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Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Abstract Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS‐CoV‐2 causing the global COVID‐19 outbreak. Here, we study the binding of two SARS‐CoV‐2‐like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin‐converting enzyme 2 (hACE2), the receptor of SARS‐CoV‐2. We find that the spike protein receptor‐binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS‐CoV‐2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2‐expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS‐CoV‐2. Additionally, cryo‐EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS‐CoV‐2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS‐CoV‐2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.


Introduction
Emerging and re-emerging viruses pose a serious threat to global public health (Gao, 2018). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 2019  that has recently emerged worldwide (The 2019-nCoV Outbreak Joint Field Epidemiology Investigation Team Tan et al, 2020;Wu et al, 2020b;Aluko et al, 2021), resulting in approximately 153.9 million laboratory-confirmed cases and over 3.2 million deaths worldwide as of 5 May 2021 (https://covid19. who.int/). Global economy is also being devastated by COVID-19 pandemic throughout the world. However, the origin of the SARS-CoV-2 and its path of transmission remain elusive, which requires better understanding of the cross-species transmission and evolutionary relationship between the SARS-CoV-2 and its highly related coronaviruses (CoVs).
CoVs are the largest group of positive-sense single-stranded RNA viruses (Adams & Carstens, 2012). Based on genotypic and serological characteristics, CoVs can be subdivided into four genera, namely alpha, beta, gamma, and delta CoVs (Lu et al, 2015;Su et al, 2016). So far, all identified CoVs that can infect humans belong to the first two genera. Phylogenetic analysis of the SARS-CoV-2 and its closely related CoVs known to date indicate that RaTG13, which is identified in bat, displays the highest whole-genome sequence identity (96.2%) with the SARS-CoV-2. Additionally, RmYN02 is another bat CoV displaying slightly lower sequence identity (93.3%) (Zhou et al, 2020a;Zhou et al, 2020b). Therefore, the SARS-CoV-2 is likely to have originated from bats and jumped to an extended host range via an intermediate host (Damas et al, 2020;Zhou et al, 2020b).
Here, we evaluated the interaction between the two pangolin CoV RBD proteins and 27 ACE2s from 10 orders in Mammalia class and chicken in the Galliformes order of Aves class. The two pangolin CoV RBDs were found to bind to ACE2s from human and some other animals with similar binding affinity as the SARS-CoV-2 RBD. In contrast, they have a boarder host range than the SARS-CoV-2 that includes some rodents. To further study the binding mechanism of the pangolin CoVs with hACE2, we solved the complex structures of the two pangolin CoV RBDs with hACE2 at the same resolution of 3.4 A using cryo-electron microscopy (cryo-EM). Our data reveal similar binding modes between hACE2 and the RBD of each one of the three viruses. A key residue in the RBDs that determines the interaction between pangolin CoVs and rodent ACE2s is also identified, indicating that the two pangolin CoVs possess a broader host range comparing to the SARS-CoV-2. Together with the functional and structural data presented in this A B Figure 2. Binding affinity assay between ACE2s and the RBDs of the SARS-CoV-2, GD/1/2019 and GX/P2V/2017 by SPR.
A The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD, and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. B The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD, and GD/1/2019 RBD) and the 13 ACE2s is shown. Mean AE SD represents the mean and standard deviation of three independent experiments.
