key: cord-0707179-xzn11n74
authors: Meng, Bo; Kemp, Steven A.; Papa, Guido; Datir, Rawlings; Ferreira, Isabella ATM.; Marelli, Sara; Harvey, William T.; Lytras, Spyros; Mohamed, Ahmed; Gallo, Giulia; Thakur, Nazia; Collier, Dami A.; Mlcochova, Petra; Duncan, Lidia M.; Carabelli, Alessandro M.; Kenyon, Julia C.; Lever, Andrew M.; De Marco, Anna; Saliba, Christian; Culap, Katja; Cameroni, Elisabetta; Matheson, Nicholas J.; Piccoli, Luca; Corti, Davide; James, Leo C.; Robertson, David L.; Bailey, Dalan; Gupta, Ravindra K.
title: Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the variant of concern lineage B.1.1.7
date: 2021-06-08
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109292
sha: 5da2191d09f6a52acfeac9396b82e71f44cbab3f
doc_id: 707179
cord_uid: xzn11n74

We report SARS-CoV-2 spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of the receptor binding motif replacements such as N439K and Y453F known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that whilst ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of S proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of H69 and V70 residues in B.1.1.7 spike (where ΔH69/V70 naturally occurs) impairs spike incorporation and entry efficiency of B.1.1.7 spike pseudotyped virus. B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.

modelled in silico. The H69/V70 deletion was predicted to alter the conformation of a 135 protruding loop comprising residues 69 to 76, pulling it in towards the NTD ( Figure 2B ). In 136 the post-deletion structural model, the positions of the alpha carbons of residues either side of 137 the deleted residues, Ile68 and Ser71, were each predicted to occupy positions 2.9Å from the 138 positions of His69 and Val70 in the pre-deletion structure. Concurrently, the positions of 139 Ser71, Gly72, Thr73, Asn74 and Gly75 are predicted to have changed by 6.5Å, 6.7Å, 6.0Å, 140 6.2Å and 8Å, respectively, with the overall effect of these residues moving inwards, resulting 141 in a less dramatically protruding loop. 142 143 This predicted change in the surface of spike could be consistent with antibody evasion. To 144 test this we explored whether H69/V70 conferred reduced susceptibility to neutralising 145 antibodies in sera from fifteen recovered individuals ( Figure 2C , D). We performed serial 146 dilutions of sera before mixing with lentiviral particles pseudotyped with Spike proteins with 147 and without H69/V70 (with virus input normalised for infectivity). We plotted infection of 148 target cells as a function of serum dilution ( Figure 2D ). All but two sera demonstrated clear 149 titratable neutralisation of both wild type and H69/V70 virus. There was no overall change 150 in susceptibility to serum neutralisation for H69/V70 relative to wild type ( Figure 2C ), but 151 there was a proportion of individuals with slightly increased neutralisation sensitivity of 152 H69/V70 ( Figure 2C , D). To further explore the role for H69/V70 in inducing immune 153 escape, we tested the binding of 12 NTD-mAbs to wildtype and H69/V70 NTD by biolayer 154 interferometry ( Figure 2E -G). All the NTD-mAbs showed less than 2-fold decrease in 155 binding to H69/V70 compared to WT. These data suggest that H69/V70 does not 156 represent an important antibody escape mechanism. 157 158

