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Evolutionary pathways to SARS-CoV-2 resistance are opened and closed by epistasis acting on ACE2

['Gianni M. Castiglione', 'Department Of Ophthalmology', 'Johns Hopkins University School Of Medicine', 'Baltimore', 'Maryland', 'United States Of America', 'Lingli Zhou', 'Zhenhua Xu', 'Zachary Neiman', 'Chien-Fu Hung']

Date: 2022-01 

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

If natural variation across species has evolved to shape ACE2 function, then the evolution of ACE2 sequences due to differences in physiology may alter the latent capacity for viral receptor usage and susceptibility. We therefore investigated whether SARS-CoV-2 binding to ACE2 could be abolished without disrupting ACE2 enzyme function. To expedite this, we took an evolutionary approach that leveraged the natural sequence variation found in mouse ACE2, which SARS-CoV-2 is unable to bind and gain infection [ 1 , 21 , 26 ]. Here, we identify a specific combination of mutations unique to rodents which fully abolishes RBD binding when inserted into human and dog ACE2, but which in isolation, significantly decreases ACE2 enzyme activity. These detrimental intermediates would likely severely compromise the cardio- and renoprotective functions of ACE2 activity, explaining why these mutations are rare across mammalian species.

In mammals, ACE2 evolved to serve an essential reno- and cardioprotective role in vivo, mediated through its regulation of the renin–angiotensin–aldosterone system (RAAS) in conjunction with ACE. The signaling peptide Angiotensin-II (Ang-II) generated by ACE—a major clinical target for hypertension—stimulates vasoconstriction, inflammation, and fibrosis responses through the Ang-II/AT 1 R axis [ 41 , 42 ]. ACE2 carboxypeptidase activity counteracts these effects through conversion of Ang-II to Ang-(1–7), a peptide that induces vasodilatory, anti-inflammatory, and antifibrotic effects through MAS signaling [ 42 , 43 ]. Loss of ACE2 in mice worsens cardiac dysfunction in obesity, increases diabetic kidney dysfunction, increases mortality rates after myocardial infarction, and can have severe effects on cardiac contractility [ 41 , 43 – 45 ]. This protective role of ACE2 is largely mediated through its enzymatic processing of Ang II to Ang-(1–7), where exogenous delivery of either ACE2 or Ang-(1–7) can protect against pathogenic features of multiple cardiovascular and kidney diseases [ 41 , 45 , 46 ]. Given the myriad protective roles of ACE2 enzymatic activity, it may be expected that ACE2 function is highly conserved across species. However, mice display approximately 50% higher ACE2 activity relative to humans [ 47 ] and are insensitive to the vasodilatory effects of Ang-(1–7) [ 43 ], suggesting that increased ACE2 activity in rodents may have evolved to serve nonvasodilatory protective functions. However, rodents depend on ACE2-mediated degradation of Ang II to main normal blood pressure and cardiovascular homeostasis [ 44 , 48 – 52 ]. These observations suggest that major physiological differences between species can drive differences in ACE2 function, as seen in the evolution of other protein systems [ 34 , 40 , 53 ]. Organismal sensitivity to mutations affecting ACE2 activity may be especially pronounced due to the X-chromosome location of the ACE2 gene [ 54 ], where even heterozygous ACE2 knockout females (−/x) display increased susceptibility to heart and kidney injury [ 43 ].

