Blots were probed with mouse anti-human influenza hemagglutinin tag (HA) primary antibody (Miltenyi Biotech, Germany) for ACE2-transfected cells at 1:1,000 or with rat anti-FLAG primary antibody (BioLegend, USA) for Spike transfected cells at 1:1,000 in PBS-Tween 20 (PBS-T, 0.1%) with 5% (w/v) milk powder overnight at 4°C. However, how widely this host range or receptor tropism extends and the molecular factors defining atypical transmission to nonhuman hosts remain the subject of intense investigation. Our findings are supported by the results of experimental SARS-CoV-2 infections, which showed that cats [22,34], hamsters [27], and fruit bats [34] are susceptible to infection, while chickens are not [22]. aminopeptidase N; BHK-21, ACE2, angiotensin-converting enzyme 2; NE, non-enveloped; RLU, relative light units; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001016.s003. Results are representative of protein expression experiments performed in duplicate, with a GAPDH loading control also shown. https://doi.org/10.1371/journal.pbio.3001016, Academic Editor: Bill Sugden, University of Wisconsin-Madison, UNITED STATES, Received: August 7, 2020; Accepted: November 17, 2020; Published: December 21, 2020. Identifying the animal reservoir of SARS-CoV-2, and any intermediate hosts via which the virus ultimately spread to humans, may help to understand how, where, and when this virus spilled over into people. As discussed above, the lack of similarly closely related SARS-CoV-2 isolates from this outbreak’s origin in Hubei makes detailed interpretation of this virus’s adaptation to human ACE2 difficult at this time. Indeed, we observed that pangolin, dog, cat, horse, sheep, and water buffalo all sustained higher levels of entry than was seen with an equivalent human ACE2 construct (Fig 2A; left heatmap, first column). The data in each row are normalised to the signal seen for human ACE2 (top), with results representing the mean percentage calculated from 3 separate experiments performed on different days. 4. multiplicity of infection; NE, The single letter amino acid (aa) code is used with the vertical height of the amino acid representing its prevalence at each position in the polypeptide (aa 18–46, 78–91, 324–358, and 392–394 are indicated). These assays demonstrate correlation between ACE2 protein sequence and fusion by SARS-CoV or SARS-CoV-2 Spike protein, plus evidence of a low affinity of SARS-CoV Spike proteins for bird or rat ACE2 and varying levels of bat ACE2 utilisation. The T27S substitution would remove the threonine side chain methyl group that sits in a hydrophobic pocket formed by the side chains of RBD residues F456, Y473, A475, and Y489, and substitution of residue D30 (which is acidic in all ACE2 proteins efficiently utilised by SARS-CoV-2 Spike) to asparagine would remove the salt bridge formed with K417 of the RBD (Fig 5B). It uses the tree drawing engine implemented in the ETE toolkit, and offers transparent integration with the NCBI taxonomy database. Writing – review & editing, Roles The tree is drawn to scale, and support was provided with 500 bootstraps. The same raw RLU receptor usage values plotted in F were used for these calculations, with pDISPLAY values removed prior to calculation. SARS-CoV pseudoparticles; SARS-CoV-2, (E) Expression of the same mutants was analysed by western blot targeting the flag tag fused to these Spike proteins (S, full-length Spike; S1/S2, cleaved variant). Therefore, all bat ACE2 proteins have substitutions that impair SARS-CoV-2 Spike binding to different degrees, but it seems likely that the E30N substitution (shared only by rat ACE2) or introduction of a charged lysine residue at position 27 (unique to horseshoe bats) are the most likely causes of the severely impaired binding of SARS-CoV-2 Spike to horseshoe bat ACE2. However, concurrent sequence analysis identified a number of variable residues within the RBD, which interact directly with ACE2 (Fig 4A, inset panel). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (A) A phylogenetic tree of ACE2 proteins assembled using the neighbor-joining method [51] conducted in MEGA7 (Temple University, USA) [52] with ambiguous positions removed. It may be that the intracellular environment of specific hosts, e.g., pigs, cannot sustain SARS-CoV-2 infection, either through the absence of an important virus–host interaction or the presence of effective mechanisms of innate immune restriction. Samples were resolved on 7.5% acrylamide gels by SDS-PAGE, using semidry transfer onto nitrocellulose membrane. Little brown bats, horseshoe bats, pangolins, and horses all share a serine as ACE2 residue 34, suggesting that serine in this position does not abolish Spike binding, and it is likely that the threonine at this position (fruit bat ACE2) would likewise be tolerated. (D) Cell–cell fusion assay signals are higher when TMPRSS2 is co-expressed in target cells, including in target cells expressing non-cognate ACE2 proteins, e.g., turkey and chicken. (B) Four of the same cell lines were infected again, this time at high MOI (1). Comma-separated values (CSV) files were exported onto a universal serial bus (USB) flash drive for analysis. In our experiments, the chicken DF-1 cell line was refractory to infection, even when a cognate ACE2 (human) was overexpressed in these cells. Phycoerythrin; PHE, All protein samples were generated using 2× Laemmli buffer (Bio-Rad, USA) and reduced at 95°C for 5 min 48 h post-transfection. Additional supplements and cell culture medium for each cell line are summarised in S1 Table. The other substitutions between bat ACE2 proteins and other mammals are likely to be benign. For pseudotype and cell–cell fusion assays, luciferase assays were performed in duplicate and triplicate, respectively, with the error bars denoting standard deviation. ACE2, However, furin and cathepsin B mRNAs were both present. In this case, SARS-CoV-2 may enter cells efficiently but fail to replicate to significant levels to support onward transmission, bring about clinical signs, or induce immunopathological sequelae. Optional. This suggests that these receptors, whose structures are clearly not optimal for SARS-CoV-2 entry, are still bound by the Spike protein. BHK-21 cells were transfected using Transit-X2 transfection reagent (Mirus Bio), as per the manufacturer’s instructions with 500 ng of each ACE2-expressing construct (S2 Table) or mock-transfected with empty plasmid vector (pDISPLAY) for 48 h. Cells were resuspended in cold PBS and washed in cold stain buffer (PBS with 1% BSA (Sigma-Aldrich), 0.01% NaN3 and protease inhibitors (Thermo Fisher Scientific, USA)). ACE2, angiotensin-converting enzyme 2; RLU, relative light units; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001016.s008, https://doi.org/10.1371/journal.pbio.3001016.s009, https://doi.org/10.1371/journal.pbio.3001016.s010, https://doi.org/10.1371/journal.pbio.3001016.s011, https://doi.org/10.1371/journal.pbio.3001016.s012, https://doi.org/10.1371/journal.pbio.3001016.s013, https://doi.org/10.1371/journal.pbio.3001016.s014, https://doi.org/10.1371/journal.pbio.3001016.s015. For the pseudotype assays NE lentiviral particles were generated, i.e., vector plasmid in place of a viral glycoprotein, to examine background levels of pseudoparticle entry. Detailed Usage: All are optional. Comparison of the hamster and rat sequences (Fig 5A) identified multiple substitutions at the RBD interaction interface that might explain this variable receptor tropism (listed as hamster to rat): Q24K, T27S, D30N, L79I, Y83F, and K353H. Quantification of cell–cell fusion was measured based on Renilla luciferase activity, 24 h later by adding 1 μM of Coelenterazine-H (Promega) at 1:400 dilution in PBS. Phylogenetic analysis of the final dataset was inferred using the neighbor-joining method [51] conducted in MEGA7 [52] with ambiguous positions removed. ACE2, angiotensin-converting enzyme 2; BHK-21, baby hamster kidney; FSC-A, forward scatter area; HA, human influenza hemagglutinin tag; PE, Phycoerythrin. We also acknowledge the support of Nadine Lewis (BioBasic) for help with gene synthesis as well as The Pirbright Institute’s Flow Cytometry Facility and The Pirbright Institute Cell Servicing Unit. Specifically, coronaviruses closely related to SARS-CoV that were isolated directly from bats were shown to inefficiently use either human or civet ACE2 [30]. Q24K and Y83F substitutions would both result in the loss of hydrogen bonds with the side chain of SARS-CoV-2 RBD residue N487 (Fig 5B). Membranes were exposed to Clarity Western ECL substrate (Bio-Rad Laboratories) according to the manufacturer’s guidelines and exposed to autoradiographic film. In our pseudoparticle entry experiments, overexpression of TMPRSS2 had a negligible impact on entry when cognate ACE2 receptors were expressed on target cells (S4A Fig; human, dog, cat, pig, and goat). The next day, cells were infected with SARS-CoV-2 pp/SARS-CoV pp equivalent to 106 to 107 relative light units (RLU), or their respective NE controls at the same dilution and incubated for 48 h at 37°C, 5% CO2. Of note, the described mutants were constructed