key: cord-0859347-80t2ovkk
authors: Shiliaev, Nikita; Lukash, Tetyana; Palchevska, Oksana; Crossman, David K.; Green, Todd J.; Crowley, Michael R.; Frolova, Elena I.; Frolov, Ilya
title: Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage
date: 2021-06-29
journal: bioRxiv
DOI: 10.1101/2021.06.28.450274
sha: 72644c2088e917d37aca9fa95f6dce5de9df4eda
doc_id: 859347
cord_uid: 80t2ovkk

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1‘ position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has recently emerged in 77

Wuhan, China and then demonstrated an unprecedented spread all over the world. To 78 date, it is circulating on all continents, and the associated COVID-19 disease has already 79 caused millions of deaths worldwide (1, 2). It is also required for synthesis of G RNA during viral replication. M and E proteins are 91 imbedded into the viral lipid envelope and determine virion assembly and release. The 92 structural S protein forms trimeric spikes on the surface of virions and is a major 93 determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, 94 fusion of the viral envelope and cellular membranes, resulting in release of G RNA-95 containing nucleocapsid into the cytoplasm. SARS-CoV-2 utilizes two entry 96 mechanisms. In the first (early) pathway, the transmembrane serine protease(s), such as 97 TMPRSS2, mediates cleavage of the S2' site, which releases the fusion peptide, and this 98 ultimately leads to fusion of viral envelope and the plasma membrane. In the cells lacking 99 TMPRSS proteases, the S2' processing is achieved by cathepsins, and fusion takes place 100 in the endosomes (5, 6). The distinguishing feature of the SARS-CoV-2-specific S 101 protein is that it contains an additional furin cleavage site, which mediates processing of 102 this protein into S1/S2 subunits before particle release from infected cells (7). 103

Human ACE2 is a high affinity receptor of SARS-CoV-2 and determines 104 specificity of the infection. However, similar to numerous other viral infections, 105 interaction with the receptor may not be the only determinant of virus attachment and 106 entry (8). Other attachment factors at the plasma membrane may also play an important 107 role(s). Despite the low affinity of this binding to additional factors at the membrane, the 108 high abundance of such molecules may promote the primary attachment of viral particles 109 before their further interactions with receptors. In many cases, these additional 110 attachment factors have been identified as glycosaminoglycans (GAGs), most frequently 111 heparin and heparan sulfate (HS). They are ubiquitously expressed at the plasma 112 membrane of most of vertebrate cell lines and in the extracellular matrix (9). It appears 113 that SARS-CoV-2 is not an exception. The receptor-binding domain of its S protein was 114 found to interact with HS (10). This binding changes the S protein receptor-binding 115 domain (RBD) from a closed to an open conformation, thereby promoting more efficient 116 binding to ACE2. 117 The ability of the viruses to interact with HS does not exclude their further 118 evolution in developing a more efficient binding of their structural proteins to HS and 119 other GAGs. For example, serial passaging of alphaviruses and flaviviruses in cultured 120 cells during development of live attenuated vaccines results in accumulation of a very 121 few point mutations, which increase a number positively charged amino acids (aa) on the 122 glycoprotein surface (11) (12) (13) (14) (15) . These aa substitutions increase affinity of the virus to HS, 123 viral infectivity in vitro and, compared to natural isolates, strongly decrease the genome 124 equivalent to plaque forming units (GE:PFU) ratio. On the other hand, these mutations 125 usually result in variants that are dramatically less pathogenic, but the mechanism of 126 attenuation remains incompletely understood. However, in some cases, HS-binding 127 alphaviruses exhibit high neurovirulence in mice (15, 16) . 128

The results of our new study show that within very few passages in vitro, SARS-129

