JOURNAL OF VIROLOGY, Feb. 2008, p. 1899–1907 Vol. 82, No.

4
0022-538X/08/$08.00⫹0 doi:10.1128/JVI.01085-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Difference in Receptor Usage between Severe Acute Respiratory

Syndrome (SARS) Coronavirus and SARS-Like
Coronavirus of Bat Origin䌤
Wuze Ren,1† Xiuxia Qu,2† Wendong Li,1‡ Zhenggang Han,1 Meng Yu,3 Peng Zhou,1 Shu-Yi Zhang,4
Lin-Fa Wang,3* Hongkui Deng,2 and Zhengli Shi1*
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China1; Key Laboratory of
Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, China2;
CSIRO Livestock Industries, Australian Animal Health Laboratory and Australian Biosecurity Cooperative Research Center for
Emerging Infectious Diseases, Geelong, Australia3; and School of Life Science, East China Normal University, Shanghai, China4

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Received 20 May 2007/Accepted 15 November 2007

Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV),
which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs
(SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organiza-
tions and high sequence identities, with the main exception of the N terminus of the spike protein (S), known
to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV
S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the
ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a
series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S
backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use
any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the
bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to
enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert
region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to
human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in
structure and in function. The significance of these findings in relation to virus origin, virus recombination,
and host switching is discussed.

The outbreaks of severe acute respiratory syndrome (SARS) veillance studies among different bat populations revealed the
in 2002–2003, which resulted in over 8,000 infections and close presence in several horseshoe bat species (genus Rhinolophus)
to 800 deaths, was caused by a novel coronavirus (CoV), now of a diverse group of CoVs, which are very similar to SARS-
known as the SARS-associated CoV (SARS-CoV) (12, 25, 33, CoV in genome organization and sequence. These viruses are
36). The association of SARS-CoV with animals was first re- designated SARS-like CoVs (SL-CoVs) or SARS-CoV-like vi-
vealed by the isolation and identification of very closely related ruses, (26, 29). Such discoveries raised the possibility that bats
viruses in several Himalayan palm civets (Paguma larvata) and are the natural reservoirs of SARS-CoV (26, 29, 38) and trig-
a raccoon dog (Nyctereutes procyonoides) at a live-animal mar- gered a surge in the search for CoVs in different bat species in
ket in Guangdong, China. A very high genome sequence iden- different geographic locations (39, 43, 44a).
tity (more than 99%) exists between the SARS-CoV-like virus Phylogenetic analysis based on different protein sequences
from civets and SARS-CoV from humans, supporting the no- suggested that SL-CoVs found in bats and SARS-CoVs from
tion that SARS-CoV is of animal origin (18). However, subse- humans and civets should be placed in a separate subgroup
quent studies showed that palm civets on farms and in the field (group b) in CoV group 2 (G2b) to differentiate them from
were largely free from SARS-CoV infection (23, 40). These other group 2 CoVs in the genus Coronavirus (17, 26, 29, 43).
results suggested that palm civets played a role as an interme- G2b CoVs display major sequence differences in the N-termi-
diate host rather than as a natural reservoir. Subsequent sur- nal regions of their S proteins. The S proteins of CoVs play a
key role in virus entry into host cells, including binding to host
cell receptors and membrane fusion (4, 10, 24). Angiotensin-
* Corresponding author. Mailing address for Z. Shi: State key Lab- converting enzyme 2 (ACE2) has been identified as the func-
oratory of Virology, Wuhan Institute of Virology, Chinese Academy of tional receptor of SARS-CoV, and the molecular interaction
Sciences, Wuhan, Hubei 430071, China. Phone: (86-27)-87197240.
Fax: (86-27)-87197240. E-mail: [email protected]. Mailing address for
between ACE2 and the SARS-CoV S protein has been well
L.-F. Wang: CSIRO Livestock Industries, Australian Animal Health characterized (27, 28, 31, 42). A 193-residue fragment (amino
Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia. Phone: acids [aa] 318 to 510) in the SARS-CoV S protein was dem-
(61-3)-52275121. Fax: (61-3)-52275555. E-mail: [email protected]. onstrated to be the minimal receptor-binding domain (RBD)
† W.R. and X.Q. contributed equally to this work.
‡ Present address: School of Life Science, Heilongjiang University,
which alone was able to efficiently bind to ACE2 (1, 42a, 45).
Harbin, 150080, China. Furthermore, it was shown that minor changes in amino acid
䌤
Published ahead of print on 12 December 2007. residues of the receptor-binding motif (RBM) of SARS-CoV S