We hypothesised that the deletion might alternatively enhance virus infectivity. In the 160 absence of virus isolates we used a lentiviral PV approach to test the impact of H69/V70 on 161 virus spike protein mediated entry. A D614G bearing Wuhan-1 spike expressing DNA 162 plasmid (WT) was co-transfected in HEK 293T producer cells along with plasmids encoding 163 lentiviral capsid and genome for luciferase. Infectivity was adjusted for input reverse 164 transcriptase activity; we observed a two-fold increase in PV infectivity of H69/V70 as 165 compared to WT in HeLa cervical epithelial cells stably expressing human ACE2 ( Figure 3A , 166 B). We observed similar fold increases with H69/V70 in a range of other target cells, both 167 in context of over-expression of ACE2/TMPRSS2 (HEK 293T cells transiently transfected  168 with ACE2, or ACE2 and TMPRSS2 and A549 lung cells stably expressing ACE2 and 169 TMPRSS2(Rihn et al., 2021) ) or endogenous levels of receptors in Calu-3 lung 170 adenocarcinoma cells ( Figure 3A ). 171 172 Western blotting for S2 spike indicated that a higher amount of cleaved spike in H69/V70 173 bearing virions and in the 293T producer cell lysates. We also noted a corresponding 174 reduction in uncleaved full length (FL) spike ( Figure 3C ). Densitometric analysis of spike 175 and p24 from western blots in multiple experiments showed almost a two-fold increase in 176 spike:p24 ratio as well as an increased ratio in S2:FL cleavage for the H69/V70, indicating 177 increased spike incorporation into virions might explain the increase in infectivity ( Figure  178 3D, E). To verify that this increased in S from producer cells was not specific to HEK 293T 179 cells we also transfected the human lung epithelial cell line H1299 (Zhang et al., 2020) with 180 spike and lentiviral packaging plasmids. We again observed that viruses from these cells had 181 a two fold increased infectivity in target cells ( Figure 3F ). In addition the increased total and 182 cleaved S levels were recapitulated both in the cell lysates and purified virions from these 183 lung cells ( Figure 3G , H). Therefore we conclude that the increased S cleavage and its 184 incorporation observed in producer cells and pseudotyped virions is a generalised 185 phenomenon for H69/V70 S. In order to explore whether D614G was required for this 186 enhanced spike cleavage and infectivity, we generated PV bearing D614 spike with and 187 without the H69/V70 followed by infection in HEK293T cells. We observed a similar two 188 fold enhancement of infection and a proportional increase in spike incorporation as we did 189 for D614G spike pseudotyped viruses (Supplementary Figure 1A, B) . Finally, to exclude the 190 possibility that increased incorporation of S was specific for pseudotyped lentiviral particles, 191 we generated coronavirus-like particles by co-transfection of WT or H69/V70 S with 192 SARS-CoV-2 (M)embrane, (E)nvelope and (N)ucleocapsid proteins as previously described 193 (Swann et al., 2020; Yurkovetskiy et al., 2020) . Enhanced infectivity of H69/V70 spike is not correlated with cleavage or entry route 198 SARS-CoV-2 entry into target cells is thought to take place by two distinct routes following 199 binding to ACE2 ( Figure 4A ). Firstly an endosomal route where cathepsin is able to cleave 200 spike with pH dependent fusion in the endosome. The second route involves fusion at the 201 plasma membrane with cleavage via the plasma membrane associated protease TMPRSS2. 202 In order to determine the mechanism by which increased spike cleavage in the context of 203 H69/V70 might impact entry, we used inhibitors of furin cleavage (CMK) and protease 204 inhibitors specific to endosomal (ED64D) and plasma membrane fusion (camostat) entry 205 routes ( Figure 4A ). We firstly treated producer cells with CMK and found that indeed CMK 206 inhibits spike S1/S2 cleavage in the producer cells transfected with S ∆H69/V70 plasmid, and 207 that the spikes with altered S1/S2 cleavage are incorporated onto the virions ( Figure 4B ). We 208 found that CMK treatment, whilst reducing S1/S2 cleavage, did not decrease the PV infection 209 in a variety of target cells ( Figure 4C ) suggesting the increased infectivity in H69/V70 is not 210 due to more efficient cleavage of spike. To confirm our findings, we generated a spike 211 lacking the polybasic cleavage site with or without ∆H69/V70 and tested PV infectivity on 212 293T cells overexpressing ACE2 and TMPRSS2. We found that deletion of the PBCS led to 213 increased infectivity of the PV, as observed previously for mutated PBCS 20 . As expected, 214 deletion of the PBCS did not alter the enhancing effect of ∆H69/V70 on PV infectivity 215 ( Figure 4D ). 216

