis

MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based 973 proteomics

Skyline: an open source document editor for creating and analyzing 975 targeted proteomics experiments