only in RaTG13 Spike expression plasmids, and assays were only performed in surrogate receptor usage assays, an approach we have previously used to safely interrogate the zoonotic potential of other viruses [15]. Furthermore, the K353H substitution would remove hydrogen bond interactions with the side chain of RBD Q498 and the backbone carbonyl oxygen of RBD G496, and neither of these could be formed by the shorter histidine side chain (Fig 5B). These studies have identified a high affinity interaction between the receptor binding domain (RBD) of Spike and the N-terminal peptidase domain of ACE2, which for SARS-CoV was shown to determine the potential for cross-species infection and, ultimately, pathogenesis [13]. Samples from both uninfected [Control] and ILTV DF-1 cells (4 biological replicates [Rep′] per condition) were examined and FPKM were calculated. While these studies did not perform specific examination of cell–cell fusion, there is a strong correlation between their findings and ours, namely, the broad tropism of SARS-CoV-2 Spike, albeit with specific examples of restriction in rodent [33], bat [32], and bird [31] species. During the SARS-CoV epidemic, where civets were identified as the intermediate reservoir of infection, a shifting pattern of increasing and decreasing ACE2 usage was observed in individual isolates of SARS-CoV taken from civets and humans (although they shared approximately 99% similarity to each other), providing evidence for adaptation to individual host receptors [13,20] with a particular focus on differential adaptation to human ACE2 residues K31 (T31 in civets) and K353. (D) The mRNA expression levels of 3 proteases relevant for SARS-CoV-2 entry, Furin, Cathepsin B, and TMPRSS2, were evaluated in DF-1 cells by analysis of the publicly available gene expression database series GSE138648 ([18], dataset and methodology (GSM4114984) available at GEO; https://www.ncbi.nlm.nih.gov/geo/). The Pirbright Institute, Woking, Surrey, United Kingdom, BHK-21 target cells were co-transfected with 500 ng of different ACE2-expressing constructs (S2 Table) and 250 ng of rLuc-GFP 8–11 plasmid. Investigation, Codon-optimised ACE2-expressing plasmids from a range of animal species were synthesised and cloned into pDISPLAY (BioBasic, Canada) (S2 Table). To this end, and in order to examine the permissiveness of nonhuman cell lines in our cell culture collection to SARS-CoV-2, we experimentally infected a range of animal cells including those established from birds, canids, rodents, ruminants, and primates (see S1 Table for details on species and cell type) with SARS-CoV-2 isolated from a patient in the United Kingdom (SARS-CoV-2 England-2/2020). The Y505H substitution in SARS-CoV-2 Spike also reduced human ACE2 receptor usage, with a concordant increase in RaTG13 activity when the reciprocal H505Y substitution was introduced (Fig 4C and 4D). Differences between closely related species that may impact RBD binding are highlighted. The β-coronavirus SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, causing a large epidemic of respiratory disease in the Hubei Province of China, centred in the city of Wuhan [1]. Methodology, Competing interests: The authors have declared that no competing interests exist. During the optimisation of our ACE2 receptor screening experiments, we also examined how transient expression of TMPRSS2 affects SARS-CoV-2 particle entry and cell–cell fusion. For the receptors where we had previously seen high levels of cell–cell fusion (hamster, pig, and rabbit), we observed robust viral replication (Fig 3C). Another example of different receptor usage between closely related species can be seen with bat ACE2 (Figs 2A and 5A). In the separate cell–cell fusion assay, which provides both luminescence and fluorescence-based monitoring of syncytium formation, a similar trend was observed with expression of chinchilla, cat, pig, sheep, goat, water buffalo, and cattle ACE2 proteins on target cells all yielding higher signals than target cells expressing human ACE2 (Fig 2A; left heatmap, second column). Hazard group 3; MAFFT, Crystallographic Object-Oriented Toolkit; CSV, The DL for the TCID-50 is indicated. Coronavirus entry into host cells is initiated by direct protein–protein interactions between the virally encoded homo-trimeric Spike protein, a class I transmembrane fusion protein found embedded in the virion envelope, and proteinaceous receptors or sugars on the surface of host cells [7]. Since the exact origin of SARS-CoV-2 is currently unknown, but widely accepted to be a Chiroptera species, we included ACE2 proteins from a broad range of bats in our study. Similarly, the Y41H substitution present in little brown bat ACE2 is also present in horse ACE2, suggesting that it does not prevent binding. 