receptor utilization of angiotensin converting enzyme 2 ... · 6/13/2020 · 31 transmission....

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Receptor utilization of angiotensin converting enzyme 2 (ACE2) 1

indicates a narrower host range of SARS-CoV-2 than that of SARS-2

CoV 3

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Ye Qiu1#, Qiong Wang1#, Jin-Yan Li1, Ce-Heng Liao1, Zhi-Jian Zhou1, Xing-Yi Ge1* 5

Hunan Provincial Key Laboratory of Medical Virology, Institute of Pathogen Biology 6

and Immunology, College of Biology, Hunan University, 27 Tianma Rd., Changsha, 7

Hunan, 410012, China 8

# These authors contributed equally to this work. 9

* Correspondence to Dr. Xing-Yi Ge, [email protected] 10

Short title：receptor utilization and host range of SARS-CoV-2 11

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted June 13, 2020. ; https://doi.org/10.1101/2020.06.13.149930doi: bioRxiv preprint

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Abstract 24

Coronavirus pandemics have become a huge threat to the public health worldwide in the 25

recent decades. Typically, SARS-CoV caused SARS pandemic in 2003 and SARS-26

CoV-2 caused the COVID-19 pandemic recently. Both viruses have been reported to 27

originate from bats. Thus, direct or indirect interspecies transmission from bats to 28

humans is required for the viruses to cause pandemics. Receptor utilization is a key 29

factor determining the host range of viruses which is critical to the interspecies 30

transmission. Angiotensin converting enzyme 2 (ACE2) is the receptor of both SARS-31

CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the 32

viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-33

utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-34

CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of 35

SARS-CoV-2. Meanwhile, pangolin CoV, another SARS-related coronavirus highly 36

homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile 37

with SARS-CoV rather than SARS-CoV-2. To clarify the mechanism underlying the 38

receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 39

5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 40

20th and 42nd amino acids that may determine the different receptor utilization of SARS-41

CoV, SARS-CoV-2 and pangolin CoV. Our studies promote the understanding of 42

receptor utilization of pandemic coronaviruses, potentially contributing to the virus 43

tracing, intermediate host screening and epidemic prevention for pathogenic 44

coronaviruses. 45

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Keywords: SARS-CoV; SARS-CoV-2; coronavirus; angiotensin converting enzyme 2 47

(ACE2); receptor utilization; interspecies transmission; host range. 48

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Introduction 50

Coronaviruses are enveloped non-segmented positive sense RNA viruses. In the recent 51

two decades, human coronaviruses (HCoVs) have caused to at least three major 52

pandemics and pose a huge threat to the public health worldwide (1). Until now, seven 53

different HCoVs have been identified and all of them belong to the family 54

Coronaviridae of the order Nidovirales. Especially, the species Severe acute respiratory 55

syndrome-related coronavirus (SARSr-CoV) of the genus Betacoronavirus contains the 56

most pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 57

(SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2). 58

SARS-CoV caused the SARS pandemic in years of 2002 – 2003, resulting in more than 59

8,000 clinical cases with a mortality of 10% (3, 4). After the SARS pandemic, plenty of 60

research and measurements have been done to prevent the emergence and re-emergence 61

of coronavirus epidemics, including transmission tracing, pathogenesis studies and 62

therapy development (5). Nevertheless, 17 years later, a novel human coronavirus, 63

SARS-CoV-2, brought a much more severe and widespread pandemic of Coronavirus 64

Disease 2019 (COVID-19) to the world and this pandemic is lasting until now (6, 7). Up 65

to May 25, 2025, about 5 month after the first reported case of COVID-19, SARS-CoV-66

2 has caused 5,432,302 confirmed infections and 342,318 deaths worldwide, far 67

surpassing the SARS pandemic. So far, most countries around the world are suffering 68

from the public health crisis, medical system overload and economic loss due to the 69

pandemic. Among common pathogenic viruses, SARS-CoV-2 emerges a high 70

transmissibility whose R0 value is currently estimated as 2.3 but could be as high as 5.7 71