Infection by SARS-CoV-2 is mediated by the binding of the viral S protein receptor-binding domain (RBD) to ACE2 [ 1 , 21 ], displaying a nanomolar affinity higher than that of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1) [ 22 ]. Structural analyses have identified that the SARS-CoV-2 RBD targets multiple binding “hotspots” within the ACE2 ectodomain, presenting a diffuse and multifaceted binding strategy [ 23 – 25 ]. Thus, it remains unclear why so few species are resistant to infection, since there is extensive sequence diversity within the viral binding domain of ACE2 [ 18 ]. The degeneracy of ACE2 sequence requirements for SARS-CoV-2 binding raises the question as to why sequences capable of blocking viral binding have not evolved more frequently by chance; mice (Mus musculus) are one of the only mammalian species known to be resistant to SARS-CoV-2 infection [ 1 , 21 , 26 ]. This exception is striking, as mice do not have naturally circulating SARS-related viruses that may be expected to drive the evolution of ACE2-mediated viral resistance, as observed during sarbecovirus arms races within bat ACE2 [ 27 , 28 ]. One explanation may be that other physiological constraints exist, potentially limiting this amino acid combination to be functionally tolerable only in mice. Genetic context or starting point can determine which sequence combinations ultimately evolve [ 29 ], where intramolecular epistasis, a form of context dependence, enables amino acids to have different functional effects depending on residues at other sites [ 30 ]. Epistasis can permit compensatory interactions to evolve following an “original” mutation [ 31 – 34 ], ultimately generating completely different amino acid combinations converging on the same structural/functional “solution” [ 29 , 35 – 38 ]. Conversely, intramolecular epistasis can limit the number of possible amino acids combinations that will confer a given function [ 39 , 40 ]. The opening and closing of evolutionary trajectories by epistasis and pleiotropy could potentially reconcile the dearth of sequences capable of blocking SARS-CoV-2 binding with the wide diversity of ACE2 sequences.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is closely related to a virus (RaTG13) isolated from the Chinese horseshoe bat (Rhinolophus affinis) [ 1 ], with circulation of related viruses in bat populations [ 2 – 4 ] and potential spillover into other species susceptible to infection by SARS-CoV-2–related coronaviruses (e.g., pangolin; Manis javanica) [ 5 ]. These zoonotic origins ultimately led to the evolution of a virus that is highly transmissible among humans [ 2 ], causing an unprecedented public health emergency [ 6 , 7 ]. A wide phylogenetic range of mammalian species have been demonstrated to be susceptible to SARS-CoV-2 infection, including nonhuman primates, dogs, cats, ferrets, hamsters, and minks [ 8 – 15 ]. Characterizing the entire host range of SARS-CoV-2 is important for identifying the risks of anthroponosis and zoonosis between humans and other species, which can pose a major health risk by forming novel viral reservoirs where new mutations can evolve [ 16 , 17 ]. A recent example is mink farms, where infection by humans led to the evolution of novel viral strains, which have since reinfected human populations [ 14 ]. Attempts to predict the host range of SARS-CoV-2 have greatly underestimated the extent to which SARS-CoV-2 can infect certain nonprimate species, especially nonfelid carnivores including mink, dog, and ferret [ 8 – 14 , 18 ]. These predictive methods depend on comparative sequence analyses of angiotensin converting enzyme 2 (ACE2)—the cellular receptor for SARS-CoV-2—and scoring based on sequence and structural homology to the human ACE2 viral binding interface [ 18 – 20 ]. The difficulty of predicting the SARS-CoV-2 host range through these methods demonstrates that the virus can bind a wide range of ACE2 orthologs despite extensive sequence divergence.

Results

We investigated ACE2–RBD binding as well as ACE2 enzymatic function across a range of boreoeutherian mammals either susceptible or resistant to SARS-CoV-2 infection [human (Homo sapiens; XM_017650263.1), dog (Canis lupus familiaris; XM_019746337.1), and pangolin (M. javanica; XM_017650263.1) versus mouse (M. musculus; XM_017650263.1) and Chinese horseshoe bat (Rhinolophus sinicus; XM_019746337.1)] [1,10,12,21]. Using flow cytometry, we found a trend of significantly stronger association of the SARS-CoV-2 RBD with both human and pangolin ACE2 relative to that of SARS-CoV-1, consistent with previous studies [22] (Fig 1A–1D). Notably, SARS-CoV-1 and SARS-CoV-2 RBD association was strongest with human ACE2. We also found evidence that the RBD of both SARS-CoV-1 and SARS-CoV-2 S protein could not bind mouse nor bat (R. sinicus) ACE2 (Fig 1B–1D), consistent with previous studies [5,10,12,21,55]. To complement these binding assays, we next characterized the ability of ACE2 orthologs to enable SARS-CoV-2 S pseudovirus entry into cells. We transfected ACE2 orthologs into human cells (HEK293T) and exposed these cells to pseudotyped murine leukemia virus (MLV) particles containing the SARS-CoV-2 S protein. Consistent with our flow cytometry data and with previous studies, this assay largely recapitulated the host range of wild-type (WT) SARS-CoV-2 [21], displaying significant infection of cells expressing human, dog, and pangolin ACE2, but not that of mouse or R. sinicus bat (Fig 1E). To investigate whether this variation in ACE2–SARS-CoV-2 S binding is mirrored by functional variation in ACE2 enzyme activity, we measured the carboxypeptidase activity of ACE2 orthologs in vitro using a well-characterized fluorometric biochemical assay [47] (Materials and methods; S1 Fig). Importantly, the Ang-II peptide substrate of ACE2 is conserved among all species investigated here (S2 Fig). Furthermore, all ACE2 protein orthologs displayed highly similar expression levels in HEK293T cells (S3 Fig). Strikingly, we found significant variation in ACE2 hydrolysis rates across all mammalian species, suggesting tuning of enzymatic function across evolutionary history (Fig 1F and 1G), perhaps in response to interspecies RAAS variation. Interesting, dog, R. sinicus bat, and pangolin all displayed low ACE2 hydrolysis relative to human and mouse. Mice displayed the highest ACE2 hydrolysis rates, consistent with previous reports [47]. This demonstrates that ACE2 function varies considerably across species and suggests that ACE2 sequence variation could potentially reflect diversification of ACE2 enzymatic function.