1899
1900 REN ET AL. J. VIROL.

protein could abolish the entry of SARS-CoV into cells ex- plasmid, only the newly inserted sequences between the BamHI (at the 5⬘ end)
pressing human ACE2 (huACE2) (7, 31). In the corresponding and EcoRI (at nucleotide 1825) sites were confirmed by direct sequencing. The
amino acid numbering of BJ01-S was used for all chimeric constructs in this
RBD region of the SL-CoV S proteins, there is significant study.
sequence divergence from those of the SARS-CoV S proteins, Cloning of ACE2 genes and establishment of ACE2-expressing cell lines. The
including two deletions of 5 and 12 or 13 aa. From crystal- coding region for the ACE2 homolog in the horseshoe bat R. pearsonii was
structural analysis of the S-ACE2 complex, it was predicted obtained from the intestine of a bat infected with SL-CoV by PCR using the
following primers: 5⬘-CGGATCCGCCACCATGTCAGGCTCTTTCTGGC-3⬘
that the S protein of SL-CoV is unlikely to use huACE2 as an
(upstream primer containing a BamHI site [underlined]) and 5⬘-CGTCGACCT
entry receptor (30), although this has never been experimen- AAAAGGAGGTCTGAACATCATCA-3⬘ (downstream primer with a SalI site
tally proven due to the lack of live SL-CoV isolates. Whether [underlined]). Cell lines expressing huACE2 and palm civet (pcACE2) proteins
it is possible to construct an ACE2-binding SL-CoV S protein were established in a previous study (37). The bat ACE2 coding region was
by replacing the RBD with that from SARS-CoV S proteins is cloned into a retroviral vector, pBabe-pure, and then transduced into HeLa cells
to generate stable cell lines expressing the bat ACE2 by following the same
also unknown. procedures described previously (37). The stable expression of ACE2 in the
In this study, a human immunodeficiency virus (HIV)-based transduced HeLa cells was confirmed by Western blotting, immunofluorescence,
pseudovirus system was employed to address these issues. Our and ACE2 activity assays.
results indicated that the SL-CoV S protein is unable to use Construction and purification of pseudoviruses. HIV-based pseudoviruses