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when more infection cases are identified (8). The high transmissibility of SARS-CoV-2 72

is probably a major reason for the rapid development of COVID-19 epidemic. In order 73

to relieve the current pandemic and prevent HCoV epidemics in the future, more studies 74

on the transmission of pathogenic HCoV is urgently required. 75

Interspecies transmission from wide animals to humans is a major cause leading to 76

the epidemics of highly pathogenic coronaviruses, such as SARS-CoV and SARS-CoV-77

2. Previous virus tracing studies have shown that Chinese horseshoe bats are natural 78

reservoirs of SARS-CoV (9, 10), and a recent phylogenetic analysis has revealed that 79

SARS-CoV-2 might also be originated from bat-SARSr-CoV (11). Some small 80

mammals, such as civets (Paguma larvata) and raccoon dogs (Nyctereutes 81

procyonoides), can serve as the intermediate hosts of SARS-CoV and might be he direct 82

sources of the SARS epidemic in early 2003 due to the successful isolation of SARS-83

CoV from these animals (12). Similarly, SARS-CoV-2-like CoVs were detected in 84

Malayan pangolins, indicating pangolins might serve as an intermediate host for SARS-85

CoV-2 (13, 14). The intermediate host animals usually play critical roles in the 86

interspecies transmission from natural virus reservoirs to humans and community 87

transmission of viruses. The host range of a virus is an essential factor determining its 88

intermediate hosts. Thus, research on the host range of viruses is of great importance for 89

virus tracing and epidemic control. 90

A main factor determining the host range of viruses is the recognition and 91

binding between viral particles and their receptors on the host cells. Angiotensin 92

converting enzyme 2 (ACE2) is utilized by SARS-CoV and SARS-CoV-2 as their 93

cellular receptor (1, 15). Discovered in the year of 2000, ACE2 was initially identified 94

as an exopeptidase expressed in vascular endothelial cells in the heart and the kidney 95

that catalyses the conversion of angiotensins (16, 17). ACE2 is highly conserved and 96


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ubiquitously expressed in most vertebrates, but not all ACE2s can serve as the receptor 97

for SARS-CoV and SARS-CoV-2, which is a key determinant of the host range of these 98

two viruses. For instance, SARS-CoV can use mouse ACE2 as its receptor but SARS-99

CoV-2 cannot, indicating that mouse is a potential host for SARS-CoV but not for 100

SARS-CoV-2 (1). Our previous study predicted the ACE2 utilization of SARS-CoV-2 101

and showed that SARS-CoV-2 could potentially utilize most mammalian ACE2s except 102

murine ACE2s and even some avian ACE2s. Meanwhile, we also predicted 9 amino 103

acid (aa) residues in ACE2 critical for SARS-CoV-2 utilization (18). However, this 104

study was mainly based on the aa analysis and lacked experimental evidence, which 105

could be hardly referred to clarify the host range of SARS-CoV-2. 106

In this study, we ectopically expressed ACE2 of 20 different animals in HeLa cells, a 107

cell line lacking of ACE2 expression naturally, and then infected the cells with HIV-108

based pseudoviral particles carrying coronavirus spike proteins to test their utilization of 109

these ACE2s. The result showed that both SARS-CoV and SARS-CoV-2 could use 110

most mammalian ACE2s as their receptors but not fish or reptilian ACE2s. Interestingly, 111

similar to mouse ACE2, SARS-CoV but not SARS-CoV-2 was capable of using 112

chicken ACE2, indicating a narrower host range of SARS-CoV-2, especially in murine 113

and birds. We also tested pangolin CoV pseudovirus with spike highly similar to SARS-114

CoV-2. To our surprise, pangolin CoV showed an ACE2 utilization profile similar to 115

SARS-CoV rather than SARS-CoV-2. By alignment of the aa sequence of the 20 ACE2 116

orthologs, we further confirmed several aa residues (among the 9 residues predicted 117

previously) critical for SARS-CoV-2 utilization, including T20, K31, Q42 and Y83, and 118

excluded some critical aa residues, such as Y41 and K68. Especially, T20 of ACE2 119

probably played critical roles in spike-ACE2 binding by interacting with S477 and T478 120

within the receptor-binding motif (RBM) of SARS-CoV-2 spike protein, but it was not 121