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TIFF original image Download: Fig 1. Natural variation in SARS-CoV-2 binding is mirrored by diversity in ACE2 enzyme activities. (A–D) Flow cytometry was used to quantify SARS-CoV-1 and SARS-CoV-2 RBD-Fc association with human cells (HEK293T) expressing ACE2–eGFP orthologs from various mammalian species (MFI [22]). N = 3 biological replicates. Standard deviation is shown. (E) SARS-CoV-2 S pseudovirus infection of HEK293T cells expressing ACE2 orthologs was quantified using a luciferase reporter system. N = 4 biological replicates. Standard deviation is shown. (F) The carboxypeptidase activity of ACE2 was quantified using a fluorometric peptide incubated with solubilized HEK293T cells transfected with ACE2 orthologs. N = 4 biological replicates. (G) Hydrolysis rate of ACE2 orthologs (fluorescence units/minute). N = 3 to 5 biological replicates. Standard error is shown. All data are available in S1 Data. ACE2, angiotensin converting enzyme 2; MFI, mean fluorescence intensity; RBD, receptor-binding domain; SARS-CoV-1, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001510.g001

To test this, we first took a bioinformatic approach. We reasoned that since ACE2 has evolved under pressures related to its enzymatic processing of Ang-II/Ang-(1–7), signatures of natural selection at sites within the viral binding interface may reflect a potential role of those sites in mediating ACE2 catalytic activity [56]. We therefore searched for shifts in ACE2 mutational rates (d N /d S ) beyond what may be expected from neutral evolutionary pressures alone [57,58]. To do this, we constructed a phylogenetic dataset representing full-length mammalian ACE2 sequences encompassing residues 22 to 742 of human ACE2 (S4 Fig, S1 Table). This dataset represents all major mammalian lineages [59], with a high sampling of bats in particular (Chioptera) since they are known to display high ACE2 sequence diversity [27,28]. We conducted a codon-based statistical phylogenetic analysis on the entire tree (PAML, HyPhy) and found significant evidence that mammalian ACE2 is under positive selection, outperforming models assuming neutral molecular evolution (S2 Table). Since synonymous variation can confound tests of positive selection, we ran a test that explicitly accounts for it in model parameters [60]. This analysis also detected significant evidence of positive selection across ACE2 (S3 Table). As a further control, we excluded bat ACE2 from the analysis, since they are known to display positive selection reflecting an arms race with sarbecovirus binding and infection [27,28]. Even when bats were excluded from the analysis, we still detected evidence of positive selection in mammalian ACE2 (S4 Table). This provides evidence that mammalian ACE2 function may be a target of natural selection that shifts in response to physiological constraints unrelated to sarbecovirus arms races.