We then chose ACE2 orthologs to evaluate their binding affinity with either of the RBDs of GX/P2V/2017 and GD/1/2019 by SPR (Fig 2A and B). hACE2 was included to evaluate the potential crosstransmission of the two pangolin CoVs to human beings. Since the two CoVs were isolated from pangolin, this animal was included. Bats carry many SARS-related CoVs and are suspected to be the natural host of SARS-CoV-2, and thus, bat ACE2s from six species were chosen. Civet is thought to play a role in the transmission of SARS-CoV, and thus, we also chose this animal. We further included mouse, rat, and hedgehog ACE2s due to their different binding features with pangolin CoVs from SARS-CoV-2. Lesser hedgehog tenrec was used as negative control.
Mouse Fc (mFc)-tagged ACE2s from the abovementioned 13 species were then prepared and immobilized on the chip. The two serially diluted soluble pangolin CoV RBD proteins were sequentially flew through the chip. SARS-CoV-2 RBD was used as control. As shown in Fig 2, all the three viral RBDs were incapable of binding to ACE2s from greater horseshoe bat, least horseshoe bat, civet, and lesser hedgehog tenrec, which were consistent with the flow cytometry results. The binding affinity between hACE2 and the three RBDs is similar, with the equilibrium dissociation constants (K D ) calculated to be 11.2 AE 0.5 nM for SARS-CoV-2 RBD, 11.9 AE 0.1 nM for GX/P2V/2017 RBD, and 13.6 AE 0.7 nM for GD/1/2019 RBD. In addition, the binding affinity between the ACE2s from 4 animals (Malayan pangolin, little brown bat, fulvous fruit bat, and big-eared horseshoe bat) and either one of GX/P2V/2017 RBD and GD/1/2019 RBD are also similar. In contrast, the binding affinity between the SARS-CoV-2 RBD and the ACE2s from Malayan pangolin and big-eared horseshoe bat is~2-fold stronger, and the binding affinity of the SARS-CoV-2 RBD to the little brown bat and fulvous fruit bat is 2-fold weaker. In terms of the ACE2 from the intermediate horseshoe bat that carries RaTG13, the GD/1/2019 RBD has a 2-fold weaker binding affinity than GX/P2V/2017 RBD and the SARS-CoV-2 RBD. As previously reported (Wu et al, 2020a), no interaction is detected between the SARS-CoV-2 RBD and mouse, rat, or European hedgehog ACE2s, while both GX/P2V/2017 RBD and GD/1/2019 RBD could bind to all the three ACE2s. GD/1/2019 RBD even displayed stronger binding affinity with these three ACE2s than the GX/P2V/2017 RBD. To determine whether GX/P2V/2017 and GD/1/2019 infect cells via binding to hACE2, three VSV-based pseudotyped CoVs (SARS-CoV-2, GX/P2V/2017, and GD/1/2019) were prepared. Similar amounts of three pseudoviruses (as determined by quantitative real-time PCR) were used to transduce HeLa cells, with or without the expression of hACE2. All three pseudoviruses were unable to transduce HeLa cells, but readily transduced Hela cells expressing hACE2 (HeLa-hACE2; Fig 3A). Moreover, The SARS-CoV-2 and GD/1/2019 pseudoviruses displayed similar transduction efficiency, but pseudotyped GX/P2V/2017 showed lower efficiency ( Fig 3B).
Out of the contacts in patch 2, the three groups contain 9, 8, and 5 H-bounds, respectively (Table 1). Compared to the binding surface of SARS-CoV-2 RBD, 2 residues (R417 and H498) and 7 residues (V417, V484, L486, Y490, E493, H498, and T501) are different in GD/1/2019 RBD and GX/P2V/2017 RBD, respectively (Fig 5B). In particular, the Q498H mutation found in both pangolin CoV RBDs may strengthen the interaction with the helix a1 of hACE2 by enhancing positive charge (Fig EV5B), and the K417R mutation from GD/1/2019 RBD may also enhance the contacts with hACE2 by forming one extra H-bond. In addition, comparing to the SARS-CoV-2 RBD, the variant residues V417, L486, and Y490 of GX/P2V/2017 RBD result in loss of multiple vdw interaction with hACE2. Together, these clues suggest that the variant residues in the pangolin CoV RBDs contribute to the differences between the two complex structures involving pangolin CoVs and that of the SARS-CoV-2 RBD-hACE2.