The altered level of S1/S2 cleavage in SARS-CoV-2 has been linked to its dependence on 218 viral entry through either membrane fusion or endocytosis in 293T and A549 cells (Peacock et 219 al., 2020; Winstone et al., 2021) . We therefore hypothesised that the increased spike cleavage 220 of H69/V70 S could influence the route of entry. To probe this, spike pseudotyped 221 lentiviruses bearing either WT spike, ∆H69/V70 spike or VSV-G were next used to transduce 222 293T-ACE2 or 293T-ACE2/TMPRSS2 cells in the presence of either E64D or camostat at 223 different drug concentrations ( Figure 4E ). As expected, the VSV-G pseudotyped particles 224

were not affected by addition of either E64D or camostat. Consistent with previous 225 observations, WT PV utilised endocytosis in the absence of TMPRSS2(Peacock et al., 2020) 226 but where TMPRSS2 was expressed, plasma membrane fusion became the dominant 227 route (Papa et al., 2021; Winstone et al., 2021) . However, there were no differences between 228 WT and ∆H69/V70 in relative utilisation of the endosomal versus plasma membrane entry 229 routes. We conclude that the enhanced spike cleavage, whilst notable in ∆H69/V70, does not 230 appear responsible for the increased infectivity of ∆H69/V70 spike observed in these cell line 231 based experiments. 232 233 J o u r n a l P r e -p r o o f H69/V70 spike compensates for reduced infectivity of RBD escape mutants 234

We next examined in greater detail the SARS-CoV-2 lineages where S mutations in the RBD 235 were identified at high frequency and where H69/V70 co-occurs. For example, N439K, an 236 amino acid replacement reported to be defining variants increasing in numbers in Europe and 237 other regions (Thomson et al., 2020) (Figure 1 and 5A) now mostly co-occurs with 238 H69/V70. N439K appears to have reduced susceptibility to some convalescent sera as well 239 as monoclonal antibodies targeting the RBD, whilst increasing affinity for ACE2 in 240 vitro (Thomson et al., 2021) . The first lineage possessing N439K (and not H69/V70), 241 B.1.141 is now extinct (Thomson et al., 2020) . A second lineage with N439K, B.1.258, later 242 emerged and subsequently acquired H69/V70 leading to the initial rapid increase in the 243 frequency of viruses possessing this deletion, spreading into Europe ( Figure 1A ) (Brejová et 244 al., 2021) . 245

The second significant cluster with H69/V70 and RBD mutants involves Y453F, another 247 spike RBD mutation that increases binding affinity to ACE2(Starr et al., 2020b) and has been 248 found to be associated with mink-human infections (Munnink et al., 2020) . Y453F has also 249 been described as an escape mutation for mAb REGN10933 and shows reduced susceptibility 250 to convalescent sera (Baum et al., 2020; Hoffmann et al., 2021) , and is possibly a T cell 251 escape mutation (Motozono et al., 2021) . The H69/V70 was first detected in the Y453F 252 background on August 24 th 2020 and thus far appears limited to Danish sequences ( Figure 1 , 253 Figure 5B ), although an independent acquisition was recently reported along with H69/V70 254 in an immune compromised Russian individual with chronic infection (Bazykin et al., 2021) . 255