To focus our analysis on ACE2 sites responsible for mediating SARS-CoV-2 binding, we took an evolutionary approach leveraging natural sequence variation found in mouse ACE2, which SARS-CoV-2 is unable to bind [1,21]. We identified a set of 6 residues unique to mouse ACE2 that were of particular interest due to their proximity to viral RBD residues implicated in the ACE2–RBD structure (Fig 2A). These sites contact opposite ends of the viral RBD, facilitating binding through a combination of hydrogen bonds (Q24 and K353), as well as van der Waals forces within a hydrophobic pocket of ACE2 (L79, M82, Y83, and P84) [23,25]. These ACE2 residues are located well outside the ACE2 active site mediating catalysis of Ang-II/Ang-(1–7) (Fig 2A; yellow), suggesting that any role of these sites in mediating ACE2 function could be through indirect effects modulating the protein structure, as seen in other protein systems [56,61]. Phylogenetic analysis of evolutionary rates across the ACE2 gene with or without the inclusion of bat ACE2 sequences (S2 and S4 Tables, respectively) identified several of these sites deviating significantly from neutral expectations (Fig 2C; sites 24, 79, dN/dS>1; site 353, dN/dS<1; M8, Bayes empirical Bayes; S5 Table). This finding held even when synonymous rate variation was incorporated into the model (FUBAR; Fig 2C). Interestingly, these sites are less variable in primates relative to other mammalian lineages [19], displaying significantly decreased ACE2 evolutionary rates relative to bats (Chiroptera) and rodents (Fig 2D, S6 Table) (CmD; dN/dS). Interestingly, 2 of these sites (24 and 79) were previously identified as being under positive selection in bats, likely due to evolutionary arms race between bat ACE2 and sarbecovirus binding and infection [27,28]. The fact that these sites remain under positive selection even after bats are excluded from the analysis (S5 Table) strongly suggests that these sites are evolving in response to physiological variables directly related to ACE2 function, rather than viral binding alone.

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TIFF original image Download: Fig 2. Interacting amino acids mediating ACE2 activity also govern SARS-CoV-2 binding and cellular infection. (A) SARS-CoV-2 gains cellular entry through the viral spike protein RBD (red), which targets binding hotspots on the ACE2 receptor (blue) distal to the ACE2 active site (yellow) [6M17; [25]]. (B) Mouse ACE2 displays unique amino acid residues at positions within the RBD binding interface relative to other mammals (S1 Table). (C) Gene-wide statistical phylogenetic analyses (dN/dS averages [dots] and ranges [gray]; PAML, HyPhy) of an alignment of mammalian ACE2 coding sequences (residues 22 to 742, human ACE2 numbering; uniport ID Q9BYF1) reveals positive (*) and negative selection (θ) on RBD binding hotspots. Alignment of ACE2 residues across boreoeutherian mammals is shown. (D) Evolutionary rates of the 6 ACE2 sites that display rare variants in mice. (E) Flow cytometry was used to quantify RBD association with human cells (HEK293T) expressing WT human and mouse ACE2, as well as mutant mouse ACE2 containing all 6 human substitutions. N = 3 biological replicates; standard deviation is shown. (F) Infection of HEK293T cells transfected with either WT or mutant ACE2 exposed to VSV-G pseudotyped with SARS-CoV-2 S protein. Cellular infection was measured as a function of luciferase luminescence. N = 4 biological replicates. Standard error is shown. (G) The effect of ACE2 mutations on ACE2 hydrolysis rates was measured using a fluorometric peptide. ACE2 activity was measured as fluorescence units per minute. N = 5 biological replicates. Standard error is shown. (H) Flow cytometry analysis of RBD association with human cells (HEK293T) expressing WT and mutant human ACE2. N = 3 biological replicates. Standard error is shown. (I) The effect of human ACE2 mutations on ACE2 hydrolysis rates. N = 3 to 5 biological replicates. Standard error is shown. (J, K) Flow cytometry analysis of RBD association with human cells (HEK293T) expressing WT and mutant human and dog ACE2. N = 2 to 3 biological replicates. Standard error is shown. (L, M) Pseudovirus infection of HEK293T cells transfected with either (L) human or (M) dog ACE2, containing the indicated mutations. N = 4 biological replicates. Standard deviation is shown. (N, O) ACE2 hydrolysis activity of dog ACE2 with indicated mutations. N = 3 to 5 biological replicates. Standard error is shown. All data are available in S1 Data. ACE2, angiotensin converting enzyme 2; LRT, likelihood ratio test; RBD, receptor-binding domain; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001510.g002