Identification of key residues responsible for the broader host range of pangolin CoVs
Next, we explored the mechanism that leads to the differences in binding patterns among two pangolin CoVs and the SARS-CoV-2, especially the amino acids vital to the binding of mouse, rat, hedgehog, and human ACE2s. Therefore, we introduced the K417R, Q498H, or K417R-Q498H mutation to the SARS-CoV-2 RBD to mimic the key variant residues of GD/1/2019 RBD.
ª 2021 The Authors The EMBO Journal 40: e107786 | 2021 To verify the impact of the abovementioned mutations to the SARS-CoV-2 RBD, we examined the binding between each SARS-CoV-2 RBD mutant and the ACE2 orthologs from human, mouse, rat, and European hedgehog. The three species besides human were chosen because of their distinct binding characteristics between the SARS-CoV-2 and the pangolin CoVs. Flow cytometry and SPR data showed that both the SARS-CoV-2 RBD and the K417R mutant have no detectable interaction with mouse, rat, or European hedgehog ACE2, but the Q498H and K417R-Q498H mutants acquired high-binding affinity with the ACE2s from mouse (287.7 AE 23.7 nM and 248.0 AE 21.9 nM), rat (338.7 AE 74.9 nM and 319.8 AE 70.0 nM), and European hedgehog (258.3 AE 9.5 nM and 484.0 AE 42.9 nM) (Fig 6A-C). In addition, the Q498H mutant displayed 5-fold stronger binding affinity with hACE2 than the wild-type SARS-CoV-2 RBD (Fig 6C).
To explore the mechanism of the changes in binding affinity caused by Q498H mutation, we identified 4 conserved residues (D38, Y41, Q42, and S45) on the surface of human, mouse, and rat ACE2s that interact with Q498H mutation, and a Q42E mutation on European hedgehog ACE2 interacting with Q498H. The mutation from glutamine (Q) to histidine (H) leads to a drastic increase in positive charges, which enhanced the affinity between Q498H mutants with human, mouse, or rat ACE2.
In agreement with observation above, while the N38 residue on European hedgehog ACE2 is neutral as D38, the Q42E mutation introduced negative charges which further attract the Q498H mutant (Fig EV5A and  B). Together, these evidences suggest the H498 residue could play an important role in the cross-species transmission of pangolin CoVs.

Discussion
The recent pandemic of COVID-19 has led to serious global crisis in public health and economy. Bat CoVs are likely the ancestors of the SARS-CoV-2, and RaTG13 has common ancestor with the SARS-CoV-2 (Zhou et al, 2020b). Due to the detection and isolation of pangolin CoVs with high sequence homology to the SARS-CoV-2, the possibility of pangolins as the intermediate host has also been proposed (Lam et al, 2020;Xiao et al, 2020;Zhou et al, 2020a). However, no SARS-CoV-2 has yet been detected in pangolins. In addition, a recent report showed that pangolins could be infected by the pangolin CoV GD/1/2019 and showed clinical symptoms and histological changes (Xiao et al, 2020). Although not universally true, natural reservoir or the intermediate hosts tend to have A C B Figure 6. Binding affinity assay between different SARS-CoV-2 RBD mutants and ACE2s by FACS and SPR.
A Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H, and K417R-Q498H) binding to human, mouse, rat, and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control. B The mFc-tagged ACE2s from human, mouse, rat, and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H, and K417R-Q498H). C The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean AE SD represents the mean and standard deviation of three independent experiments.