CoV-2 rapidly evolves, and variants demonstrating more efficient spread in cultured cells 130 are selected. These variants acquire higher affinities to HS, which largely result from the 131 insertion of a short positively charged peptide into the N-terminal domain (NTD) of the S 132 protein. Another means of adaptation to cell culture is inactivation of SARS-CoV-2-133 specific furin cleavage site by substitution of a single aa. This mutation appears rapidly 134 during virus rescue from infectious cDNA in Vero E6 cells and becomes prevalent. The 135 positively charged amino acids in the uncleaved furin site also increase the affinity of the 136 virus to HS. However, the decrease in cleavage efficiency does not prevent further 137 evolution that results in accumulation of additional positively charged amino acids in the 138 NTD. Both, the insertion of the peptide and point mutation in the furin cleavage site, 139 strongly increase infectious titers and plaque size of SARS-CoV-2 on Vero cells and 140 accelerate viral spread. They also decrease GE:PFU ratio by several orders of magnitude. 141 and SfiI, whose site is located downstream of the poly(A) tail, and the 3' fragment of the 202 viral sequence was also isolated by agarose gel electrophoresis. The original WA1/2020 isolate that we received from WRCEVA (UTMB) had 264 already been passaged 4 times (P4). Similar to another previously described P4 stocks 265 (28, 29), it produced heterogeneous plaques on Vero E6 cells ( Similar ACE2-expressing stable cell lines were also generated on human Huh7.5, 278

hamster BHK-21 and mouse NIH 3T3 cells. All these cell lines became highly 279 susceptible to SARS-CoV-2 and, upon infection, rapidly developed a cytopathic effect 280 (CPE). However, in contrast to Vero/ACE2, infection of these cells with WA1/2020 281 induced formation of very large syncytia with hundreds of cells involved. This resulted in 282 relatively low virus production, and thus, limited application of the latter cell lines. 283

Attempts to isolate variants from the pinpoint and small plaques (Fig. 1A ) 284 developed by the WA1/2020 P4 virus on Vero/ACE2 cells were unsuccessful. As it had 285 been previously shown (29), plaque-purified variants immediately evolved during 286 amplification in both Vero E6 and Vero/ACE2 cells, and harvested stocks produced 287 heterogeneous plaques. This was an indication of rapid evolution of SARS-CoV-2 during 288 its passaging on Vero cells. 289

In order to better understand the mechanism of SARS-CoV-2 evolution in 290 cultured cells, virus from the original stock was passaged 5 times on Vero E6 with 291 gradual decrease of the inoculum volume used for infection. Infectious titers of the 292 harvested stocks increased from 1.5x10 6 PFU/ml at starting passage 5 to 1.5-2x10 8 293 PFU/ml at passage 9. After this selection, the harvested viral pool became homogeneous 294 in terms of plaque size ( Fig. 2A) , and, compared to the original stock, the plaques formed 295 became dramatically larger (Figs. 1A and 2A). Comparison of particle concentrations 296 released into the media [genome equivalents per ml (GE/ml)] demonstrated that 297 passaging did not result in a profound increase in virus release, because concentrations of 298 GE in the media at different passages were similar (Fig. 2B ). This passaging drastically 299 decreased the GE:PFU ratio, suggesting that the ~100-fold higher infectious titers 300 resulted from a higher infectivity of the released virions. However, it remained unclear 301 whether the more infectious variant(s) was present in the originally received, 302 heterogeneous pool of SARS-CoV-2, or there was viral evolution and selection during 303 the above-described passaging. 304 305 Evolution of the S protein in WA1/2020 variant. Next, the S protein-coding sequence 306 of the P9 viral pool was amplified by RT-PCR and sequenced. The furin cleavage site 307 (FCS), which was frequently deleted in prior studies (29), was found to be intact, 308

indicating that deletion of this sequence is not the only means of increasing infectious 309 titers in vitro. A major mutation identified in the S protein of passage 9 virus was an in-310 frame insertion of 21 nt into the NTD-coding sequence (Fig. 2C ). This insertion added a 311 peptide GLTSKRN between aa 214 and 215. A second dominant mutation, H655Y, was 312 in the CTD2 domain. It has been found in many natural isolates and became dominant in 313 Brazilian lineage P.1 (https://www.gisaid.org/hcov19-variants). 314

Further analysis of viral pools, which were harvested at different passages, by 315