0. phyloT: a tree generator based on NCBI taxonomy which can visualize trees directly in iTOL; table2itol: an R script that makes it easy to generate iTOL annotations from spreadsheet files; Gotree: GoTree is a set of command line tools to manipulate phylogenetic trees, implemented in Go language. That being said, for some animals such as mice [26], it is now clear that inefficient ACE2 receptor usage is the primary barrier to infection. Structural studies of SARS-CoV-2 [6], and more recently RaTG13 [12], help to shed light on the interactions underpinning these phenotypic changes (Fig 6). In addition, SARS-CoV-2 entry was shown only with human ACE2, but not with aminopeptidase N (APN) or dipeptidyl peptidase 4 (DPP4), the β-coronavirus group I and MERS-CoV receptors, respectively (S2 Fig), indicating high specificity in both assays. ... Topo-phylogeny is a new approach to displaying phylogenetic relationships. The Pearson correlation was calculated, and a linear line of regression fitted together with 95% confidence intervals. Fruit bats conserve a T27, whereas little brown bats have the bulkier isoleucine residue and horseshoe bats have a bulky charged lysine residue in this position, both of which are likely to clash with the F456-Y473-A475-Y489 hydrophobic pocket of the RBD, with the lysine substitution likely to be more deleterious due to the introduction of the positive charge. secondary structure mapping; TMPRSS2, Although data analysis of this type between related viruses might represent a mechanism for identifying intermediate reservoirs, similar outliers that favoured SARS-CoV-2 entry were not evident in our study. The apparent lack of tropism for bat ACE2 proteins we observed was surprising as there is previous evidence of SARS-CoV-2 infection of bat ACE2 expressing cells in vitro [1], and in vitro binding experiments suggest that the SARS-CoV-2 RBD binds bat ACE2 with high affinity [29]. Positive cells were gated as represented in S1 Fig, and the same gating strategy was applied in all experiments. However, TMPRSS2 co-expression significantly enhanced the use of non-cognate receptors (S4D Fig; fruit bat, ferret, turkey, and chicken), when compared with ACE2 or ACE2 and trypsin treatment. bovine viral diarrhoea virus; -COVID-19, Miles Carroll). As discussed earlier, the closest known relative of SARS-CoV-2, RaTG13, was isolated from intermediate horseshoe bat (R. affinis). For SARS-CoV, this problem was circumvented with the development of transgenic mice expressing human ACE2 [23] or mouse-adapted SARS-CoV [24,25], research that has recently been extended to SARS-CoV-2 [26]. This research has identified vertebrate species where cell entry is most efficient, allowing prioritisation of in vivo challenge studies to assess disease susceptibility. The aa sites bound by SARS-CoV and SARS-CoV-2 Spike [11] are indicated by red arrows. 2 years 14. Building on the hypothesis that the progenitor of SARS-CoV-2 was RaTG13-like, we next developed pseudotype and cell–cell fusion assays for RaTG13 Spike (S8A–S8C Fig) to examine the biological properties of this protein. Yes Cells were fixed with formaldehyde for 30 min and then stained with 0.1% Toluidine blue (Sigma-Aldrich). However, our finding that hamster ACE2 allows the entry of SARS-CoV-2 (Fig 2A) indicates this animal is a suitable model for infection, consistent with recent in vivo studies demonstrating experimental infection of these animals [27]. Filename extensions are usually noted in parentheses if they differ from the file format name or abbreviation. Importantly however, receptors, which showed little evidence of supporting particle entry when expressed on their own, supported much more robust levels of entry when TMPRSS2 was co-expressed (S4A Fig; turkey and chicken), despite TMPRSS2 protein alone not supporting entry (S4B Fig). Is the Subject Area "SARS CoV 2" applicable to this article? For an optimal output of the phylogenetic tree, we randomly selected a subset of 2000 samples by using a random number generator in Python. The detection limit for the TCID-50 (DL) is indicated. Conceptualization, The predicted guinea pig mRNA sequence was more divergent than expected and contained a premature stop codon. receptor binding domain; RLU, Recent structural and functional data have shown that SARS-CoV, SARS-CoV-2, and other β-coronavirus (lineage