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necessary for the binding of ACE2s with SARS-CoV and pangolin CoV spikes. These 122

aa residues might partially determine the unique receptor utilization and host range of 123

SARS-CoV-2. In summary, our study provides a more clear view of ACE2 utilization 124

by SARS-CoV-2, which may contribute to a better understanding about the virus-125

receptor interaction and the host range of SARS-CoV-2. 126

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Results 128

Validation of pseudovirus preparation and ACE2 expression 129

In this study, we investigated the ACE2 utilization of three SARSr-CoVs, including 130

SARS-CoV-BJ01, SARS-CoV-2 and Pangolin CoV. Pangolin CoV was tested since 131

pangolins were suspected to be a potential intermediate host of SARS-CoV-2 (14). 132

Notably, the RBM of Pangolin CoV spike is almost identical with SARS-CoV-2 spike 133

except for the 498th aa residue, but they are different from SARS-CoV spike on multiple 134

aa sites (Figure 1A). 135

In order to validate the successful preparation of the three pseudovirus, the HEK293T 136

cells for virus package were lysed after virus harvest the cells were lysed and subjected 137

to Western blot detection of HA-tagged spike proteins. As shown in Figure 1B, samples 138

from all three package cells showed typical bands (about 180 kDa) of SARSr-CoV 139

spike proteins, indicating the success of pseudovirus preparation. 140

Then, we continued to validate the ectopic expression of 20 ACE2s from different 141

animals in HeLa cells, an ACE2-negative human cell line. We transfected the HeLa 142

cells with plasmids harboring the coding gene of ACE2s from different animals. At 48 h 143

post transfection, he cells were lysed and subjected to Western blot detection of 6*His-144

tagged ACE2 protein. As shown in Figure 1C, bands of all ACE2s (about 150 kDa) 145

could be observed, indicating the successful expression of all ACE2s in HeLa cells. 146


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Difference in the ACE2 utilization by SARS-CoV and SARS-CoV-2 148

We transfected the HeLa cells with the 20 plasmids expressing different ACE2s 149

individually or empty vector as a control. At 48 h post transfection, the cells were 150

infected with SARS-CoV-BJ01, SARS-CoV-2 or Pangolin CoV pseudovirus. After 48 h 151

of infection, the cells were lysed and subjected to luciferase assay to evaluate the cell-152

entry efficiency of the pseudoviruses mediated by different ACE2s. As shown in Figure 153

2, little luminescence signals could be observed in samples from HeLa cells transfected 154

with empty vector and infected by any of the three pseudoviruses, indicating that native 155

HeLa without ACE2 could not mediate the pseudovirus entry. Luminescence signals 156

from cells expressing crucian, crocodile or viper snake ACE2 were also low, indicating 157

that fish and reptilia ACE2s could barely mediate the pseudovirus entry. Cells 158

expressing chicken or mouse ACE2 showed a strong luminescence signal when infected 159

by the SARS-CoV-BJ01 pseudovirus but not by the SARS-CoV-2 pseudovirus, 160

indicating that SARS-CoV-BJ01 could use chicken or mouse ACE2 for cell entry but 161

SARS-CoV-2 could not. Pangolin CoV was capable of utilizing both chicken and 162

mouse ACE2s, but its utilizing efficiency of chicken ACE2 was much lower than 163

SARS-CoV-BJ01. On the contrary, SARS-CoV-2 pseudovirus ignited a stronger 164

luminescence than SARS-CoV-BJ01 or Pangolin CoV pseudovirus in cells expressing 165

bat ACE2, indicating the highest utilizing capability of bat ACE2 by SARS-CoV-2. For 166

the other ACE2s, infection of all the three pseudoviruses led to strong luminescence 167

signals, implying that all the three SARSr-CoV were capable of utilizing a broad range 168

of ACE2s. 169

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Phylogenetic analysis of ACE2s and key amino acids for SARS-CoV-2 utilization 171