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ACE2 proteins of different species for cell entry and that were prepared as described previously (37). In brief, 12 ␮g each of pHIV-Luc
(pNL4.3.Luc.R⫺E⫺-Luc) and the S-expressing plasmids (or empty vector con-
SARS-CoV S protein also failed to bind the ACE2 molecule of trol) was cotransfected into 2 ⫻ 106 293T cells in 10-cm dishes. After 8 h, the
the horseshoe bat, Rhinolophus pearsonii. However, when the medium was replaced with fresh medium. Supernatants were harvested at 48 h
RBD of SL-CoV S was replaced with that from the SARS-CoV posttransfection, clarified from cell debris by centrifugation at 3,000 ⫻ g, and
S, the hybrid S protein was able to use the huACE2 for cell filtered through a 0.45-␮m-pore-size filter (Millipore). Pseudoviruses in the cell
supernatant (4 ml) were purified by ultracentrifugation through a 20% sucrose
entry, implying that the SL-CoV S proteins are structurally and
cushion (4 ml) at 55,000 ⫻ g for 60 min using a Ty90 rotor (Beckman). The
functionally very similar to the SARS-CoV S. These results pelleted pseudoviruses were dissolved in 100 ␮l of phosphate-buffered saline
suggest that although the SL-CoVs discovered in bats so far are (PBS) and stored at ⫺80°C in aliquots until they were used. To evaluate the
unlikely to infect humans using ACE2 as a receptor, it remains incorporation of S proteins and HIV p24 into the pseudotyped viruses, 20 ␮l of
to be seen whether they are able to use other surface molecules purified viruses was subjected to sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and Western blotting as detailed below.
of certain human cell types to gain entry. It is also conceivable Analysis of S protein expression and pseudovirus packaging by Western blot-
that these viruses may become infectious to humans if they ting. Lysates of 293T and HeLa cells or pelleted pseudovirions were separated by
undergo N-terminal sequence variation, for example, through SDS-PAGE (using 8% gels), followed by transfer to a nitrocellulose membrane
recombination with other CoVs, which in turn might lead to a (Millipore). For Western blotting, incubation with different antibodies was done
at room temperature for 1 h. For detection of S expression, the membrane was
productive interaction with ACE2 or other surface proteins on
incubated with MAb F26G8 (1:1,000), and bound antibodies were detected using
human cells. the AP-conjugated goat anti-mouse IgG, also diluted at 1:1,000. To detect HIV
p24 in pseudoviruses, a MAb against HIV p24 (p24 MAb) was used as the first
antibody at a dilution of 1:1,000, followed by the same AP-conjugated antibody
MATERIALS AND METHODS described above. Expression of ACE2 in HeLa cells was detected using either the
Cell lines and antibodies. The human cell lines 293T and HeLa were grown in goat antibody against the huACE2 ectodomain (1:500) or the rabbit antibody
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum against bat RpACE2 (1:100) as a primary antibody, followed by incubation with
(Gibco). Goat polyclonal antibody against the huACE2 ectodomain was pur- the AP-conjugated protein-A/G mix (1:1,000) or AP-conjugated goat anti-rabbit
chased from R&D Systems. Monoclonal antibody (MAb) F26G8, which recog- IgG (1:1,000).
nized a linear epitope (aa 615 to 620 of the SARS-CoV S protein) conserved in Examination of pseudovirus virions by EM. Purified pseudoviruses were
all known S proteins of SARS-CoVs and SL-CoVs (M. Yu, unpublished results), checked by electron microscopy (EM) using Formvar- and carbon-coated copper
was kindly provided by Jody Berry (3). A MAb against p24 of HIV was generated grids (200 mesh), negatively stained with 2% phosphotungstic acid (pH 7.0), and
by the HIV group of the Wuhan Institute of Virology (unpublished results). examined at 75 kV in a Hitachi H-7000FA transmission electron microscope.
Rabbit polyclonal antibodies against ACE2 of the bat R. pearsonii (RpACE2) Immunofluorescence confocal microscopy. ACE2-expressing and control
was generated using a recombinant RpACE2 protein expressed in Escherichia HeLa cells were grown on coverslips in 24-well plates (Costar, Corning, NY).
coli at our laboratory at the Wuhan Institute of Virology, following standard The following day, the cells were washed with PBS and fixed with 4% formal-
procedures. Alkaline phosphatase (AP)-conjugated goat anti-mouse immuno- dehyde in PBS (pH 7.4) for 30 min at 4°C, followed by overnight incubation with
globulin G (IgG) antibodies were purchased from Santa Cruz Biotechnology, goat anti-huACE2 antibody (at 1:150 dilution in PBS) at 4°C. The next day,
AP-conjugated goat anti-rabbit IgG from Chemicon (Australia), AP-conjugated incubation with FITC-conjugated donkey anti-goat IgG (at 1:100 dilution in
protein-A/G mix from Pierce, and fluorescein isothiocyanate (FITC)-conjugated PBS) was carried out at room temperature for 30 min. For nuclear staining, cells
donkey anti-goat IgG from PTGLab (Chicago, IL). were incubated with Hoechst 33258 for 5 min at room temperature. Each incu-
Construction of expression plasmids. The construction of a codon-optimized bation step was followed by washing with PBS five times. After the final wash, the
spike (S) protein gene of SARS-CoV BJ01 (BJ01-S) in pcDNA3.1(⫹) was de- slides were mounted with 50% glycerol and observed under a Leica confocal
scribed previously (34, 46). The full-length S gene of a bat SL-CoV (Rp3) was microscope (Leica, Germany).
cloned by PCR amplification from cDNA prepared using fecal samples from an ACE2 activity assay. The cell membrane fraction was prepared as described
R. pearsonii bat positive for SL-CoV (29). After codon optimization for the first previously (11). Briefly, HeLa cells (with or without ACE2 expression) were
400 aa at the N terminus, the modified S gene was cloned into pcDNA3.1(⫹). collected by centrifugation at 10,000 ⫻ g for 5 min. The cell pellet was washed
For introduction of the RBM of SARS-CoV S into the SL-CoV S, the coding with ice-cold PBS and suspended in Tris-buffered saline (TBS) (100 mM Tris and
region from aa 424 to 494 of BJ01-S was used to replace the corresponding 500 mM NaCl, pH 6.5). The cells were subjected to three cycles of freeze/thaw,
regions of Rp3-S, resulting in a chimeric S (CS) gene designated CS424-494. Using followed by brief sonication on ice. The cell lysate was centrifuged at 15,000 ⫻
the same strategy, a series of CS genes with BJ01-S sequences were constructed g for 30 min, and the supernatant was taken as the soluble protein fraction. The
by stepwise replacement. To facilitate the construction of S chimeras, a point pellet, which contained the membrane fraction, was washed with ice-cold TBS
mutation (A to G) at nucleotide 1825 (the A residue of the ATG codon was and recentrifuged as described above. After resuspension in TBS, the total
designated nucleotide 1) was introduced to generate a unique EcoRI site in the protein contents of both the membrane and soluble fractions were determined
S open reading frame. The chimera CS424-494 was confirmed by full-length with a BCA Protein Assay Kit (Chenergy Biocolor, China) using bovine serum
sequencing to ensure that no unexpected mutation was introduced during the albumin as a standard. The activity of the ACE2 assay was determined as
PCR processes. For other chimeras constructed using CS424-494 as a donor described by Douglas et al. (11) using the ACE2-specific quenched fluorescent
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FIG. 1. Multiple amino acid sequence alignment of the N-terminal regions (aa 1 to 400) of different ACE2 proteins. The sequence alignment
was conducted using the Clustal W program (39a). Potential N glycosylation sites are indicated by the # sign above the corresponding asparagine
residues (N). Asterisks indicate the crucial amino acid residues that are involved in direct contact between the huACE2 and the SARS-CoV S
protein. The accession numbers for the ACE2 proteins are as follows: human, NM 021804; palm civet, AY881174; RpACE2, EF569964; mouse,
NM 027286; and rat, NM 001012006. Black shading, 100% identity; gray shading, 75% identity.