Next Generation Sequencing (NGS) demonstrated that the insertion was likely present in 316 the originally received WA1/2020 P4 sample and rapidly became dominant in the viral 317 pool by passage 9 during further passaging (Fig. 2D) . Thus, the insertion-containing 318 variant was selected from the original heterogeneous pool, as it had higher infectivity for 319

Vero E6 cells and, thus was able to spread more efficiently than others. The 320 distinguishing characteristic of the inserted peptide was the presence of 2 positively 321 charged aa, lysine and arginine. Their presence could potentially increase affinity of the 322 virus to GAG(s), which are abundantly present at the plasma membrane, and mediate 323 more efficient primary viral attachment to the cells before interaction with the ACE2 324 receptor. The H655Y mutation was already abundant in P5 virus, and thus, could not 325 directly increase viral infectivity, at least while being alone. In addition, we have also 326 detected an S247R substitution in all the samples of passaged WA1/2020 (Fig. 2D ). It 327 was less abundant than the above insertion and H655Y mutation, present in smaller 328 fraction of viral genomes, and its frequency was not increasing. However, the possible 329 benefit of S247R for viral infectivity and spread cannot be completely ruled out. 3C). Its further passaging led to rapid evolution to the large plaque-forming phenotype 345 (Fig. 3C) . By passage 5, viral infectious titers increased by two orders of magnitude and 346 approached 2x10 8 PFU/ml. As with the above experiments using WA1/2020, the detected 347 evolution did not result in more efficient accumulation of released particles, but rather 348 increased their infectivity, which led to a strong decrease in the GE:PFU ratio (Fig. 3D) . 349

A preliminary Sanger sequencing of the S gene in the high passage viral pool 350 identified a single nucleotide mutation resulting in S686G substitution, which was the 351 first aa downstream of the FCS (Fig. 4A) cleavage. However, it was eliminated by passage 7, suggesting that the latter mutation 361 was less advantageous than S686G for viral spread. In the course of this study, 362 electroporation of the in vitro-synthesized SARS-CoV-2/GFP RNA was repeated 363 multiple times, and there was always evolution of the rescued virus to a large plaque-364 forming phenotype within very few passages. In addition, we have also sequenced 365 genomes in the virions pooled together from three independent electroporations of the in 366 vitro-synthesized CoV-2/GFP RNAs and found that rescued viruses also contained 367 mutations in FCS site (S686G at 59% and R683P at 30%). Thus, as in the case of the 368 above described natural isolate WA1/2020, serial passaging of the cDNA-derived virus in 369 vitro was also leading to rapid selection of variants demonstrating higher infectivity and 370 formation of large plaques. Albeit this adaptation was achieved by accumulation of 371 different mutations in the spike-coding gene. In the case of recombinant virus, the 372 mutations in FCS became dominant by passage 2 and were likely selected from those 373 randomly generated by the SP6 polymerase during RNA synthesis. analysis of viral replication rates in Vero/ACE2 cells (Fig. 5C ) showed that at any times 404 p.i., the mutations led to higher infectious titers identical to those of the originally 405 selected, late passage viruses. The recombinant viruses, but not CoV-2/GFP P0 and 406 WA1/2020 P4, also caused complete CPE by 40 h p.i. The double mutant CoV-407 2/GFP/ins/G replicated more efficiently than the single mutants (Fig. 5C ). Mutations in 408 the S proteins of WA1/2020 P9, CoV-2/GFP P5 and CoV-2/GFP/ins/G P0 did not 409 abrogate infectivities of the viruses for Calu3 cells (Fig. 5C) , which are widely used in 410 SARS-CoV-2 research. Variants containing the S686G mutation (the late passage CoV-411 2/GFP, CoV-2/GFP/G and CoV-2/GFP/ins/G) demonstrated less efficient processing of 412 the S protein (Fig. 5D ). Taken together, the data indicated independent functions of the 413 mutations introduced into S protein in viral replication to higher infectious titers, at least 414 in Vero cells. However, it remained unclear whether these changes in the S protein 415 function via the same or different mechanisms. 416