We hypothesised that H69/V70 might have arisen after Y453F and N439K in order to 257 compensate for potential loss of infectivity that has been reported for these RBD mutants 258 previously (Motozono et al., 2021; Sungnak et al., 2020) . We therefore generated mutant 259 spike plasmids bearing RBD mutations Y453F and N439K ( Figure 5C ) both with and without 260 H69/V70 and performed infectivity assays in the lentiviral pseudotyping system. RBD 261 mutations reduced infectivity of Spike relative to WT by around 2 fold ( Figure 5D ) and was 262 partially rescued by H69/V70. Based on observations of the impact of H69/V70 on spike 263 incorporation in WT ( Figure 3D ), we predicted that the mechanism of increased infectivity 264 for H69/V70 in context of RBD mutations might be similar. The analysis of virions from 265 cell supernatants and cell lysates indeed showed increased ratio of spike:p24 ( Figure 5E , F). 266 J o u r n a l P r e -p r o o f As observed for WT, we also observed increased cleaved S2:FL when H69/V70 was 267 present along with the RBD mutants in PV ( Figure 5E , G). 268 269 H69/V70 is required for optimal B.1.1.7 spike S2 incorporation and infectivity 270 A lineage containing the H69/V70 deletion was first detected in the UK with the RBD 271 mutation N501Y along with multiple other spike and other mutations (Figure 1 , 272

Supplementary Figure 2) . These UK sequences were subsequently named as B.1.1.7, termed 273 a variant of concern (VOC), as they are associated with higher transmission rate (Volz et al., 274 2021b Table 2 ). We found that 7 out of 12 NTD-specific mAbs (58%) showed a 289 marked decrease or complete loss of neutralising activity to both B.1.1.7 and B.1.1.7 290 H69/V70 (>30 fold-change reduction), suggesting that in a sizeable fraction of NTD 291 antibodies the H69/V70 deletion is not responsible for their loss of neutralising activity 292 (Supplementary Figure 3) . The remaining 5 mAbs showed a partial reduction (2-to-10 fold) 293 in B.1.1.7 neutralisation that was not rescued by reversion of H69/V70 deletions. 294 295 Given our data on introduction of H69/V70 into WT (Figure 3 ), we hypothesised that 296 H69/V70 was selected in the evolution of B.1.1.7 in order to increase viral entry. We 297 predicted that the replacement of H69 and V70 would impair the infectivity of B.1.1.7 PV 298 and reduce total spike levels. To examine this, we compared the infectivity of B.1.1.7 spike 299 J o u r n a l P r e -p r o o f PV versus B.1.1.7 PV with H69 and V70 restored to B.1.1.7 spike. We observed that 300 infectivity of B.1.1.7 infectivity was slightly lower than WT ( Figure 6B ). As expected, we 301 observed a significant reduction in infectivity for viruses where the H69 and V70 had been 302 re-inserted across a number of cell types, including H1299 expressing endogenous levels of 303 ACE2 and TMPRSS2 receptors ( Figure 6B , C). When we measured spike incorporation into 304 virions we found that the reduced infectivity of the B.1.1.7 with replaced H69 V70 was 305 associated with reduced spike:p24 and S2:FL ratio as expected ( Figure 6D -G). 306 307 B.1.1.7 spike mediates faster syncytium formation and is H69/V70 dependent 308

Previous reports have shown that SARS-CoV-2 spike protein localises to the cell host plasma 309 membrane and possesses high fusogenic activity, triggering the formation of large multi-310 nucleated cells (named syncytia) in vitro and in vivo potentially providing an additional and a 311 more rapid route for virus disseminating among neighbour cells (Bussani et al., 2020; Cattin-312 Ortolá et al., 2020; Papa et al., 2021) . The role of syncytium formation in viral replication 313 and pathogenesis of severe COVID-19 has been reported and may be druggable process to 314 treat COVID-19 pathology (Braga et al., 2021) . We expressed B.1.1.7 spike and a B.1.1.7 315 with restored H69 and V70 together with the mCherry fluorescent protein in 293T cells and 316 labelled Vero cells with a green fluorescent dye ( Figure 7A ). All spike constructs showed 317 similar protein expression and achieved similar cell-cell fusion by 16 hours. B.1.1.7 appeared 318 to mediate more cell-cell fusion events over earlier time points with the colour overlap area 319 being 2-3 times greater for B.1.1.7 as compared to wild type at 6 hours post mixing. 320

