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Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

['Wenlin Ren', 'Center For Infectious Disease Research', 'School Of Medicine', 'Tsinghua University', 'Beijing', 'Jun Lan', 'Beijing Advanced Innovation Center For Structural Biology', 'School Of Life Sciences', 'Xiaohui Ju', 'Mingli Gong']

Date: 2021-12

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as “Cluster 5” that contained mutations in the spike protein. However, the functional properties of these “Cluster 5” mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

Employees infected with SARS-CoV-2 transmitted the virus to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant “Cluster 5” with four amino acid changes in the spike protein. We demonstrated that the one of the mutations-Y453F as an adaptive mutation increased virus interaction with mink ACE2 receptor, without compromising its utilization of human ACE2 receptor. In addition, miSARS-CoV-2 exhibited resistance to convalescent sera from COVID-19 patients and still sensitive to ACE2-based therapeutic strategy. Therefore, it is urgently needed to identify the potential zoonotic reservoirs, and monitor the evolution of virus in the zoonotic host to prevent future outbreaks and develop countermeasures accordingly.

Funding: National Natural Science Foundation of China (32070153 to QD), Tsinghua University Spring Breeze Fund (2021Z99CFY030 to QD), Beijing Municipal Natural Science Foundation (M21001 to QD), the Postdoctoral Science Foundation of China (2021TQ0182 to XJ), Shuimu Tsinghua Scholar Program (XJ), Start-up Foundation of Tsinghua University (53332101319 to QD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Ren et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we demonstrate that the Y453F mutation in the miSARS-CoV-2 spike is an adaptive mutation that increases interaction with mink ACE2 without compromising utilization of human ACE2. In addition, the miSARS-CoV-2 exhibited partial resistance to neutralization with convalescent serum. Our study not only provides critical insights into viral adaption and evolution but also highlights the importance of surveillance of viral variants in animals to reduce the risk of the emergence of new viral variants and prevent future outbreaks.

Recently, the first animal-to-human transmission of SARS-CoV-2 was reported[ 14 , 15 ]. In April 2020, SARS-CoV-2 was transmitted to minks at two farms in the Netherlands by infected employees, and the virus subsequently circulated among the minks[ 16 , 17 ]. On 5 November 2020, the Danish public health authorities reported 214 cases of humans infected with mink-associated SARS-CoV-2 variants (miSARS-CoV-2) containing a combination of mutations not previously observed[ 15 ]. Genetic analysis grouped the miSARS-CoV-2 variants into 5 clusters with seven mutations[ 18 ]. Of note, the “Cluster 5” variant with four amino acid changes in the spike protein was identified in mink and isolated from 12 human cases in North Jutland[ 14 , 18 ]. The implications of the mutations in this variant are not yet well characterized. Preliminary results suggested that the “Cluster 5” miSARS-CoV-2 strain has moderately decreased sensitivity to neutralizing antibodies[ 14 , 19 , 20 ]. Further investigations are urgently needed to dissect the biological significance of these mutations and to understand the implications of these identified changes for diagnostics, therapeutics and vaccine development.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein binds its cellular receptor angiotensin-converting enzyme 2 (ACE2), thus mediating viral entry[ 4 , 5 ]. It has been demonstrated that the interaction of a virus with (a) species-specific receptor(s) is a primary determinant of host tropism and constitutes a major interspecies barrier at the level of viral entry[ 6 ]. Our previous study found that numerous mammalian ACE2 orthologs could function as receptors to mediate virus entry in vitro[ 7 ], and other studies have demonstrated that rhesus macaques, dogs, cats, cattle, hamsters, ferrets, minks and other animals are susceptible hosts[ 8 – 13 ]. Together, these findings suggest that SARS-CoV-2 has a broad host range, with many species that could serve as potential reservoirs and thus pose a risk for spillover to humans in the future[ 7 ].

Coronaviruses are enveloped, positive-stranded RNA viruses that circulate broadly among humans, other mammals, and birds and can cause respiratory, enteric, and hepatic disease[ 1 ]. In the last two decades, coronaviruses have caused three major outbreaks: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and the recent Coronavirus Disease 2019 (COVID-19)[ 2 , 3 ]. COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat.