Although these bioinformatic signatures suggest that these sites are functionally important, these predictions could have been influenced by the presence of multinucleotide substitutions among ACE2 orthologs [62]. Thus, we experimentally tested whether ACE2 sequence variation at these sites plays a role in the diversification of ACE2 function. We also investigated whether these sites mediate viral binding and infection. First, we substituted the rare residues found in mouse ACE2 with the residues found in human ACE2 (Fig 2B). Using flow cytometry, we found that these mutations were sufficient to confer mouse ACE2 with binding to SARS-CoV-2 S RBD, at nearly 80% of that of human WT ACE2 (Fig 2E). Using the MLV-SARS-CoV-2 pseudovirus system, we found that mutant mouse ACE2 containing these 6 substitutions was sufficient to confer pseudovirus infection of HEK293T cells, even beyond that conferred by WT human ACE2 (Fig 2F). This demonstrates that these rare mouse mutations likely play a key role in the resistance of mice to SARS-CoV-2 [1,21]. Interestingly, we find that these 6 mutations altogether also significantly decreased the catalytic activity of mouse ACE2 by over 25% (Fig 2G), suggesting that at least some of these sites are functionally important. Next, we investigated whether these sites also mediate the enzymatic activity and the binding of SARS-CoV-1/2 S RBD to human ACE2. Using co-immunoprecipitation and flow cytometry, we systematically characterized the effect of these rare variants from mouse on the binding of SARS-CoV-1/2 S RBD to human ACE2. The single mutation with the largest effect on the RBD binding of both SARS-CoV-1 (S5 Fig) and SARS-CoV-2 to ACE2 was K353H (Fig 2H). The other single mutations had only minor effects on SARS-CoV-2 RBD binding (Fig 2H). However, each of the 6 single mutations displayed considerable effects on human ACE2 hydrolysis activity (Fig 2I). This demonstrates that ACE2 residues within the RBD binding interface can indirectly modulate ACE2 activity.

Next, we attempted to abolish RBD binding to both human and dog ACE2 by making multiple substitutions at these ACE2 sites. Consistent with the reciprocal experiment in mice described above (Fig 2E), RBD binding to the ACE2 of both human and dog was abolished with just these 6 mouse substitutions (Fig 2J and 2K). This is surprising given the numerous other sites implicated in mediating human and dog ACE2 interactions with the viral RBD [23,25,63]. In both human and dog ACE2, abolition of RBD binding depended on mutating sites 24 and 82 (Fig 2J and 2K), despite displaying different residues in human (Q24; M82) and dog (L24; T82) ACE2 (Fig 2C). This demonstrates that SARS-CoV-2 utilizes the same combination of ACE2 positions to bind both human and dog ACE2 despite amino acid variation at these sites. This may explain why bioinformatic predictions of SARS-CoV-2 host range based on human ACE2 sequence homology have tended to underestimate the infection risk of species such as dogs, ferrets, and minks [9,10,12–14,18]. Using the MLV-SARS-CoV-2 pseudovirus system, we found that these 6 mouse substitutions also significantly reduced pseudovirus infection of HEK293T cells expressing WT and sextuple mutant human and dog ACE2 (Fig 2L and 2M). Although infection was not completely abolished in mutant human and dog ACE2, the reciprocal human substitutions into mouse ACE2 were sufficient to confer infection (Fig 2E). This altogether demonstrates that these rare mouse mutations are likely sufficient for resistance to SARS-CoV-2.

We had observed that these rare mouse substitutions are partly responsible for the high enzymatic activity of mouse ACE2 relative to that of human (Fig 2G). Yet, when substituted into human ACE2, single mutations drive down ACE2 activity (Fig 2I). The dependence of mutational effects on genetic background implies epistasis in ACE2 function. Consistent with this, we observed in both human and dog ACE2 a context dependence (epistasis) of mutational effects between the distal domains of the RBD binding interface. Specifically, we found that the detrimental functional effects of the quadruple mutant in both human and dog ACE2 (L79T; T/M82S; Y83F; P84S) as well as the K353H single mutant were significantly reversed when combined—a phenomenon known as sign epistasis—displaying partial compensation for each other’s detrimental effects on ACE2 hydrolysis rates (Fig 2N and 2O). Further evidence of epistasis in this ACE2 domain is seen in the sextuple mutant, where introduction of L24N in dog ACE2 fully rescued ACE2 activity, whereas Q24N decreased activity in human ACE2 relative to the quintuple mutant (Fig 2N and 2O). This discrepancy is likely attributable to amino acid interactions between site 24 and other sites not investigated. These results demonstrate that SARS-CoV-2 binding and infection depend on functionally important ACE2 sites that are highly sensitive to background effects (intramolecular epistasis).

