Acquisition of the L452R mutation in the ACE2-binding interface of Spike protein triggers recent massive expansion of SARS-Cov-2 variants

The recent rise in mutational variants of SARS-CoV-2, especially with changes in the Spike protein, is of significant concern due to the potential ability for these mutations to increase viral infectivity, virulence and/or ability to escape protective antibodies. Here, we investigated genetic variations in a 414-583 amino acid region of the Spike protein, partially encompassing the ACE2 receptor-binding domain (RBD), across a subset of 570 nasopharyngeal samples isolated between April 2020 and February 2021, from Washington, California, Arizona, Colorado, Minnesota and Illinois. We found that samples isolated since November have an increased number of amino acid mutations in the region, with L452R being the dominant mutation. This mutation is associated with a recently discovered CAL.20C viral variant from clade 20C, lineage B.1.429, that since November-December 2020 is associated with multiple outbreaks and is undergoing massive expansion across California. In some samples, however, we found a distinct L452R-carrying variant of the virus that, upon detailed analysis of the GISAID database genomes, is also circulating primarily in California, but emerged even more recently. The newly identified variant derives from the clade 20A (lineage B.1.232) and is named CAL.20A. We also found that the SARS-CoV-2 strain that caused the only recorded case of infection in an ape - gorillas in the San Diego Zoo, reported in January 2021 - is CAL.20A. In contrast to CAL.20C that carries two additional to L452R mutations in the Spike protein, L452R is the only mutation found in CAL.20A. According to the phylogenetic analysis, however, emergence of CAL.20C was also specifically triggered by acquisition of the L452R mutation. Further analysis of GISAID-deposited genomes revealed that several independent L452R-carrying lineages have recently emerged across the globe, with over 90% of the isolates reported between December 2020 – February 2021. Taken together, these results indicate that the L452R mutation alone is of significant adaptive value to SARS-CoV-2 and, apparently, the positive selection for this mutation became particularly strong only recently, possibly reflecting viral adaptation to the containment measures or increasing population immunity. While the functional impact of L452R has not yet been extensively evaluated, leucine-452 is positioned in the receptor-binding motif of RBD, in the interface of direct contact with the ACE2 receptor. Its replacement with arginine is predicted to result in both a much stronger binding to the receptor and escape from neutralizing antibodies. If true, this in turn might lead to significantly increased infectivity of the L452R variants, warranting their close surveillance and in-depth functional studies.


Abstract. 1 5
The recent rise in mutational variants of SARS-CoV-2, especially with changes in the 1 6 Spike protein, is of significant concern due to the potential ability for these mutations to 1 7 increase viral infectivity, virulence and/or ability to escape protective antibodies. Here, 1 8 we investigated genetic variations in a 414-583 amino acid region of the Spike protein, 1 9 partially encompassing the ACE2 receptor-binding domain (RBD), across a subset of 2 0 570 nasopharyngeal samples isolated between April 2020 and February 2021, from 2 1 Washington, California, Arizona, Colorado, Minnesota and Illinois. We found that 2 2 samples isolated since November have an increased number of amino acid mutations in 2 3 the region, with L452R being the dominant mutation. This mutation is associated with a 2 4 recently discovered CAL.20C viral variant from clade 20C, lineage B.1.429, that since 2 5 November-December 2020 is associated with multiple outbreaks and is undergoing 2 6 massive expansion across California. In some samples, however, we found a distinct 2 7 L452R-carrying variant of the virus that, upon detailed analysis of the GISAID database 2 8 genomes, is also circulating primarily in California, but emerged even more recently.