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To evaluate the phylogenetic relationship of the 20 ACE2s assessed above, we built a 172

phylogenetic tree based on the aa sequences of all the ACE2s (Figure 3A). In the tree, 173

we observed the obvious branches of mammalian, bird, reptilia and fish ACE2s. 174

However, with the mammalian ACE2s, there were no significant branches 175

corresponding to the ACE2 utilization by SARS-CoV-BJ01 or SARS-CoV-2 176

pseudoviruses, indicating that the whole sequence analysis could hardly reveal the key 177

factors underlying the receptor utilization by the viruses. 178

In our previous study, we predicted 9 key aa sites on human ACE2 potentially 179

critical for the receptor utilization, including T20, K31, Y41, K68, Y83, K353, D355, 180

R357 and M383, based on the SARS-CoV-2 utilization of human, bat, civet, swine and 181

mouse ACE2s (18). Here we tested 15 more species, and then we checked the 9 sites of 182

the 20 ACE2 orthologs to validate the role of the nine sites in SARS-CoV-2 utilization. 183

As shown in Figure 3A, Y/H41, D355, R357 and R383 were conserved in all ACE2s, 184

indicating that these sites were not determining the receptor utilization by SARS-CoV-2. 185

Similarly, K68 was conserved in both mouse and chicken ACE2s that could not be used 186

by SARS-CoV-2 and was probably not important for SARS-CoV-2 utilization either. 187

Q42 was conserved in mouse ACE2 but was substituted by E42 in chicken ACE2 188

whose role in SARS-CoV-2 might be complicated. On the contrary, T20, K31 and Y83 189

in usable ACE2s were distinct from the corresponding aa in unusable ACE2s, indicating 190

their cirtical role in determining SARS-CoV-2 utilization. 191

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Discussion 193

In history, most pandemic viruses originated from wild reservoir animals and 194

transmitted directly or indirectly to humans, such as Ebola virus from bats (19) and 195

human immunodeficiency viruses from chimpanzees (20). The reason for this 196


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phenomena is probably that native human viruses have undergone long-term 197

coevolution with humans and are unlikely to cause severe diseases to their hosts after 198

multiple natural selections. Therefore, interspecies transmission is usually a prerequisite 199

for a virus to cause a pandemic. A broad host range is supposed to lead to effective 200

interspecies transmission of virus and such virus is more likely to cause a pandemic. 201

The COVID-19 pandemic caused by SARS-CoV-2 is the most severe worldwide 202

pandemic in the recent years surpassing the SARS pandemic in 2003, so it is likely to 203

speculate that SARS-CoV-2 has a broad host range. Surprisingly, our cell-entry result 204

showed that SARS-CoV-2 had a smaller range of ACE2 utilization than SARS-CoV. 205

SARS-CoV-2 could not utilize mouse or chicken ACE2 which could be used by SARS-206

CoV, indicating a narrower host range of SARS-CoV-2, especially in murine and birds. 207

The reason is probably that the host range of SARS-CoV-2 is broad enough to support 208

its transmission from bats to humans, and lack of infection to some kinds of animals 209

does not affect such transmission due to redundant routes. Thus, it is not suggested to 210

over-interpret the determination of the host range on the possibility of a virus to cause 211

pandemics, especially for the viruses with broad host ranges. 212

Notably, SARS-CoV-2 has a better utilization of bat ACE2 than SARS-CoV. Though 213

SARS-CoV originate from bat-SARSr-CoV, the utilization of bat ACE2 by SARS-CoV 214

is quite limited which is supported by the previous reports (10, 21). According our 215

current results, SARS-CoV-2 utilizes Chinese horseshoe bat ACE2 much better than 216

SARS-CoV, indicating a higher homology between SARS-CoV-2 and its ancestor. This 217

speculation is supported by the phylogenetic analysis of viral genomes in the previous 218

study (1). 219

Our cell-entry result showed that SARS-CoV and SARS-CoV-2 could use a wide 220

variety of mammalian ACE2s, which is further supported by the reports about the 221


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susceptibility of various mammals to SARS-CoV-2 infection (22). However, the 222

utilization of fish and reptilian ACE2s was quite poor for both SARS-CoV and SARS-223