Since normal ACE2 activity is essential for blood pressure regulation and cardiac homeostasis (Fig 3A and 3B; [43]), the deleterious functional effects of these mutations may have closed this human ACE2 evolutionary trajectory leading to SARS-CoV-2 resistance [31,40]. Indeed, we found extremely low human allele frequencies for missense polymorphisms at these ACE2 sites (gnomAD), with most at zero (Fig 3C). It is notable that the 2 sites with polymorphisms (82 and 84) are the only 2/6 where we detected no evidence of positive or negative selection (Fig 2C). We investigated the predicted pathogenicity of these polymorphisms and compared them to the hypothetical polymorphism K353H (Fig 3D)—a substitution required to abolish SARS-CoV-2 binding at the cost of large detrimental effects on human ACE2 activity. We employed PolyPhen—a machine learning–based Bayesian method for estimating pathogenicity of nonsynonymous human mutations ([64]). Consistent with its relatively high allele frequency (2.44 × 10−5), the M82I polymorphism had a zero PolyPhen score, indicating it as benign (Fig 3D). The nearly 5-fold less common polymorphism P84T (5.47 × 10−6) had a slightly higher pathogenicity score but was still predicted as benign (Fig 3D). By comparison, the hypothetical human mutation (K353H) produced a “possibly damaging” PolyPhen score (Fig 3D), consistent with the 2-fold decrease in human ACE2 activity caused by the K353H mutation (Fig 2H), the absence of human polymorphisms at this site (Fig 3C), and strong purifying selection across mammals (Fig 2C). Interestingly, a human ACE2 polymorphism directly adjacent to site 353 (G352V) produced a “probably damaging” PolyPhen score (Fig 3D), consistent with its low allele frequency (5.75 × 10−6). This analysis suggests that polymorphisms at sites 352 to 353 may be unlikely to evolve in human populations due to functionally deleterious effects. This functional constraint likely blocks evolutionary escape of human ACE2 from SARS-CoV-2 binding, which depends on K353H (Fig 3E). In dog ACE2, many single and combinatorial mutations at these sites are also functionally detrimental, including K353H (Fig 3F). Unlike human ACE2, however, in the dog ACE2 background, the sign epistasis of K353H induces fully compensatory effects, such that simultaneously substituting all 6 mutations becomes functionally nearly neutral (Fig 3F, dashed yellow arrow). This suggests that unlike humans and other primates, evolutionary escape from CoV-2 binding may be possible in canines along future evolutionary trajectories.

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TIFF original image Download: Fig 3. Human polymorphisms sufficient for SARS-CoV-2 resistance are blocked by deleterious functional effects related to cardiovascular constraints. (A, B) Hydrolysis of ANG-II by ACE2 generates ANG-(1–7), a vasodilatory peptide with protective effects against cardiac hypertrophy [43]. (C) Allele frequencies for human missense polymorphisms at these positions are lower than the protein-wide average frequency at a given ACE2 site (black line). Site 352 is included for comparison to site 353, which had no polymorphisms. (D) Predicted pathogenicity of human ACE2 polymorphisms and the hypothetical substitution (K353H) sufficient for resistance to SARS-CoV-2 (PolyPhen). (E) Substitution of mouse residues into human ACE2 abolishes ACE2–RBD binding, but with large trade-offs on ACE2 hydrolytic activity due to K353H. (F) Unlike human ACE2, dog ACE2 can theoretically reach an alternative sequence that abolishes viral binding without deleterious trade-offs on ACE2 activity. For comparison, each species ACE2 hydrolysis rates are normalized to respective WT values. All data are available in S1 Data. ACE2, angiotensin converting enzyme 2; RBD, receptor-binding domain; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001510.g003