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The EMBO Journal 40: e107786 | 2021 ª 2021 The Authors coevolved with their viruses and usually do not display clinical symptom. For example, bats are natural reservoirs for a variety of emerging viruses yet rarely cause clinical disease in bats (Wang et al, 2006;Shi & Hu, 2008). Dromedary camels are thought to be the intermediate host for MERS-CoV; they could carry the virus without showing any severe disease (Peck et al, 2015). Thus, pangolins are more likely to be another victim of SARS-CoV-2-like viruses, rather than to be the natural reservoir or intermediate host.
However, due to the lack of sampling and detection of CoVs in pangolin, we cannot rule out the possibility that this animal carries other SARS-CoV-2-like viruses but without clinical symptoms. Very few pangolins are present in China, and the detection results were all negative for SARS-CoV-2 (Deng et al, 2020;Wacharapluesadee et al, 2021). The two CoVs studied in this report were identified from the smuggled pangolins, with unknown source (Lam et al, 2020;Xiao et al, 2020). Thus, retrospective studies on animals and humans in the countries with pangolins, such as Southeast Asia, are highly needed.
Recent studies reported that many cofactors are involved in the SARS-CoV-2 infection, but ACE2 is still the most important receptor (Cantuti-Castelvetri et al, 2020;Daly et al, 2020;preprint: Gu et al, 2020). By evaluating the interaction between SARS-CoV-2 RBD and multiple species ACE2 orthologs, the possible broad host range of the SARS-CoV-2 has been well identified by us and other groups (Damas et al, 2020;Shi et al, 2020;Wu et al, 2020a). Here, using a similar strategy, two pangolin CoVs (GD/1/2019 and GX/P2V/2017) are revealed to bind to hACE2 as efficient as the SARS-CoV-2. The VSV-based pseudoviruses incorporating GD/1/2019 or GX/P2V/ 2017 S protein also enter into cells by the mediation of hACE2, suggesting the two pangolin CoVs may enter hACE2-expressing cells and cause human infection. Furthermore, two pangolin CoV RBDs associate with a panel of ACE2 orthologs at a similar level to that of the SARS-CoV-2, but present a broader host range, with binding capability to ACE2 orthologs from three additional animals (mouse, rat, and European hedgehog). These results also evoke concerns about the potential cross-species transmission of the two pangolin CoVs, and further spill-over to humans. Thus, not only the SARS-CoV-2, but also the two pangolin CoVs could be serious threats to the broad hosts, especially mammals (Damas et al, 2020). Therefore, continuous surveillance of pangolin CoVs is needed.
Mutational analysis in this study suggests that the residue H498 rather than R417 is crucial for the binding between the RBD and the ACE2s from mouse, rat, and European hedgehog which are closely related to human life. The substitution of Q498 with histidine shifts the electrostatic feature to the opposite and triggers the capacity of the RBD to interact with the mouse, rat, and European hedgehog ACE2s. Currently, the Q498H substitution has been found in the SARS-CoV-2 in two human samples (SA-lsf-27 and SA-lsf-37) from Iran. Notably, a recent report showed that HRB-26m, a mouseadapted strain of the SARS-CoV-2, contains A81T in the nsp8, Q498H, and N969S, together with the deletion of QTQTN 675-679 in the S protein. According to our data, the SARS-CoV-2 RBD with Q498H increases the binding strength to hACE2 by 5-fold, suggesting the Q498H mutant is more ready to interact with human receptor than the wild type and highlighting the necessity for more strict control of virus and virus-infected animals. Further studies are essential for the binding analysis of different CoVs to human ACE2 and their molecular basis as they will help narrow down the revelation of SARS-CoV-2 origin.
The pEGFP-N1 plasmids expressing 27 species ACE2 proteins for FACS and the pCAGGS plasmids expressing 13 species ACE2 proteins for SPR were constructed in our recent work (Wu et al, 2020a).
For HEK293F cell expression, the coding sequences of SARS-CoV-2 RBD and its three mutations (K417R, Q498H, K417R-Q498H) tagged with a C-terminal 6× His tag were cloned into the pCAGGS expression vector using the EcoRI and XhoI restriction sites.