heparin. Next, we evaluated interaction of different viral variants with heparin. Samples 419 of all of the viruses indicated in Fig. 6 were prepared in serum-free media. This set 420 included the UAB clinical isolate CoV-2/UAB P2, WA1/2020 P5, early passage CoV-421 2/GFP P2, late passage WA1/2020 P10 and CoV-2/GFP P7. Viral samples were diluted 422 to 0.1 M concentration of NaCl, loaded to the heparin sepharose column and eluted using 423 a step gradient of NaCl (see Materials and Methods for details). The results presented in 424 Passaged viruses WA1/2020 P10 and CoV-2/GFP P7 bound to heparin sepharose more 431 efficiently, and less than 1 % of the infectious viruses were found in the FT fraction. 432 WA1/2020 P10 and CoV-2/GFP P7 were eluted by 0.35 M and 0.3 M NaCl, respectively. 433

Thus, the mutations accumulated in the S protein of the high passage viruses, which 434 became more infectious for Vero cells, also increased their affinities to heparin. 435

The pools of the high passage viruses still contained variants with a variety of 436 mutations in the S protein-coding sequence. Therefore, it was difficult to make definitive 437 conclusions about the effects of GLTSKRN insertion and S686G mutation on virions' 438 affinity to heparin. Therefore, we also evaluated binding of the above-described designed 439 recombinant viruses to heparin sepharose. Based on sequencing data, they had no 440 additional aa substitutions in the S proteins. Their samples were also prepared in serum-441 free media. Binding and elution patterns of CoV-2/GFP/ins P1 and CoC-2/GFP/G P1 442 (Fig. 7) correlated with those of the late passage WA1/2020 and CoV-2/GFP (Fig. 6) , 443 respectively. In the FT fraction, both recombinant viruses were present at very low levels. 444

The peak of CoV-2/GFP/ins variant elution was at 0.35 M NaCl, and CoV-2/GFP/G was 445 eluted by 0.3M NaCl. Thus, both the insertion and S686G mutation increased affinities of 446 the viruses to heparin sepharose. The S686G mutation did not increase the positive 447 charge of the S protein surface, but it likely transformed the unprocessed furin cleavage 448 site into the heparin-binding site. Interestingly, the double mutant CoV-2/GFP/ins/G, 449 which contained both the insertion in the NTD and S686G mutation, was also eluted by 450 0.35 M NaCl. Thus, the combination of both modifications in the spikes did not increase 451 viral affinity above that of the high-passage natural isolates. This was suggestive that 452 although binding to heparin was likely determined by two separate sites, the higher 453 affinity was mostly determined by the insertion in the NTD and played the dominant role. 454

Taken together, the results of these experiments demonstrated that passaging of 455 SARS-CoV-2 leads to its rapid evolution towards a more efficient binding to heparin. 456

These adaptive mutations include insertion of the short positively charged peptide after aa 457 214 in the S protein and transformation of the furin cleavage cite into a heparin-binding 458 aa sequence. However, additional contributions of other mutations in the NTD of the 459 spike protein, such as S68R or N74K, which may additionally increase its affinity to HS, 460 are also possible. 461

Variants having the S686G mutation exhibited one more interesting characteristic. 462

They lost the ability to form syncytia on Vero/ACE2 cells (Fig. 8) . In our experimental 463 conditions, SARS/UAB, WA1/2020 P9 and CoV-2/GFP/ins formed very large syncytia. 464

The early passage CoV-2/GFP P2, which had a mixed population of viral variants, was 465 still capable of forming at least small syncytia, but by passage 7, it completely lost this 466 ability. Both CoV-2/GFP/G and double mutant CoV-2/GFP/ins/G were unable to produce 467 syncytia at all. 468