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The newly identified variant derives from the clade 20A (lineage B.1.232) and is named 3 0 CAL.20A. We also found that the SARS-CoV-2 strain that caused the only recorded 3 1 case of infection in an ape -gorillas in the San Diego Zoo, reported in January 2021 -is 3 2 CAL.20A. In contrast to CAL.20C that carries two additional to L452R mutations in the 3 3 Spike protein, L452R is the only mutation found in CAL.20A. According to the 3 4 phylogenetic analysis, however, emergence of CAL.20C was also specifically triggered 3 5 by acquisition of the L452R mutation. Further analysis of GISAID-deposited genomes 3 6 revealed that several independent L452R-carrying lineages have recently emerged 3 7 across the globe, with over 90% of the isolates reported between December 2020 -3 8 February 2021. Taken together, these results indicate that the L452R mutation alone is 3 9 of significant adaptive value to SARS-CoV-2 and, apparently, the positive selection for 4 0 this mutation became particularly strong only recently, possibly reflecting viral 4 1 adaptation to the containment measures or increasing population immunity. While the 4 2 functional impact of L452R has not yet been extensively evaluated, leucine-452 is 4 3 positioned in the receptor-binding motif of RBD, in the interface of direct contact with the 4 4 ACE2 receptor. Its replacement with arginine is predicted to result in both a much 4 5 RBD (L452R, T470N, T478K, G482V, E484K and S494P) or in the 570-572 loop of  1  0  4 CTD1 (A570V and T572I). All of the amino acid mutations had been reported previously 1 0 5 and could be identified in nCoV sequences deposited to GISAID. Though the notorious 1 0 6 N501Y mutation was not identified in our samples, five samples contained mutation 1 0 7 E484K that is found in lineages from various countries and present in the 'Brazil' variant 1 0 8 B.1.1.28. E484K is located in the receptor-binding ridge epitope and was shown to 1 0 9 provide marked resistance to neutralizing antibodies in multiple studies [8,11,12]. 1 1 0 The most frequent mutation found in the region 414-583 was by far L452R, occurring in 1 1 1 19 out of 39 (48.7%) total and 13 out of 26 (50.0%) non-linked samples, isolated in 9 out 1 1 2 of 18 (50%) separate collection sites. Three samples with L452R also had separate 1 1 3 silent changes and two samples (from the same site) had an additional amino acid 1 1 4 change T572I. L452R was found mostly in samples (14) and sites (6) from California, 1 1 5 though 5 samples from 3 sites came from Washington. L452R is part of the 443-450 1 1 6 loop epitope and is located on the edge of the receptor-binding motif of RBD formed by 1 1 7 residues in direct contact with ACE2 [8,13,14]. Its occurrence appeared to be only 1 1 8 sporadic in most of 2020 and has received significant attention only very recently due to 1 1 9 the report of the California Department of Public Health released January 17, 2021 and 1 2 0 a follow-up publication [15]. It was noted that there has been a recent sharp rise in 1 2 1 isolation of nCoV variants with L452R across multiple outbreaks in California, 1 2 2 accounting for more than a third of all isolates. According to the GISAID database, only 1 2 3 six nCoV genomes with L452R were deposited in September-October 2020 (all from 1 2 4 California), but since then additional genomes with the mutation have been deposited 1 2 5 including 142 in November (95.7% from California), 488 in December (79.1%) and 619 1 2 6 in January 2021 (69.2%). This expansion has been linked to a single viral variant from 1 2 7 clade 20C according to the Nextstrain nomenclature of nCoV (lineage B.1.429 1 2 8 according to the PANGO nomenclature of nCoV) and was designated as CAL.20C 1 2 9 (20C/S:452R; B.1.429). 1 3 0 According to genomic analysis, CAL.20C has also been defined by 4 additional amino 1 3 1 acid mutations, including two Spike protein mutations, S13I and W152C, located in the 1 3 2 signal peptide and N-terminal domain, respectively [15]. Using an approach similar to 1 3 3 our sequencing of the region 414-583, we amplified and sequenced the aa1-250 coding 1 3 4 region in all samples with the L452R mutation. Both S13I and W152C mutations were 1 3 5 found in 15 total (11 non-linked) samples suggesting their identity with CAL.20C. 1 3 6 (Among those samples, one contained an additional silent mutation, while in two 1 3 7 samples from one site a four-amino acid deletion (141-144 LGVY) was found.) 1 3 8 Surprisingly, in 4 samples with L452R the additional mutations were absent. Those 1 3 9 samples originated from two separate sites (two samples in each) in California. One 1 4 0 sample pair carried the T572I mutation in the 414-583 region mentioned above. To compare the clonal diversity of CAL.20A and CAL.20C strains, we build a 1 6 5 phylogenetic tree of the January isolates of CAL.20A and 50 randomly selected 1 6 6 genomes of CAL.20C also deposited in January 2021. We also included into the 1 6 7 analysis some strains that we have identified as the most closely related to the L452R 1 6 8 variants, but without this mutation. Based on the shorter branches overall, the CAL.20A 1 6 9 cluster appeared to be less diverse than the CAL.20C cluster (Figure 2B). The pairwise 1 7 0 difference in the number of silent, presumably neutrally accumulating mutations per 1 7 1 genome was 2.56±1.41 and 8.22±4.19 mutations, respectively (P<.01), indicating that 1 7 2 CAL.20A has emerged much more recently than CAL.20C. 1 7 3 In contrast to CAL.20C, L452R was the only omnipresent amino acid mutation in the 1 7 4 Spike protein of CAL.20A, relatively to the Wuhan-Hu-1 reference strain. Other 1 7 5 mutations, like T572I in our samples, were found only sporadically and in few strains 1 7 6 (with most mutations in the S2 region). In fact, L452R was the only amino acid mutation 1 7 7 in the entire genome that separated CAL.20A from the closest non-L452R strains within 1 7 8 the B.1.232 lineage (GISAID # 636127, Figure 2B). Thus, acquisition of L452R appears 1 7 9 to be the primary evolutionary event that led to emergence of CAL.20A. Though it was 1 8 0 originally reported that, besides L452R, all CAL.20C strains carry 4 more specific amino 1 8 1 acid mutations, including S13I and W152C in the Spike protein, we found that few 1 8 2 closely related strains in the B.1.429 lineage carry either S13I alone (GISAID # 977963) 1 8 3 or both S13I and W152 but not L542R (GISAID # 847642 and # 977918, Figure 2B). strains each (Figure 3). The strains were isolated from over 20 countries across all 1 9 2 continents, with no apparent dominance of any geographic area. In one deposited 1 9 3 strain, from lineage B.1.74, an L452Q mutation was present instead of L452R. There is 1 9 4 a clear temporal trend in the number of deposited genomes, with 2 genomes only from 1 9 5 July-September, 28 from October-November, 127 from December 2020 and 238 from 1 9 6 January 2021. Thus, the L452R mutation has been acquired independently in a variety 1 9 7 of clonally diverse nCoV strains, with 92.6% of L452R strains reported after November 1 9 8 2020, indicating a very recent emergence of all those lineages. 1 9 9 Discussion 2 0 0 Taken together, our results show that two independent nCoV variants recently emerged 2 0 1 in the state of California that carry the L452R mutation in the Spike protein, the already 2 0 2 defined and currently dominant CAL.20C [15] as well as the more recently emerged 2 0 3 CAL.20A identified here. The fact that, according to our analysis, emergence of both 2 0 4 CAL.20A and CAL.20C was triggered by the L452R mutation alone provides direct 2 0 5 evidence for the adaptive significance of this mutation specifically and, also, creates a 2 0 6 potential opportunity to isolate and functionally compare naturally occurring isogenic 2 0 7 variants of nCoV with and without L452R.