Protein expression and purification
Protein expression and purification of different expression system have been well described in our recent study . Briefly, for Bac-to-Bac baculovirus expression system, the bacmid was first transfected into Sf9 insect cells (Invitrogen, 11496015). The cell culture supernatants, which contain the packaged recombinant baculoviruses, were harvested about 72 h post-transfection. The baculovirus was then passaged in Sf9 cells for 2-3 times, while Hi5 cells (Invitrogen, B85502) were used for protein production. Soluble proteins were purified by 5-ml HisTrap HP column (GE Healthcare) after the supernatant was collected and filtration by 0.22-lm filter membrane. The samples were then pooled and further purified by Superdex TM 200 Increase 10/300 GL column (GE Healthcare) with a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl.
For the mFc-fusion protein expression used for SPR, pCAGGS plasmids containing coding sequences for the 27 ACE2 proteins were transiently transfected into HEK293T cells (ATCC, CRL-3216). The culture supernatants containing the indicated proteins were collected and concentrated about 72 h after transfection.
For protein expression in HEK293F cells (ATCC), the pCAGGS plasmid containing the coding sequences of the SARS-CoV-2 RBD and its three mutations (K417R, Q498H, and K417R-Q498H) was transiently transfected into HEK293F cells. After 72 h, the supernatant was collected and soluble proteins were purified by using a 5-ml HisTrap excel column (GE Healthcare). The sample was further purified via gel filtration chromatography with a Superdex TM 200 Increase 10/300 GL column (GE Healthcare) with a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl.

Surface plasmon resonance (SPR) analysis
The binding affinities of indicated mFc-fusion protein with three RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) or the mutations (K417R, Q498H and K417R-Q498H) of SARS-CoV-2 RBD were evaluated with SPR as previously described . SPR-based measurements were performed by BIAcore8000 system (GE healthcare) with CM5 chips (GE Healthcare) at 25°C in single-cycle mode. All proteins used for kinetic analysis were exchanged to the HBST buffer (20 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Tween 20). The CM5 biosensor chip was first immobilized with anti-mIgG antibody (ZSGB-BIO, ZF-0513). Concentrated supernatants containing different ACE2-mFc proteins were then individually captured by the antibody immobilized on the CM5 chip at more than 400 response units. Gradient concentrations of the RBD proteins were then run across the chip surface, with another channel set as control. After each cycle, regeneration of the sensor chips was performed using Glycine pH 1.7. The affinity value K D for each pair of interaction was calculated with BIAcore_8K evaluation software (GE Healthcare). The graphics were prepared using OriginPro 9.1.

Production and quantification of pseudoviruses
The SARS-CoV-2, GX/P2V/2017, and GD/1/2019 pseudoviruses were constructed with a GFP encoding replication-deficient vesicular stomatitis virus (VSV) vector backbone (VSV-DG-GFP) and the coding sequence of corresponding spike proteins, as previously described Muik et al, 2021). Briefly, HEK 293T cells were transfected by 30 lg of spike protein expression plasmids. The VSV-DG-GFP pseudovirus was added 24 h post-transfection. The inoculum was removed after incubation for 1 h at 37°C. The culture medium was then changed into DMEM supplemented with 10% FBS and 10 lg/ml of anti-VSV-G antibody (I1-Hybridoma ATCC â CRL2700 TM ) after washing cells with PBS. The pseudoviruses were harvested 20 h post-inoculation, passed through a 0.45-lm filter (Millipore, Cat#SLHP033RB) before aliquoted, and stored at À80°C.
All pseudoviruses were treated with 0.5 U/ll BaseMuncher Endonuclease (Abcam, ab270049) for 1.5 h at 37°C to remove unpackaged RNA before quantification. Viral RNA was extracted (Bioer Technology, Cat# BYQ6.6.101711-213) and quantitated by quantitative RT-PCR (qPCR) using 7500 fast real-time PCR system (Applied Biosystems) with the primers and probe for detecting the P protein coding sequence of VSV.