CoV-2. This can be explained by the remote phylogenetic relationship between 224

fish/reptilian ACE2s and mammalian ACE2s. By comparison, bird ACE2s have closer 225

phylogenetic relationship with mammalian ACE2s, and thus, bird ACE2s could be used 226

by some but not all SARSr-CoVs, such as SARS-CoV. This indicates that SARSr-CoVs 227

are more likely to be transmitted by mammals and birds but not fish and reptiles, and 228

more attention should be paid to domestic mammals and birds to prevent CoV pandemic. 229

Our previous study predicted 9 critical aa residues on ACE2 orthologs based on the 230

aa sequence comparison of Chinese horseshoe bat, civet, swine and mouse ACE2s, 231

since these were the only ACE2s tested by SARS-CoV-2 utilization at that time (18). In 232

this study, we have tested 16 more ACE2s which could help to confirm or exclude 233

critical aa residues found previously. K31 and Y83 in human ACE2 were confirmed by 234

this study as critical aa residues for SARS-CoV-2 utilization. Substitutions of K31D and 235

Y83F were reported to abolish or strongly inhibit SARS-CoV binding (23, 24). Here we 236

have found more K31 substitutions abolishing SARS-CoV-2 utilization that chicken 237

ACE2 with K31E, crocodile and viper snake ACE2s with K31Q and mouse ACE2 with 238

K31N could not be utilized by SARS-CoV-2, matching our previous prediction. 239

Considering both chicken and mouse ACE2s could be utilized by SARS-CoV, K31 may 240

be a key aa residue leading to the different receptor utilization of SARS-CoV and 241

SARS-CoV-2. Also, chicken, mouse and viper snake ACE2s with Y83F could not be 242

utilized by SARS-CoV-2, consistent with our previous prediction and other former 243

reports. On the contrary, though Y41A was reported to abolish SARS-CoV binding (23), 244

horse ACE2 with Y41H could be used by SARS-CoV-2 well, indicating that Y41H 245

barely affect SARS-CoV-2 utilization. However, Chinese horseshoe bat ACE2 with 246


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Y41H showed a relatively lower utilization by SARS-CoV-2 and even lower utilization 247

by SARS-CoV, indicating Y41H might decrease receptor utilization synergistically with 248

only with other unknown substitutions, especially for SARS-CoV. Substitution of K68 249

was reported to slightly inhibit SARS-CoV binding (23). Nevertheless, horse ACE2 250

with K68R could be used by SARS-CoV-2 well, indicating that K68R might not affect 251

SARS-CoV-2 utilization either. Crucian and viper snake ACE2 with K68I could not be 252

used by SARS-CoV-2, but these ACE2s contains so many other substitutions that we 253

may not attribute the abolishment of SARS-CoV-2 utilization to K68I. 254

Remarkably, our study revealed T20 of ACE2 as a potential key aa residue for 255

SARS-CoV-2 utilization. T20 has not been reported to affect SARS-CoV utilization but 256

mouse ACE2 with T20L and chicken ACE2 with T20V could not be used by SARS-257

CoV-2. T20 is located at the N terminus of most mature mammalian ACE2s (excluding 258

the signal peptide). According to the structure of SARS-CoV-2 receptor-binding domain 259

complexed with human ACE2, the N-terminal T20 of ACE2 is close to S477 and T478 260

of SARS-CoV-2 RBM (Figure 3B) (25). Both threonine and serine contain hydroxyl 261

radicals that allow them to form hydrogen bonds with each other. Thus, T20 of human 262

ACE2 is likely to bridge with S477 and T478 of SARS-CoV-2 spike via hydrogen 263

bonds, which stabilizes the ACE2-spike binding. However, L20 on mouse ACE2 and 264

V20 on chicken ACE2 are both aliphatic aa that cannot form hydrogen bonds to support 265

the ACE2-spike binding, which may impair the utilization of these two ACE2s by 266

SARS-CoV-2. In SARS-CoV spike, the two aa residues are substituted by G463 and 267

K464. K464 can form hydrogen bonds with threonine and G463 is a non-polar aa that 268

can interact with valine or leucine via hydrophobic bond together with the adjacent 269