In this report, we demonstrate that SARS-CoV-2 rapidly adapts to replication in Vero E6 471 cells, which are most commonly used for virus propagation. This adaptation results in 472 higher infectious titers in vitro, which are determined by higher infectivity of the viral 473 particles rather than by more efficient particle release. The evolved variants form 474 dramatically larger plaques on Vero cells and demonstrate more rapid infection spread. 475 Similar evolution of SARS-CoV-2 to the large plaque-forming phenotype has been 476 detected in previous publications, suggesting that this is a common effect, and likely 477 independent of experimental conditions (28). 478 Viral evolution developed by two mechanisms. The first means of adaptation was 479 determined by the insertion of a 7-aa-long peptide into the S protein of the WA1/2020 480 isolate (Fig. 2) . It was already present in the provided heterogeneous viral sample, and 481 rapidly became dominant during further passaging. This GLTSKRN insertion was 482 located in the NTD (Figs. 2, 9 and 10 contribute to an increase in the positive charge of the NTD surface (Fig. 9 ). Since 493 presence of the latter mutation did not increase during passaging, its contribution to 494 infection spread was likely lower than that of the above-described insertion. 495

The second mechanism of SARS-CoV-2 adaptation was based on the appearance 496 of the S686G substitution in P1' position of the S1/S2 cleavage site, FCS, which was 497 rapidly selected in the S protein of recombinant CoV-2/GFP. The previously published 498 data showed that the peptide representing FCS is often entirely or partially deleted during 499 virus passaging (28-30), and the same S686G mutation was also found after passaging of 500 other SARS-CoV-2 isolates (28, 29). The identified mutation strongly affected processing 501 of the S protein (Figs. 4C and 5D) and formation of the syncytia (Fig. 8) during viral 502 replication. It also increased infectivity of harvested viruses, their plaque sizes, and 503 infectious titers (Figs. 3C and 5C ). In prior studies, the FCS could be inactivated by either 504 its deletion or accumulation of point mutations eliminating the arginines essential for 505 furin cleavage (29). Viral mutants with deleted FCS, and R682L, S686G and H655L 506 mutations have been detected in the stock of WA1/2020 isolate from CDC (31). The 507 effect of the FCS deletion was experimentally evaluated in the context of pseudovirions 508 or recombinant SARS-CoV-2 (32, 33). It resulted in higher infectious titers of the viruses 509 harvested from Vero cells, but the detected increase was small. It has also been 510 demonstrated that the deletion of FCS affected viral infectivity in human cells including, 511

Calu-3, Caco2 and primary lung epithelia cells. 512

In our study, the selected recombinant viruses with the S686G mutation replicated 513 to two orders of magnitude higher infectious titers in Vero cells than did the natural 514 isolates and previously designed deletion mutants. They also remained capable of 515 infecting and reaching high titers on Calu-3 cells (Fig. 5) . These results suggest that in 516 contrast to FCS deletion, inhibition of the S1/S2 processing by S686G is likely not the 517 only advantage of the new variants. Viral spikes with S686G substitutions retained the 518 entire polybasic motif, which was originally required for cleavage by furin protease (Fig.  519 4A), and such viruses attained high affinity to heparin sepharose (Figs. 6 and 7), higher 520 infectivity and more efficient spread on Vero cells. Similar evolution of the FCS into the 521 heparin-binding motif has previously been described for some of the group 1 522 coronaviruses (34). In that study, the resultant polybasic motif of the S protein-specific 523 FCS was functioning in HS-binding. Interestingly, mutations in FCS have been shown to 524 both increase and decrease CoV pathogenicity. HCoV-OC43 encodes the RRSRG 525 cleavage site, which is similar to that in our S686G mutant, and its spike protein remains 526 unprocessed (35). However, the clinical isolates with functional FCS were viable and 527 showed lower neurovirulence (36). On the other hand, in the feline enteric coronavirus 528 (FECV), FCS mutations abolishing spike protein processing transformed the virus to 529 another more pathogenic biotype, feline infectious peritonitis virus (FIPV) (37, 38). In 530 our study, at the early passages, we were able to detect another CoV-2/GFP variant with 531 mutated positively charged aa in the furin cleavage site (R685H mutation). However, it 532 was likely less competitive than S686G mutant, and was eliminated from the viral pool 533 within a few subsequent passages. Thus, the S686G mutant apparently produced higher 534 infectious titers than other variants with mutated arginine-rich motifs. Interestingly, the 535 latter RRAR remains in the S1 subunit of the original isolates after furin-mediated 536 processing. However, in this post cleavage conformation, it likely interacts with heparin 537 less efficiency, if at all, than that in the uncleaved S protein of the selected mutant. 538