Pseudovirus infection assay
The pseudovirus particles of SARS-CoV-2, GX/P2V/2017, and GD/ 1/2019 were normalized to the same amount for quantitation by qRT-PCR. Then, 100 ll of each pseudovirus was added to each well of 96-well plate containing HeLa-hACE2 cells. Untransfected HeLa cells were used as control. Plates were imaged 15 h posttransfection. The numbers of fluorescent cells were determined on a CQ1 confocal image cytometer (Yokogawa). Each group contains 5 replicates. Statistical significance was determined by a two-sided unpaired Student's t-test.

Cryo-EM sample preparation and data collection
For cryo-EM analysis, 4 ll aliquot of the purified GX/P2V/2017 RBD-hACE2 or GD/1/2019 RBD-hACE2 complex protein at about 0.2 mg/ml was applied to glow-discharged holey carbon grids (Quantifoil Au 1.2/1.3, 300 mesh). The grids were blotted by a couple of 55-mm filter papers (TED PELLA, INC.) for 2-2.5 s at 22°C with 100% humidity and flash-frozen in liquid ethane using a FEI Vitrobot Mark IV. The data were collected on a 300 kV Titan Krios electron microscope equipped with a Gatan BioQuantum energy filter with K3 direct electron detection camera (Gatan) using AutoEMation (Lei & Frank, 2005). Micrographs were recorded in counting mode at a nominal magnification of 130,000×, resulting in a physical pixel size of 0.6625 A per pixel. Defocus values varied from À1.5 lm to À2.5 lm. The exposure rate was 12.7 electron per pixel per second. Exposures of 1.28 s were dose-fractionated into 32 sub-frames, leading to a total accumulated dose of 50 electrons per A 2 .

Image processing and 3D reconstruction
The raw dose-fractionated image stacks were 2× Fourier binned, aligned, dose-weighted, and summed using MotionCor2 (Zheng et al, 2017). Contrast transfer function (CTF) parameters were estimated using CTFFIND4 (Rohou & Grigorieff, 2015) for the summed micrographs. We used the non-dose-weighted micrographs for determination of CTF parameters and dose-weighted micrographs for the following data processing. Bad micrographs were removed manually based on the CTF parameters. Only the micrographs fitting well with simulated and actual thon ring were selected for the next steps. Subsequent processing steps were performed in RELION-3.1 (Zivanov et al, 2018) with similar process. For all the datasets, manually picked sets of about 3,000 particles were subject to 2D classification. These processes generated templates for referencebased particle picking, respectively. The automatically picked particles were extracted with a box size of 240 pixels and rescaled to 80 pixels (bin3, pixel size 1.9875 A) in RELION-3.1 for the following 2D and 3D classification. Then, one round of reference-free 2D classification (total 150 classes and 25 iterations) was performed to remove contaminants and noise in the raw automatically picked particles. All the selected particles after 2D classification were used to generate the initial model in RELION-3.1. After two rounds of reference-based 3D classification (3 classes and 30 iterations for each round), the most homogeneous particles were selected and reextracted with a box size of 240 pixels and rescaled to 160 pixels (bin1.5, pixel size 0.99375 A) for the final 3D auto-refinement. A soft edge mask was generated from the auto-refinement volumes for the final post-processing step in RELION-3.1. More details related to data processing are summarized in Table EV1.

Model building and refinement
The crystal structure of the SARS-CoV-2 RBD-hACE2 complex (PDB: 6LZG) was docked into the two cryo-EM density maps by using UCSF Chimera (Pettersen et al, 2004). The target RBD amino acid sequence was obtained by point mutation using COOT (Emsley & Cowtan, 2004). The two models were then real-space refined using PHENIX (Adams et al, 2010) with secondary structure restraints. Finally, the models were validated with MolProbity . Statistics for model refinement and validation are shown in Table EV1.

Quantification and statistical analysis
Flow cytometry analysis All experiments were performed three times; one representative of each experiment is shown in Figs 1 and 6.
Binding affinity analysis K D values of SPR experiments were obtained with BIAcoreâ 8K Evaluation Software (GE Healthcare), using a 1:1 binding model. The values indicate the mean AE SD of three independent experiments.
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