substrate QFS (7-methoxycoumarin-4-yl)-acetyl-Ala-Pro-Lys (2,4-dinitrophe- GenBank accession number. The sequence of the R. pearsonii ACE2 coding
nyl), purchased from Auspep (Melbourne, Australia). Assays were performed in region has been deposited in GenBank under the accession number EF569964.
black 96-well plates with 50 ␮M QFS per reaction. Liberated fluorescence (in
relative fluorescence units) after incubation at 37°C for 1 h was measured at 320
to 420 nm. To demonstrate specific inhibition, ACE2 activity was also deter- RESULTS
mined in the presence of 0.1 mM EDTA.
Pseudovirus infection and infectivity assay. HeLa cells expressing huACE2, Cloning of bat ACE2 and its functional expression in HeLa
pcACE2, or bat RpACE2 were seeded in a 96-well plate at 1 ⫻ 104 cells/well 1 cells. The ACE2 gene homolog amplified from the horseshoe
day before infection. For infection, 4 ␮l of purified pseudoviruses mixed with 96
bat R. pearsonii (RpACE2) codes for an 805-aa protein that is
␮l of medium containing 8 ␮g/ml Polybrene was added to the cells. The mixture
was removed and replaced with fresh medium 8 to 12 h postinfection (p.i.). The identical in size to the ACE2 proteins of humans and civets.
infection was monitored by measuring the luciferase activity, expressed from the Sequence alignment indicated that the bat RpACE2 molecule
reporter gene carried by the pseudovirus, using the luciferase assay system is similar to the ACE2 proteins of other mammals and has an
(Promega). Briefly, cells were lysed at 48 h p.i. by adding 20 ␮l of lysis buffer amino acid sequence identity of 81% to huACE2 and pcACE2
provided with the kit, and 10 ␮l of the resulting lysates was tested for luciferase
activity by the addition of 50 ␮l of luciferase substrate in a Turner Designs
and 77% to mouse and rat ACE2 proteins. As shown in Fig. 1,
TD-20/20 luminometer. Each infection experiment was conducted in triplicate, the major sequence variation is located in the N-terminal re-
and all experiments were repeated three times. gion. For the 18 residues of huACE2 known to make direct
1902 REN ET AL. J. VIROL.

FIG. 2. Western blot analysis of bat ACE2 (RpACE2) expressed in

HeLa cells using rabbit anti-RpACE2 antibodies. Lane 1, HeLa cell
lysate used as a negative control; lanes 2 to 4, lysates from HeLa-
huACE2, HeLa-pcACE2, and HeLa-RpACE2, respectively. The mo-
lecular masses (in kDa) of prestained protein markers (Fermentas,
Canada) are given on the left.