Interestingly, this enhancement was abrogated by re-insertion of H69 and V70 residues 321 ( Figure 7B -D). We conclude that B.1.1.7 spike mediates faster fusion kinetics than wild type 322 bearing D614G Wuhan-1 spike, and that this is dependent on H69/V70. pausing/dissociation events in reverse transcriptase (Harrison et al., 1998) . Since all nucleic 367 acid polymerases have a common ancestor with homologous dNTP binding motifs and 368 similar global structures (Delarue et al., 1990; Ollis et al., 1985; Sousa et al., 1993) it is 369 probable that all RNA polymerases use similar mechanisms for transcript termination (Reeder 370 and Lang, 1994) . A recent in-cell biochemical analysis of SARS-CoV2 RNA structure 371 showed nucleotide reactivity consistent with this model within these stem-loops (Huston et 372 al., 2021) . These analyses provide a rationale for preferential emergence of H69/V70 and 373 other deletions such as the well described NTD-antibody escape deletion Y144 (Chi et al., 374 2020; McCarthy et al., 2020 McCarthy et al., , 2021 ) (in B.1.1.7 and the recently reported B.1.525) at the 375 terminal loops of helical loop motifs. H69/V70 itself has frequently followed immune 376 escape associated amino acid replacements in the RBD (eg N439K and Y453F), and is 377 specifically found in the B.1.1.7 variant known to have higher transmissibility (Volz et al., 378 2021a) and possibly pathogenicity (Davies et al., 2021) . 379

We find that H69/V70 does not significantly reduce sensitivity of spike to neutralising 381 antibodies in serum from a group of recovered individuals or binding of multiple mAb 382 directed against the NTD. In addition we have shown that repair of H69/V70 does not 383 appreciably alter the potency of NTD antibodies against the B.1.1.7 spike. Thus the deletion 384 is unlikely to represent an immune escape mechanism. Instead, our experimental results 385 demonstrate that the H69/V70 deletion is able increase infectivity of Wuhan-1 D614G spike 386 pseudotyped virus, as well as pseudotyped virus bearing the additional RBD mutations 387 N439K or Y453F, explaining why the deletion is often observed after these immune escape 388 mutations that carry infectivity cost (Motozono et al., 2021; Sungnak et al., 2020) . We show 389 that the mechanism of enhanced infectivity across the RBD mutations tested is associated 390 with greater spike incorporation into virions where the H69/V70 deletion is present. The 391 phenotype is also independent of producer cell used. Importantly, we were able to 392 recapitulate the H69/V70 phenotype in a spike protein that did not have the D614G 393 mutation, indicating that D614G is not involved in the mechanism. These observations are 394 supported by H69/V70 being observed in D614 viruses in Jan 2020 both in the US and 395

Thailand. Although we did not use a replication competent system, a recent pre-print reports 396 H69/V70 mutated Washington strain virus isolate as conferring increased replication in cell 397 lines and higher viral loads in hamsters (Liu et al., 2021) . Indeed, H69 has been observed in 398 cell culture during remdesivir selection experiments with replication competent virus, 399 consistent with a replication advantage (Szemiel et al., 2021) . 400

We have found consistent differences in S, as well as cleaved S, in the producer cell and its 402 incorporation into PV particles when comparing H69/V70 to a Wuhan-1 spike (both with 403 D614G). This could be explained by stability during intracellular trafficking or the route 404 taken to the surface, differences in post-translational modification of the S-protein, or 405 membrane characteristics at the site of virus budding or virus like particles. As the amount of 406 S incorporation into virions reflects the S in the cells, virion formation is likely unaffected by 407 H69/V70. Interestingly, although pharmacological inhibition of furin by CMK in producer 408 cells did prevent S1/S2 cleavage, and altered the balance of S2:FL spike in cells and virions, 409 PV infectivity was not reduced by drug treatment. These data suggested that the increase in 410 entry efficiency conferred by S H69/V70 is independent of spike S1/S2 cleavage. Similar 411 findings on lack of relationship between balance of S2:FL and infectivity were reported in the 412 context of furin knockout cells rather than furin inhibition with CMK(Papa et al., 2021) . In 413 addition, a recent report on the S1/S2 cleavage site mutation P681H demonstrated enhanced 414 cleavage of Spike P681H was not associated with increased PV infectivity or cell fusion 415 relative to WT (Lubinski et al., 2021) . There may however be differences in vivo. 416