Interestingly, downregulation of the S protein processing had a negative effect on the 539 ability of the recombinant viruses to induce syncytia formation at least in Vero cells (Fig.  540 8), but it remains unclear whether the mutants became less efficient in forming syncytia 541 in vivo. 542

The experimental evidences suggest that the virus with unprocessed FCS remains 543 less efficient in HS binding than the variant with the NTD insertion. First, viruses with 544 S686G are eluted from heparin sepharose by lower concentrations of NaCl (Figs. 6 and 545 7). Secondly, in contrast to the insertion mutant, the originally selected CoV-2/GFP 546 variant with the S686G mutation continued to evolve and attain additional mutations in 547 the NTD, such as I68R and N74K (Figs. 4 and 10) . Their accumulation was relatively 548 slow, but clearly detectable, and implied their additional contribution to HS binding and 549 viral infectivity. Analysis of sequences submitted to GISAR revealed that accumulation 550 of mutations that increase the positive charge of NTD upon passaging in Vero cells, is 551 quite common: N74K was detected in EPI_ISL_1039208, EPI_ISL_1190402, 552 EPI_ISL_1718321, EPI_ISL_1707039, EPI_ISL_1785073, EPI_ISL_1785076, and 553 I68R/K was found in EPI_ISL_493139, EPI_ISL_2226226. The WA1/2020 from CDC 554 was reported to contain 9 low frequency R/K substitutions in NTD including a 4-aa-long 555 insertion after D215 (31). The indicated I68R and N74K mutations, which additionally 556 increase the positive charge of the NTD surface (Fig. 9) , are also located relatively close 557 to the above-described S247R (Fig. 10) . Currently, there is not enough information to 558 speculate whether the increase of positive charge on the NTD surface attenuates the virus, 559 makes it more pathogenic or has no effect on pathogenicity. Importantly, since R/K 560 mutations in NTD are highly beneficial for virus propagation in vitro, viruses used for 561 animal studies should be thoroughly analyzed by NGS. 562

Combining the positively charged insertion and S686G mutation in the same S 563 protein did not detectably increase the affinity of infectious viral particles to heparin 564 sepharose above the level determined for CoV-2/GFP/ins (Fig. 7) . However, the 565 recombinant double mutant demonstrated significantly higher infection rates than did the 566 single site mutants (Fig. 5) . CoV-2/GFP/ins/G was reaching the peak titers above 10 8 567 PFU/ml by 24 h p.i. even at low MOI, such as 0.01 PFU/cell. This was an indication that 568 during viral infection, the introduced mutations, GLTSKRN insertion and S686G 569 substitution, were able to function in either synergistic or additive modes. The structural 570 model of the S protein supports the possibility of a combined effect. The above insertion 571 and furin cleavage site are in close proximity on the surface of the S protein (Fig. 10) and 572 may have a stimulatory effect on primary binding of the virus to GAGs at the plasma 573

membrane. An important characteristic of the double mutant is that its further evolution 574

in cultured cells appears to be unlikely. The latter virus demonstrated the lowest GE:PFU 575 ratio, and its further decrease at least in Vero cells appears to be an improbable event. 576