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contact with the SARS-CoV S protein (27), there are seven
changes in RpACE2 compared to the cognate human protein:
Q24R (Q in huACE2 and R in RpACE2 at aa 24), Y41H,
Q42E, M82D, Q325E, E329N, and G354D (Fig. 1).
A stable HeLa cell line that continuously expressed the bat
RpACE2 protein was established. Western blot analysis
showed that RpACE2 was expressed efficiently in HeLa cells
and was recognized by anti-RpACE2 antibodies (Fig. 2).
Moreover, huACE2 and pcACE2 could also be recognized by
anti-RpACE2 antibodies. The levels of expression for the
three different ACE2 molecules were very similar. On SDS-
PAGE, RpACE2 has mobility very similar to those of the other
two ACE2 proteins, with an apparent mass of approximately FIG. 3. Detection of ACE2 expression by immunofluorescence con-
90 to 100 kDa, which is within the size range of the predicted focal microscopy. Cells were incubated with goat anti-huACE2 antibody,
mass of 93 kDa. followed by probing with FITC-conjugated donkey anti-goat IgG. The top
three rows (from the top down) show HeLa cells expressing huACE2,
The localization of expressed ACE2 on the cell surface was pcACE2, and RpACE2. The bottom row shows HeLa cells as a negative
demonstrated by immunofluorescence confocal microscopy us- control. The columns (from left to right) show staining of expressed ACE2
ing a commercial antibody raised against the ectodomain of (green fluorescence of FITC), staining of cell nuclei (blue fluorescence of
huACE2 (Fig. 3). The surface location, as well as ACE2 func- Hoechst 33258), and the merged double-stained image.
tionality, was further assessed by an enzyme activity assay
using an ACE2-specific peptide substrate, QFS. As shown in
Fig. 4, membrane fractions prepared from three ACE2- was normal, the pseudovirus seemed unable to assemble the
expressing HeLa cell lines displayed substantially higher CS protein at a level detectable by Western blot analysis (Fig.
protease activities toward QFS than the control HeLa cells. 5A) and EM (Fig. 5B), whereas p24 expression and assembly
Furthermore, the protease activity was largely abolished in appeared to be normal (Fig. 5A).
the presence of 0.1 mM EDTA, a known inhibitor of ACE2
proteases (11). These data indicated that all three ACE2
molecules were expressed as a functional protease associ-
ated with cellular membranes.
Pseudovirus packaging. The expression of functional S pro-
teins and their correct incorporation into pseudoviruses were
monitored by three approaches, i.e., Western blotting of cell
lysates with an S-specific MAb, Western blotting of pelleted
virions using both S-specific and p24-specific MAbs, and direct
examination of virions by EM. The results are summarized in
Fig. 5. Both the BJ01-S and Rp3-S genes were efficiently ex-
pressed in transfected 293T cells, and the expressed S proteins
were incorporated into the respective pseudoviruses as ex-
pected (Fig. 5A). When examined by EM, the pseudoviruses
containing the Rp3-S or BJ01-S protein displayed a morphol- FIG. 4. Determination of ACE2 activity. The protease activities from
ogy characteristic of CoVs (Fig. 5B). On the other hand, con- different ACE2-expressing cell membrane fractions were determined us-
trasting results were obtained for two CS proteins. For CS14-608, ing the ACE2-specific substrate QFS (see Materials and Methods). The
liberated fluorescence (in relative fluorescence units [RFU]) determined
the expressed S protein was incorporated into the pseudovirus, at 320 to 420 nm in the absence (A) and presence (B) of EDTA is shown.
as observed for the other two nonchimeric constructs. How- HeLa cells with no exogenous ACE2 gene and PBS buffer were used as
ever, for CS424-494, although the expression of the S protein negative controls. The error bars indicate standard deviations.
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FIG. 5. Analysis of S protein expression and incorporation into
pseudovirus. (A) Western blot analysis. S proteins expressed in trans-
fected 293T cells were probed with MAb F26G8 or S-MAb (top); the
middle and bottom panels are Western blots of pelleted pseudoviruses
using MAbs F26G8 and p24, respectively. (B) EM examination of
pseudovirus morphology. The name of the S protein construct in each
pseudovirus is shown at the top left corner of the electron micrograph.