A461. Therefore, SARS-CoV spike can interact with various N-terminal aa of ACE2s, 270

and this may be the reason why SARS-CoV can utilize mouse and chicken ACE2s 271


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while SARS-CoV-2 cannot. Pangolin CoV spike shares high similarity with SARS-272

CoV-2 which keeps S477 and T478. However, Pangolin CoV spike harbors alkaline 273

H498 that can interact with acidic Q42 and E42 of mouse and chicken ACE2, 274

respectively, via ionic affinity, which may complementally support the ACE2-spike 275

binding and allow the utilization of mouse and chicken ACE2 by Pangolin CoV. On the 276

contrary, SARS-CoV-2 spike substitutes H498 with acidic Q498, leading to ionic 277

repulsion with Q42 and E42, blocking its binding with mouse and chicken ACE2s. 278

These proposed interactions provide a possible explanation for the different utilization 279

of ACE2s by SARS-CoV-2 and pangolin CoV. Nevertheless, all the speculation about 280

the aa residues on ACE2 key for spike-ACE2 binding is based on the sequence analysis, 281

and mutation validation is necessary to further verify the roles of those sites. 282

In summary, our current study has validated the ACE2 utilization by SARS-CoV-2 283

predicted in our previous analysis by pseudovirus cell-entry assay. The results showed 284

less ACE2 utilization by SARS-CoV-2 compared to SARS-CoV and pangolin CoV, 285

especially for murine and bird ACE2s, indicating narrower host range of SARS-CoV-2. 286

Meanwhile, with these data, we screend 5 key aa residues of ACE2 for SARS-CoV-2 287

utilization. Especially, the N-terminal T20 and Q42 might be critical in determining the 288

difference of ACE2 utilization by the three SARSr-CoV. Our findings deepen the 289

understanding about the receptor utilization and the host range of SARS-CoV-2, 290

providing useful information for tracing virus transmission routes and preventing 291

pandemics caused by CoVs in the future. 292

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Methods and materials 294

Cell lines and plasmids 295


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HEK293T and HeLa cells were obtained from the American Tissue Culture Collection 296

and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, USA) 297

supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO2 incubator at 298

37°C. 299

Full-length SARS-CoV-2 spike (GenBank accession number MN908947.3), 300

SARS-CoV BJ01 spike (GenBank accession number AY278488.2) and Pangolin CoV 301

GD1 spike (GISAID accession number: EPI_ISL_410721) were all synthesized by 302

Sangon and subcloned into the pcDNA3.1 vectors with a C-terminal HA tag. The 303

cDNAs encoding different ACE2 proteins (Table S1) were synthesized by Sangon and 304

subcloned into pcDNA3.1 vectors with a C-terminal His tag. All the plasmids were 305

verified by Sanger sequencing. 306

307

Western blot analysis 308

Lysates of cells or filtered supernatants containing pseudoviruses were separated by 309

SDS–PAGE, followed by transfer to a nitrocellulose membrane (Millipore，USA). For 310

detection of S protein, the membrane was incubated with anti-HA tag mouse 311

monoclonal antibody (bimake, USA, 1:2000), and the bound antibodies were detected 312

by Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG (Abbkine, China, 313

1:5,000). For detection of HIV-1 p24 in supernatants, monoclonal antibody against HIV 314

p24 (p24 MAb) was used as the primary antibody at a dilution of 1:8,000, followed by 315

incubation with HRP-conjugated goat anti-mouse IgG at the same dilution. To detect 316

the expression of 20 ACE2s in HeLa cells, Mouse anti-His tag monoclonal antibody 317

(Bioworld, USA, 1:5000) was used as the first antibody, followed by incubation with 318

HRP-conjugated goat anti-mouse IgG at the same dilution. 319

320


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Pseudovirus preparation and cell-entry assay 321

The pseudovirus cell-entry assay was performed as described previously (26). In brief, 322

HEK293T cells were co-transfected with a luciferase-expressing HIV-1 plasmid (pNL4-323

3.Luc.R-E-) and a plasmid encoding HA-tagged SARS-CoV-BJ01 spike, SARS-CoV-2 324

spike or Pangolin CoV spike. The supernatant containing pseudoviruses was collected 325