Results

The Y453F mutation in miSARS-CoV-2 spike increases interaction with mink ACE2 To test our hypothesis, we first employed a cell-based assay, using flow cytometry to assess the binding of S protein to ACE2 orthologs and variants (Fig 2A). We cloned the cDNA of ACE2 variants, each with a C-terminal FLAG tag, into a bicistronic lentiviral vector that expresses the fluorescent protein zsGreen1 via an IRES element (pLVX-IRES-zsGreen1) and can be used to monitor transduction efficiency. Next, WT or Y453F S1-Fc (a purified fusion protein consisting of the S1 domain of SARS-CoV-2 S protein and an Fc domain of human lgG) was incubated with HeLa cells transduced with the ACE2 variants. Binding of S1-Fc to ACE2 was then quantified by flow cytometry as the percent of ACE2-expressing cells positive for S1-Fc (Figs S1A and 2B). As expected, the binding of S1-Fc or S1 (Y453F)-Fc to HeLa cells expressing mouse ACE2 was very low and comparable to that of the empty vector control while S1-Fc efficiently bound to HeLa cells expressing human ACE2, which is consistent with previous reports[5]. S1 (Y453F)-Fc bound human ACE2 more efficiently than WT S1-Fc (99.1% vs 86.5%). Notably, WT S1-Fc bound mink ACE2 with limited efficiency, in contrast, S1 (Y453F)-Fc bound mink ACE2 with 77% efficiency, demonstrating that the miSARS-CoV-2 mutation enhances binding to mink ACE2. Moreover, after replacing the amino acid residue at position 34 of human ACE2 with its mink counterpart to generate human ACE2 (H34Y), binding to S1-Fc was reduced (86.5% [WT] vs 42.0%) but increased to S1 (Y453F)-Fc (99.1% [WT] and 98.5%). Performing the reverse by substituting the amino acid residue at position 34 of mink ACE2 with its human counterpart to generate mink ACE2 (Y34H) only slightly increased binding to S1-Fc (1.2% vs 2.2%) and binding to S1(Y453F)-Fc (77.0% vs 80.0%) (Figs 2B and S1B). As we used cell-surface staining of ACE2 to sort the cells used for these experiments so that they had comparable ACE2 expression, the limited or undetectable binding of certain ACE2 variants with the S1 variants was not due to low expression of ACE2 or alteration of its cell surface localization (S2 Fig). The expression level of the ACE2 variants was also assessed by immunoblotting using an anti-Flag antibody and cell surface localization by immunofluorescent microscopy (Fig 2C and 2D). Together, these results showed that all the ACE2 orthologs were expressed and localized at the cell surface at comparable levels, excluding the possibility that the limited binding efficiencies of ACE2 orthologs with S1-Fc variants was attributable to varied cell surface localization. As miSARS-CoV-2 also harbored other mutations outside of RBD in the spike (Fig 1A), we purified the spike protein (1aa-1208aa) without transmembrance domain and intracellular domain with different mutations (WT, Y453F, del69-70/I692V or del69-70/Y453F/I692V) to test their binding with human or mink ACE2 respectively (S3A and S3B Fig). S (Y453F) and S (del69-70/Y453F/I692V) could exclusively bind with mink ACE2; in contrast, other S mutants without Y453F mutation could not (S3C Fig). These results demonstrated that Y453F mutation is essential and sufficient for enhanced binding with mink ACE2. To further quantify the binding of ACE2 variants with the spike protein variants, we expressed and purified recombinant WT and Y453F SARS-CoV-2 RBD as well as ACE2 variants to assay binding in vitro by surface plasmon resonance (SPR) analysis (Figs 2E and S4). Mink ACE2 bound Y453F SARS-CoV-2 with a K D of 78.22nM but binding to WT RBD was not detectable. Human ACE2 bound SARS-CoV-2 RBD with a K D of 6.5nM, and Y453F RBD with a K D of 0.98nM. These SPR results are thus consistent with the findings of our cell-based assay. Collectively, our results demonstrate that the SARS-CoV-2 spike binds mink ACE2 with limited affinity. However, the Y453F mutation dramatically increased the binding affinity with mink ACE2 without compromising binding to human ACE2, which suggests that Y453F is an adaptive mutation to improve its fitness in a new host—mink.

The Y453F mutation in miSARS-CoV-2 spike increases its interaction with other Mustela ACE2 orthologs Mink is a member of the Mustela genus which also includes stoats and ferrets, the latter of which have been used as animal models owing to their susceptibility to SARS-CoV-2[25]. Due to the high similarity of ACE2 proteins in the Mustela genus (S5A Fig), we performed the binding experiments of the S1 variants (WT or Y453F) with ferret and stoat ACE2. Consistent with the results of mink ACE2, ferret and stoat ACE2 exhibited limited binding capability with WT S1-Fc but increased binding ability with S1 (Y453F)-Fc (Figs 4A and S6A and S6B). As before, we confirmed that the ACE2 orthologs expressed and localized at the cell surface at comparable levels to exclude the possibility that differences in binding capability were due to variation in ACE2 expression and/or mislocalization (Figs 4B and S5B). In line with our cell-based binding assay, SPR analysis did not detect the binding of WT RBD with ferret or stoat ACE2, but Y453F RBD could bind ferret or stoat ACE2 with a K D of 31.17nM and 48.06 nM, respectively (Fig 4C). Taken together, all of these results suggest that the Y453F mutation increases SARS-CoV-2 RBD binding affinity with Mustela ACE2 orthologs. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Increased binding of Y453F RBD protein to Mustelidae ACE2 orthologs. (A) HeLa cells were transduced with human or Mustelidae ACE2 orthologs as indicated. Transduced cells were incubated with WT or Y453F S1 domain of SARS-CoV-2 C-terminally fused with His tag and then stained with anti-His-PE for flow cytometry analysis. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. These experiments were independently performed three times with similar results. (B) Cell surface localization of human, ferret and stoat ACE2. HeLa cells were transduced with lentivirus expressing ACE2 orthologs as indicated. Cells were incubated with rabbit polyclonal antibody against ACE2 and then stained with goat anti-rabbit IgG (H+L) conjugated with Alexa Fluor 568 and DAPI (1μg/ml). The cell images were captured with a Zeiss LSM 880 Confocal Microscope. This experiment was independently repeated twice with similar results and the representative images are shown. (C) The binding kinetics of ACE2 proteins (ferret or stoat) with recombinant WT or Y453F SARS-CoV-2 RBD were obtained using the BIAcore. ACE2 proteins were captured on the chip, and serial dilutions of RBD were then injected over the chip surface. Experiments were performed three times with similar result, and one set of representative data is displayed. https://doi.org/10.1371/journal.ppat.1010053.g004

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