FIG. 6. Measurement of pseudovirus infectivity by determining the

Use of huACE2, pcACE2, and bat RpACE2 for cell entry by reporter luciferase activity. Cell lysates were prepared 48 h p.i. from
different pseudoviruses. When HIV/Rp3-S pseudovirus was HeLa-huACE2 (A), HeLa-pcACE2 (B), and HeLa-RpACE2 (C), and
used to infect HeLa cells expressing huACE2, pcACE2, or luciferase activity was determined as described in Materials and Meth-
ods. The error bars indicate standard deviations.
RpACE2, only a low level of luciferase activity (approximately
100 relative light units, the same as the background level gen-
erated by the vector control) was obtained (Fig. 6). In contrast,
when the BJ01-S-typed pseudovirus was used, high levels of huACE2 binding, a series of CS proteins was constructed, and
luciferase activity (more than 105 relative light units) were the exact amino acid locations are summarized in Fig. 7A. S
detected in the cell lysates of HeLa-huACE2 and HeLa- protein expression in 293T cells and its incorporation into
pcACE2 (Fig. 6A and B), but not in HeLa-RpACE2 (Fig. 6C). pseudovirus for each construct were analyzed by Western blot-
Interestingly, the pseudovirus packaged with a CS protein, ting using S-specific and p24-specific antibodies as described
HIV/CS14-608, displayed a level of luciferase activity similar to above (Fig. 7B). Although the levels of S protein expression
that of HIV/BJ01-S (Fig. 6A). As expected from the above- were similar for all constructs, significant differences in incor-
mentioned Western blot and EM analyses, HIV/CS424-494, in poration were observed. The S protein was undetectable in
which the RBM of BJ01-S was transferred into the Rp3-S pseudoviruses derived from CS371-608 and was only weakly de-
backbone, failed to produce luciferase activity above the back- tected for the chimeras CS310-608, CS45-608, and CS310-518. The
ground level in any of the three ACE2-expressing HeLa cell infectivities of all the CS constructs in HeLa-huACE2 were
lines. examined, and several observations were made (Fig. 7C). With
Mapping of the minimal BJ01-S region required to convert the exception of the chimeras CS417-608 and CS371-608, which
the non-ACE2-binding Rp3-S to a chimeric ACE2-binding S produced a background level of luciferase activity, all of the
protein. Based on the results mentioned above, it is evident chimeras exhibited significant levels of luciferase activity, sug-
that the Rp3-S protein is capable of mediating cell entry as gesting that pseudoviruses containing these CS proteins were
long as the ACE2-binding component is incorporated into the able to use huACE2 for cell entry. From these results, it was
N-terminal region of its molecule. To define the minimal re- deduced that the region from aa 310 to 518 of BJ01-S was
gion required for this conversion from non-huACE2 binding to necessary and sufficient to convert Rp3-S into a huACE2-
1904 REN ET AL. J. VIROL.

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FIG. 7. Construction and functional analysis of pseudoviruses derived from different CS protein constructs. (A) Schematic presentation of
constructs of human SARS-CoV S protein (BJ01-S), bat SL-CoV S (Rp3-S), and different CS proteins. The numbers in the subscripts indicate the
amino acid locations of the BJ01-S sequences used to replace the corresponding region of Rp3-S. The open box indicates the location of the RBM.
TPA, signal peptide from tissue plasminogen activator; TM, transmembrane domain derived from the fusion protein of Sendai virus. (B) Western
blot analysis. S proteins in transfected 293T cells (top) or purified pseudoviruses (middle) were probed with MAb F26G8 (S-MAb); at the bottom
is a blot of different pseudoviruses probed with p24 MAb as a control to determine the relative quantity of HIV pseudovirus for each construct.
(C) Measurement of pseudovirus infectivity by determining luciferase activity. Cell lysates were prepared 48 h p.i. from HeLa-huACE2 infected
with pseudoviruses containing different S proteins as indicated below the bar graph. The error bars indicate standard deviations.

binding molecule. For CS310-608, CS45-608, and CS310-518, the organization. The key difference between these two groups of
levels of luciferase activity detected were much higher than closely related viruses lies in their S protein sequences, specif-
that predicted from the level of S protein incorporated into the ically, the RBM, in which there are two deletions in the bat
virion, suggesting that the infection assay is more sensitive than SL-CoV S sequences (29). From previous published studies
Western blotting. It is also important to note that although a indicating the importance of the structural fit between the
low level of S protein was present in the virion of CS417-608, it receptor ACE2 and the S protein of SARS-CoV variants and
did not lead to a productive infection. the sensitivity of S proteins to point mutations in the RBM
region, it was speculated that SL-CoV S is unlikely to use
DISCUSSION
ACE2 as a receptor for cell entry unless the bat ACE2 ho-
The CoV spike glycoproteins are responsible for cellular- molog is significantly different from those of other mammals.
receptor recognition, cell tropism, and host specificity (7, 9, 19, To address these unanswered questions, we cloned and ex-
25a). Our previous results showed that the SL-CoVs identified pressed the bat R. pearsonii ACE2 gene and examined the
in bats are similar to SARS-CoV in genome sequence and abilities of ACE2 proteins from human, palm civet, and R.
VOL. 82, 2008 RECEPTOR USAGE BY DIFFERENT SARS CORONAVIRUSES 1905