We explored entry route of H69/V70 spike using cathepsin inhibition to block endosomal 418 entry and camostat to block entry via plasma membrane fusion. H69/V70 spike was equally 419 sensitive to camostat and the cathepsin inhibitor ED64 as WT, arguing that the efficiency of 420 entry route usage is similar despite differences in cleaved spike. Although S1/S2 cleavage 421 allows avoidance of endosome-associated IFITM restriction and appears critical for 422 transmission in animal models (Peacock et al., 2020) , cleaved spike may be less infectious if 423 S1 is shed prematurely, therefore possibly conferring a disadvantage under some 424 circumstances. In addition, Peacock et al showed that S1/S2 cleavage in the producer cell, 425 conferred by a polybasic stretch at the cleavage site, is advantageous in cells expressing 426 abundant TMPRSS2 but deleterious in cells lacking TMPRSS2(Peacock et al., 2020) . In summary we have found that a two amino acid deletion, H69/V70 promotes SARS-CoV-482 2 spike incorporation into viral particles and increases infectivity by a mechanism that 483 remains to be fully explained. This deletion has arisen multiple times and often after spike 484 antibody escape mutations that reduce spike mediated entry efficiency. Critically, B1.1.7 485 spike mediates faster syncitium formation and this enhanced cell-cell fusion activity is 486 dependent on H69/V70. In addition, B.1.1.7 spike requires H69/V70 for optimal 487 infectivity and we conclude therefore that H69/V70 enables SARS-CoV-2 to tolerate 488 multiple immune escape mutations whilst maintaining infectivity and fusogenicity. 489 Raw anonymised data are available from the lead contact without restriction. 678 679

The study was primarily a laboratory based study using pseudotyped virus (PV) with 681 mutations generates by site directed mutagenesis. We tested infectivity in cell lines with a 682 range of drug inhibitors and monoclonal antibodies. Sensitivity to antibodies in serum was 683 tested using convalescent sera from recovered individuals collected as part of the Cambridge 684 NIHR Bioresource. We also performed phylogenetic analyses of data available publicly in 685 GISAID. 

Plasmids encoding the full-length spike protein of SARS-CoV-2 D614 (Wuhan) and 762

RaTG13, in frame with a C -terminal Flag tag (Conceicao et al., 2020) , were used as a 763 template to produce variants lacking amino acids at position H69 and V70. The deletion was 764 introduced using Quickchange Lightning Site-Directed Mutagenesis kit (Agilent) following 765 the manufacturer's instructions. Viruses were purified by ultracentrifugation; 25mL of crude 766 preparation being purified on a 20% sucrose cushion at 2300rpm for 2 hrs at 4˚C. After 767 centrifugation, the supernatant was discarded and the viral pellet resuspended in 600 µL 768 DMEM (10% FBS) and stored at -80˚C. Infectivity was examined in HEK293 cells 769 transfected with human ACE2, with RLUs normalised to RT activity present in the 770 J o u r n a l P r e -p r o o f pseudotyped virus preparation by PERT assay. Western blots were performed on purified 771 virus with anti-HIV1 p24, 1:1,000 (Abcam) or anti-FLAG, 1:2,000 (Sigma) antibodies used 772 following SDS-PAGE and transfer. 773 774

The reverse transcriptase activity of virus preparations was determined by qPCR using a 777 SYBR Green-based product-enhanced PCR assay (SG-PERT) as previously 778 described (Vermeire et al., 2012) 

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