Similar rapid evolution to more efficient spread in vitro was previously described 577 for alphaviruses. As with SARS-CoV-2 in this study, chikungunya, Venezuelan equine 578 encephalitis, Ross River and Sindbis viruses acquired 1 or 2 additional basic aa in the E2 579 glycoprotein, which had strong positive effects on virus interaction with 39, 580 40) . The mutations increased plaque sizes, stimulated spread of infection and 581 dramatically decreased the GE:PFU ratio of the evolved alphaviruses. These mutant 582 viruses were usually less pathogenic in mice. Therefore, it is tempting to speculate that, 583 similar to other RNA+ viruses that adapted to binding to HS, the designed single mutants 584 and the double mutant, in particular, became attenuated in vivo, but this hypothesis needs 585 experimental support. Of note, the development of the recombinant live attenuated 586 vaccine for SARS-CoV-2 will also require its large-scale production and passaging in cell 587 culture, most likely in Vero cells. Therefore, viral evolution to a more stable HS-binding 588 phenotype through the acquisition of mutations in the NTD is likely an unavoidable event 589 and needs to be considered. The original isolates form very small plaques and have very 590 high GE:PFU ratio. Their passaging results in rapid evolution of viral pool and selection 591 of variants with higher infectivities in vitro. However, it is unlikely that they fully 592 reproduce SARS-CoV-2 infection and pathogenesis in vivo. 593

Interestingly, previously published data, which were generated on the receptor-594 binding domain (RBD) and the isolated S protein, suggested that the RBD is capable of 595 efficient interaction with heparin sepharose and HS (10). In our experiments, the early 596 passage natural isolates demonstrated relatively inefficient binding to heparin sepharose. 597

The main fraction of infectious virus remained in the FT fraction (0.1 M NaCl) and some 598 virus was also eluted as a small peak at NaCl concentration of 0.2 M. However, the 599 insertion-and/or S686G mutation-containing variants, which became adapted to cell 600 culture, were eluted by NaCl at 0.35 and 0.3 concentrations, respectively, suggesting their 601 higher affinities to heparin, which strongly correlated with viral infectivities and spread 602 in cultured cells. 603 Importantly, we observed rapid adaptation of the recombinant, cDNA-derived 604 virus, and the S686G mutation became dominant by passage 2, because it increased viral 605 infectivity by 100-fold. The SP6 RNA polymerase used in our study and the T7 606 polymerase used by other groups have the mutation rates of 1-2x10 -4 /nt (41). Thus, the in 607 vitro-synthesized viral genomes already contain a wide range of mutations, which 608 represent a ground for further selection of the most efficiently spreading variants. 609

In conclusion, previously published data and the results of this study suggest that 610 both, natural isolates of SARS-CoV-2 and the cDNA-derived recombinant variants, 611 rapidly adapt to cell culture. They evolve to higher infectivity, which results in lower 612 GE:PFU ratios, and more efficient infection spread by either acquiring mutations in the 613 NTD, suggesting an important role of this domain in viral binding to the cells, or in the 614 FCS. These mutations increase virus affinity to HS during its primary attachment to cells 615 before interaction with the ACE2 receptor. The mutations leading to more efficient 616 spread include i) substitutions of single aa by those positively charged, ii) insertions of 617 short peptides that contain basic aa, and iii) S686G substitution that likely transforms 618 FCS into the HS-interacting peptide. Such viral variants rapidly become dominant, and 619 their further evolution and adaptations are unlikely. These adapted variants appear to be 620 more convenient for CPE-based screening of the antiviral drugs. As with the HS-binding 621 mutants of other RNA+ viruses, the evolved SARS-CoV-2 may also be attenuated in 622 vivo, particularly the double mutant that demonstrates the most adapted phenotype and 623 alterations in syncytia formation. Thus, they may also be used as a basis for development 624 of stable live attenuated vaccines for COVID-19. 

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An amino acid substitution in the 869 coding region of the E2 glycoprotein adapts Ross River virus to utilize heparan 870 sulfate as an attachment moiety

Misincorporation by wild-type and mutant 872 T7 RNA polymerases: identification of interactions that reduce misincorporation 873 rates by stabilizing the catalytically incompetent open conformation

This study was supported by Public Health Service grants R01AI133159 and 717 R01AI118867 to EIF, R21AI146969 to IF and U19AI142737 for TJG and by the UAB 718Research Acceleration Funds to EIF and IF. 719 720