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On the other hand, it is possible that the lack of other mutations specific to CAL.20A or 2 0 9 shared with CAL.20C could be the reason why the former variant has not undergone as 2 1 0 extensive expansion as CAL.20C. This would indicate (and availability of CAL.20A 2 1 1 should help to affirm) that other mutations in CAL.20C might enhance the adaptive 2 1 2 impact of L452R, i.e. that the genomic background of L542R plays a significant role as 2 1 3 the target of positive selection. However, it is also possible that CAL.20A is at least as fit 2 1 4 as CAL.20C, but has not expanded as broadly because it emerged much more recently 2 1 5 and after CAL.20C has underwent extensive expansion in the same geographic niche 2 1 6 area. Co-circulation of CAL.20A and CAL.20C in the same area provides a unique 2 1 7 opportunity to study the interplay between variants in space and time in 2 1 8 demographically diverse but interconnected communities and patient populations. 2 1 9 According to the public databases, in addition to the two California strains the L452R 2 2 0 mutation has been acquired at this point by at least half a dozen independent lineages 2 2 1 across multiple countries and continents. Though detailed examination of the timing, 2 2 2 geography and genomic background of L452R emergence in different lineages is 2 2 3 beyond the scope of this study, such repeatedly emerging hot-spot mutations typically 2 2 4 indicate strong positive selection. Interestingly, it appears that the selection for L452R 2 2 5 became especially strong very recently. This is possibly reflecting adaptive evolution of 2 2 6 the virus in response to either the epidemiological containment measures extensively 2 2 7 introduced in the fall of 2020 or a growing proportion of the population with immunity to 2 2 8 the original viral variants, i.e. the reconvalescents and vaccinated individuals.