We hypothesized that the situation may have been analogous in the rodent ancestor 70 MYA; these 6 mutations may have represented a viable alternative sequence–function optimum, thus facilitating their eventual evolution in the mus genus. To test this, we constructed a large jawed vertebrate ACE2 phylogenetic dataset (Fig 4A, S6 Fig) and reconstructed the ancestral rodent (RodAnc) ACE2 using likelihood methods [65]. Since the carboxyl terminus of vertebrate ACE2 was too divergent to align, we constructed the RodAnc ACE2 coding-sequence with either mouse (-M) or human (-H) carboxyl termini and measured ACE2-specific hydrolysis activity. We found that RodAnc-M displayed high ACE2 hydrolysis activity indistinguishable from that of mouse (Fig 4B). Moreover, the RodAnc with the human carboxyl terminus displayed ACE2 activity significantly lower than mouse ACE2, but significantly higher than that of human, suggesting that the carboxyl terminus is necessary, but not sufficient to explain the high ACE2 activity seen in the RodAnc. We therefore continued our mutagenesis investigation using RodAnc-M (hereafter referred to as RodAnc). RodAnc ACE2 had none of the rare sequence variants unique to the RBD binding domain of mouse ACE2 (Fig 4C). This strongly suggests that these mutations conferring mice with resistance to SARS-CoV-2 infection appeared relatively recently in rodent ACE2 evolution. Consistent with this, RodAnc ACE2 bound SARS-CoV-2 S RBD at a level nearly identical to that of human ACE2, with viral binding abolished after introduction of the 6 mouse mutations (Fig 4D). Consistent with our hypothesis, we found that RodAnc could tolerate the introduction of all 6 mouse mutations without deleterious effects on ACE2 enzymatic activity (Fig 4E). The functional viability of this alternative sequence combination in the genetic background of RodAnc ACE2 may therefore explain why these rare variants were ultimately permitted to evolve in mice.

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TIFF original image Download: Fig 4. Alternative sequence–function optima in rodent ancestor ACE2 serendipitously confers resistance to SARS-CoV-2 in extant mice. (A) Phylogeny of jawed vertebrates used in the ancestral reconstruction of ACE2 from the last common ancestor of rodents (RodAnc). (B) Evolution of high ACE2 activity in the rodent ancestor. The carboxyl terminus of mouse (M) or human (H) was used to determine hydrolysis rates of a fluorometric peptide. N = 4 to 5 biological replicates. Standard error is shown. (C) RodAnc ACE2 displays none of the rare sequence variants unique to the RBD binding domain of mice. (D) Flow cytometry analysis of SARS-CoV-2 S RBD binding to RodAnc-M and human ACE2. Sextuple mutants of each contain the 6 mouse variants. N = 3 biological replicates. Standard deviation is shown. (E) Hydrolysis rates of WT and mutant ACE2. N = 5 biological replicates. Standard error is shown. (F) Residues conferring mice with resistance to SARS-CoV-2 are unlikely to evolve in humans due to antagonistic pleiotropy with human ACE2 hydrolysis rates. This antagonistic pleiotropy is absent from dogs and the rodent ancestor. All data points relative to human WT, except dog, for which RBD binding is shown relative to dog WT. (G) Systolic blood pressure divergence between rodents (red) and primates (blue) is correlated with differences in body size (p = 0.001; phylogenetically independent least squares linear regression). All data are available in S1 Data. ACE2, angiotensin converting enzyme 2; PIC, phylogenetic independent contrast; RBD, receptor-binding domain; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001510.g004

If the functional viability of this alternative sequence explains the traversal of this trajectory by mice, then why was this alternative sequence combination never realized in dog ACE2, where it is, a priori, equally viable? We hypothesized that mammalian differences in physiological constraints related to ACE2 function may influence these evolutionary trajectories. Although ACE2 activity plays a critical role in cardioprotection and prevention of high blood pressure in both mice and humans [43,66], a key physiological difference in rodents is small body size, which is known to result in a lower homeostatic blood pressure set point relative to larger body size mammals [67]. We conducted a phylogenetic statistical analysis on systolic blood pressure values across mammalian species and observed a significant correlation with body size (r2 = 0.26; p = 0.001; phylogenetic independent contrast least squares linear regression) (Figs 4G and S7). Blood pressures were lowest in rodent and bat species and much higher in carnivores, including dog, as well as primates (Fig 4G). Since ACE2 is critical for degrading ANG-II and preventing ANG-II-mediated vasoconstriction in rodents [44,48–52], we propose that high ACE2 activity may have evolved in the rodent ancestor to maintain the lower homeostatic blood pressure set point in rodents relative to large mammals, such as primates and carnivores. This is consistent with fossorial data suggesting that the RodAnc was among the smallest ancestral mammal 70 MYA [68]. Although preliminary, these observations suggest that low blood pressure, and by extension, high ACE2 activity, may be a prerequisite to traverse this evolutionary trajectory, even if it ultimately leads to a sequence conferring equivalent activity (Fig 4F). By contrast, high ACE2 activity may be unlikely to evolve in carnivores and primates due to blood pressure constraints, therefore closing this trajectory. Although speculative, we discuss below how these genetic and physiological contingencies may have indirectly influenced the evolution of resistance to SARS-CoV-2.

[END]

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