48 h after transfection and the remaining cell pellet was lysed for Western blot detection 326

of HA-tagged spike proteins. In cell-entry assay, pseudoviruses were incubated with 327

recipient cells at 37 °C for 6 h, the medium was changed and cells were incubated for an 328

additional 42 h. Cells were then washed with PBS buffer and lysed. Lysates were tested 329

for luciferase activity (Promega, USA). Each infection experiment was carried out on 330

for three times. 331

332

Phylogenetic analysis 333

Multiple sequence alignment was performed for the whole aa sequences of ACEs using 334

MAFFT with a local alignment strategy FFT-NS-2. The phylogenetic tree was 335

constructed by MEGA7 using the neighbor-joining (NJ) method with 1,000 bootstrap 336

replicates and visualized using FigTree. 337

338

Acknowledgements 339

This work was jointly funded by the National Natural Science Foundation of China 340

(grant number 32041001 and 81902070) and the Provincial Natural Science Foundation 341

of Hunan Province (grant number 2019JJ20004, 2019JJ50035, and 2020SK3001). 342

343

Author contributions 344


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Conceptualization, Y.Q., Q.W., and X.G.; Lab Work, Y.Q., Q.W., J.L., and C.L.; Data 345

Analysis, Y.Q., Q.W., and Z.Z.; Funding Acquisition, Y.Q. and X.G.; Writing - 346

Original Draft, Y.Q.; Writing - Review & Editing, Y.Q. and X.G. 347

348

Declaration of interest statement 349

The authors declare no competing interests. 350

351

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422

Figure legends 423

Figure 1. Validation of pseudovirues preparation and ACE2 expression. (A) 424

Western blot Detection of spike proteins of SARS-CoV-BJ01, SARS-CoV-2 and 425

Pangolin CoV in the package cells using an antibody against the HA tag conjugated to 426


https://doi.org/10.1101/2020.06.13.149930

17

the viral spike proteins. β-actin was detected as the loading control. (B) Alignment of 427

the amino acid sequences of the receptor binding motifs (RBM) of SARS-CoV, SARS 428

CoV-2 and Pangolin CoV spike proteins. (C) Detection of different ACE2 orthologs in 429

HeLa cells after transfecting the corresponding plasmids using an antibody against the 430

6XHis tag conjugated to the ACE2 proteins. β-actin was detected as the loading control. 431

432

Figure 2. Entry efficiency of SARS-CoV-BJ01, SARS-CoV-2 and Pangolin CoV 433

pseudoviruses into ACE2-expressing cells. HeLa cells expressing different ACE2 434

orthologs were infected by SARS-CoV, SARS-CoV-2 or Pangolin CoV pseudoviruses. 435

At 48 h post infection, pseudovirus entry efficiency was determined by measuring 436

luciferase activity in cell lysates. The results were presented as the mean relative 437

luminescence units and the error bars indicated the standard deviations (n = 9). 438

439

Figure 3. Phylogenetic analysis of the 20 ACE2 orthologs and the key amino acid 440

residues for SARS-CoV-2 utilization. (A) The phylogenetic tree was constructed on 441

the whole aa sequences of ACE2s using NJ method by MEGA7 with 1,000 bootstrap 442

replicates (left panel) and the amino acids on the 9 critical sites predicted previously 443

were listed (right panel). (B) The structure of the complex of SARS-CoV-2 spike and 444

human ACE2 was adapted from Protein Data Bank (PDB ID: 6VW1). S477, T478 and 445

Q498 of SARS-CoV-2 spike were labelled in green, blue and cyan, respectively. T20 446

and Q42 were labelled in red and yellow, respectively. 447


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C

A B

Figure 1

180 kDa -

150 kDa -

150 kDa -

β-Actin

HA-spike

β-Actin

His-ACE2

β-Actin

His-ACE2


https://doi.org/10.1101/2020.06.13.149930

Figure 2


https://doi.org/10.1101/2020.06.13.149930

Figure 3

A

B

T478

S477

T20

Q498

Q42

SARS-CoV-2 spike

Human ACE2


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