pearsonii to support infection by HIV-based pseudoviruses ies will be important to elucidate the molecular mechanism of
containing different S protein constructs. Our results indicated pathogenesis for SARS-CoV and related viruses. The outcome
that the bat SL-CoV (Rp3) S protein is unable to use ACE2 for of such research will also be invaluable in formulating control
cell entry regardless of the origin of the ACE2 molecule. We strategies for potential future outbreaks caused by viruses that
also demonstrated that the human SARS-CoV S cannot use are similar to, but different from, the SARS-CoVs responsible
bat RpACE2 as a functional receptor. On the other hand, we for the 2002–2003 outbreaks.
demonstrated that after replacement of a small segment (aa The RBM of SARS-CoV S protein has been localized be-
310 to 518) of Rp3-S by the cognate sequence of BJ01-S, the tween aa 424 and 494 by crystal structure reconstruction and
CS protein mimics the function of BJ01-S in regard to receptor functional studies (27). When this region alone was used to
usage in the HIV pseudovirus assay system. Although we cur- replace the cognate sequence in the SL-CoV S protein, the
rently have no way of confirming that the Rp3-S protein is chimeric protein (CS427-494) was unable to be incorporated
functional in binding its cognate receptor due to the lack of a into the pseudovirus at a level detectable by Western blotting.
horseshoe bat cell line, our chimeric-construct analysis sug- Similar results were obtained for a few other CS constructs. It
gests that the Rp3-S gene was intact and would be functional if is interesting that these CS constructs are similar to others in
an appropriate receptor was identified. It is also worth noting that the chimerization mutagenesis was located exclusively in