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Though potentially an accidental event, isolation of CAL.20A from a gorilla at the San 2 3 0 Diego Zoo is worthy of note. According to the sequence deposited in GISAID, the gorilla 2 3 1 CAL.20A variant carries two additional SNPs, both in the ORF1ab non-structural protein 2 3 2 2 (nsp2), a silent c934t and non-synonymous c810t (T183I), but with several sequence 2 3 3 stretches unfortunately missing. It is impossible to say at this point to what extent the 2 3 4 isolation of the CAL.20A variant is connected to possibly distinctive biological properties 2 3 5 of the strain and specifically the L452R mutation. However, it would be valuable to 2 3 6 determine whether the occurrence of CAL.20A infection in the gorilla is due to specific 2 3 7 features of CAL.20A with regard to viral transmissibility, infectivity or virulence.

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Despite mounting evidence for the adaptive value of L452R, the exact functional or 2 3 9 structural effect of the mutation and its impact on viral immunogenicity, pathogenesis, 2 4 0 infectivity and/or transmissibility remains to be determined. Due to only recent attention 2 4 1 to L452R variants, relatively few studies investigated the potential effects of this 2 4 2 mutation. It was found that L452R reduces the Spike protein reactivity with a panel of 2 4 3 the virus neutralizing antibodies and sera from convalescent patients [8,14]. Moreover, 2 4 4 it was found that out of 52 naturally occurring mutations in the receptor-binding motif 2 4 5 residues of RBD that form the interface of direct interaction with ACE2, L452R results in 2 4 6 the largest increase in free energy of the RBD-ACE2 binding complex, predicting 2 4 7 stronger virus-cell attachment and, thus, increased infectivity [13]. We hope that the 2 4 8 current and the original report on L452R variants of nCoV will facilitate in-depth 2 4 9 structure-functional studies of the leucine residue in position 452 position and the 2 5 0 change of hydrophobic leucine to a polar, highly hydrophilic arginine (or glutamine).

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We cannot exclude the possibility of sample collection bias in our study as it was not 2 5 2 originally designed as an in-depth surveillance study in specific geographical regions. In 2 5 3 addition, our analysis is limited to a relatively small set of samples in hand and to 2 5 4 publicly available genomes. However, we believe that the identification of CAL.20A and 2 5 5 CAL.20C, with both common and unique features relative to the other circulating nCoV 2 5 6 variant, will be useful in the optimization of real-time monitoring and the more complete 2 5 7 understanding of the biological properties of this pandemic virus with a recently 2 5 8 expanding number of genetic variations that are cause for significant public concern. 2 5 9