B clade 1) Spike proteins bind the same domain in ACE2 to initiate viral entry [5,6,8–10]. The data underlying this figure may be found in S1 Data. No glycoprotein controls (NE) were also set up using empty plasmid vectors (500 ng pcDNA3.1), and all transfected cells were incubated at 37°C, 5% CO2. Similar to the pseudotype assay, the expression of all 3 bat ACE2 proteins resulted in less cell–cell fusion than that seen with human ACE2. Receptor usage screens: BHK-21 cells were seeded in 48-well plates at 5 × 104/well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) (S2 Table) in OptiMEM and TransIT-X2 (Mirus Bio) transfection reagent according to the manufacturer’s recommendations. Results represent the raw RLU signals from 3 independent experiments with the mean signal plotted and error bars denoting standard deviation. position-specific iterative; qPCR, SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. ACE2, angiotensin-converting enzyme 2; DF-1, chicken embryonic fibroblast cell line; DL, detection limit, FPKM, fragments per kilobase of exon per million reads mapped; HA, human influenza hemagglutinin tag; ILTV, infectious laryngotracheitis virus-infected; MFI, mean fluorescent intensity; MOI, multiplicity of infection; SARS-CoV-2, SARS Coronavirus 2; TCID-50, 50% tissue culture infective dose; TMPRSS2, transmembrane protease serine 2. https://doi.org/10.1371/journal.pbio.3001016.s007. Yes While none of these bat ACE2s supported SARS-CoV-2 fusion to the same levels as humans, there were dramatic differences in the ability of SARS-CoV-2 Spike to utilise the ACE2 from horseshoe bats versus fruit bats and little brown bats (Fig 2A). However, in the cell–cell system, all of these receptors permitted Spike-mediated fusion, above the background levels seen in pDISPLAY transfected cells (Fig 2A) or in effector cells not expressing SARS-CoV-2 Spike (S3 Fig; no spike), albeit at levels significantly lower than that seen for human ACE2. Carina Conceicao, RDP News. Tosyl phenylalanyl chloromethyl ketone; USB, Department of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom, Roles Human ACE2 utilisation by SARS-CoV-2 and SARS-CoV Spike is also plotted for reference. SARS-CoV-2 England-2/2020 was isolated from a patient in the UK, and a passage 1 stock was grown and titred in Vero E6 cells by Public Health England (PHE) (kindly provided to The Pirbright Institute by Prof. In all subsequent experiments, “NE” and “No Spike” controls were compared against SARS-CoV-2 pseudoparticles or SARS-CoV-2 Spike-expressing effector cells (see S3 Fig). Accordingly, in the BHK-21 cells transfected with carrier plasmid, we saw very little evidence for virus infection and/or virus production, confirming that these cells do not natively support SARS-CoV-2 infection (Fig 3C). Binds to elements within the NEUROD1 promoter (By similarity). SCG is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (098406/Z/12/B). SARS-CoV-2 pseudoparticles; SSM, Investigation, Department of Pathology, University of Cambridge, Cambridge, United Kingdom, Roles Like rats, horseshoe bat N30 would be unable to form a salt bridge with RBD K417. Thus, multiple substitutions are predicted to inhibit Spike binding to rat ACE2 when compared with the closely related hamster protein, with K353H being of particular relevance (Fig 5B). A recent structure for RaTG13 Spike [12] allowed us to directly compare the RBDs of SARS-CoV-2 and RaTG13, identifying a high degree of structural conservation (Fig 4A). Fixed cells were resuspended in PBS before being analysed using the MACSQuant Analyzer 10 (Miltenyi Biotech), and the percentage of PE-positive cells was calculated by comparison with unstained and stained mock-transfected samples. Surprisingly, RaTG13 receptor usage of human ACE2 was still higher than that of horseshoe bat ACE2, although the human ACE2 values were significantly lower than those observed for SARS-CoV-2 and SARS-CoV (Fig 4B, blue data points). (D and E) Receptor screening experiments were performed as described for SARS-CoV-2 and SARS-CoV (see S4 Fig). transmembrane protease serine 2; TPCK, electron microscopy; FBS, (A) Various cell lines derived from birds, dogs, rabbits, rodents, pigs, ruminants, and primates were experimentally infected with SARS-CoV-2 at a MOI of 0.001. For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2. Cells were resuspended in the substrate, and 50 μL was transferred to a white-bottomed plate in duplicate. CSV files were exported onto a USB flash drive for analysis.
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