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that, although we have no experimental data to demonstrate the ectodomain. The failure of incorporation was thus most
direct binding of different S proteins to huACE2 on the HeLa likely due to conformational changes that prevented the
cell surface, we did confirm that either anti-huACE2 or anti- proper folding and cell surface presentation of the chimeric
SARS-CoV polyclonal antibodies were able to neutralize in- proteins. It is unclear at this stage whether these constructs can
fection by different pseudoviruses (data not shown). be functionally incorporated into a different pseudovirus sys-
The significance of these findings is as follows. First, the tem, such as the vesicular stomatitis virus system (16). At the
failure of SARS-CoV S protein to use bat RpACE2 as a re- same time, we also had CS constructs that showed very low
ceptor suggests that despite the presence of a diverse group of levels of incorporation into virus but a significant level of
SL-CoVs in horseshoe bats, they are unlikely to be the natural luciferase activity or infection (e.g., CS45-608). This suggests
reservoir of the immediate progenitor virus for SARS-CoV. It that there may not be a direct correlation between the effi-
is therefore important to continue the search for the reservoir ciency of incorporation and cell entry, which argues for further
of SARS-CoV in other bat and wildlife species. It is also investigation of these constructs using different systems.
important to conduct such searches in different geographical The RpACE2 protein is composed of 805 aa, the same
locations because live-animal markets in southern China number as huACE2, and the level of sequence conservation
source their animals from all over China and from foreign among these two ACE2 proteins is significant, with 81% se-
countries (26, 29, 39) and it is possible that the natural host of quence identity and 90% similarity. Several published studies
SARS-CoV was not indigenous to the site of the first SARS have identified a number of residues in huACE2 that are
outbreak. Second, as predicted, sequence variation in the N- important for the S-ACE2 interaction (20, 27, 31). From crys-
terminal region of the SL-CoV S protein rendered it incapable tal-structural analysis of an ACE2-S complex (20, 27, 31), 18
of using ACE2 as a receptor for cell entry. However, the amino acid residues have been identified as being in direct
ACE2-binding activity of SL-CoVs was easily acquired by the contact with the SARS-CoV S protein. Among them, only
replacement of a relatively small sequence segment of the S seven are different in RpACE2 (Fig. 1). Residues Tyr-41 and
protein from the SARS-CoV S sequence, highlighting the po- Lys-353 have been shown to interact with amino acid residues
tential dangers posed by this diverse group of viruses in bats. It of SARS-CoV S proteins that are most sensitive in S-ACE2
is now well documented that bat species, including horseshoe interaction (20, 27, 31). Since Lys-353 is conserved in
bats, can be infected by different CoVs. Coinfection by differ- RpACE2, it would be interesting to see whether the Tyr-to-His
ent CoVs in an individual bat has also been observed (26, 29, change (T41H) in RpACE2 was mainly responsible for the
39). Knowing the capability of different CoVs to recombine failure to act as a SARS-CoV receptor. It should be empha-
both in the laboratory (2, 14, 15, 32) and in nature (22, 41, 44), sized that, in addition to amino acid sequence variation, there
the possibility that SL-CoVs may gain the ability to infect are also differences in the numbers and locations of potential
human cells by acquiring S sequences competent for binding to N glycosylation sites between huACE2 and RpACE2 (Fig. 1).
ACE2 or other surface proteins of human cells can be readily It remains to be seen whether different glycosylation patterns
envisaged. This could occur if the same bat cells carry re- also play a role in the utilization of ACE2 proteins of different
ceptors for both types of viruses. In this context, it could be species by SARS-CoVs.
concluded that the particular horseshoe bat species investi- Bats have been identified as natural reservoirs for many
gated in the current study is unlikely to be the putative emerging zoonotic viruses, such as Hendra virus, Nipah virus,
mixing host. However, one cannot rule out the possibility and lyssaviruses (6). Although an intermediate host is neces-
that a different horseshoe bat or a different bat species sary to amplify most bat viruses and deliver them to human
might have a functional ACE2, thus giving it the ability to populations, it has been demonstrated that the Nipah virus in
act as a mixing host. Bangladesh is capable of direct bat-to-human transmission (re-
Our results indicated that the overall structure of the SL- viewed in reference 13). Since the discovery of SL-CoVs in
CoV S protein is very similar to that of the SARS-CoV S bats, a large number of CoVs have been discovered in different
protein. It remains to be seen whether a recombinant SL-CoV bat species (26, 29, 35, 39, 43, 44a). It is now clear that bats are
containing a CS protein (e.g., CS14-608) will be capable of reservoirs of a diverse group of CoVs. Considering the docu-
infecting experimental animals and causing disease. Such stud- mented observations of coinfection of the same bat species by
1906 REN ET AL. J. VIROL.

different CoVs, the same CoVs infecting different bat species 16. Fukushi, S., T. Mizutani, M. Saijo, S. Matsuyama, N. Miyajima, F. Taguchi,
S. Itamura, I. Kurane, and S. Morikawa. 2005. Vesicular stomatitis virus
(26, 29, 39), the high density of bat habitats, and the propensity pseudotyped with severe acute respiratory syndrome coronavirus spike pro-
for genetic recombination among different CoVs, it is not un- tein. J. Gen. Virol. 86:2269–2274.
reasonable to conclude that bats are a natural mixing vessel for 17. Gorbalenya, A. E., E. J. Snijder, and W. J. Spaan. 2004. Severe acute
respiratory syndrome coronavirus phylogeny: toward consensus. J. Virol.
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before some of them cross species barriers into terrestrial 18. Guan, Y., B. J. Zheng, Y. Q. He, X. L. Liu, Z. X. Zhuang, C. L. Cheung, S. W.
mammal and human populations. The findings presented in Luo, P. H. Li, L. J. Zhang, Y. J. Guan, K. M. Butt, K. L. Wong, K. W. Chan,
W. Lim, K. F. Shortridge, K. Y. Yuen, J. S. Peiris, and L. L. Poon. 2003.
this study serve as the first example of host switching achiev- Isolation and characterization of viruses related to the SARS coronavirus
able for G2b CoVs under laboratory conditions by the ex- from animals in southern China. Science 302:276–278.
19. Haijema, B. J., H. Volders, and P. J. Rottier. 2003. Switching species tropism:
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ACKNOWLEDGMENTS
potent entry inhibitor. Virology 350:15–25.
We thank Bryan Eaton for critical reading of the manuscript. We 21. Reference deleted.
thank Rongge Yang for kindly providing the p24 MAb. 22. Jia, W., K. Karaca, C. R. Parrish, and S. A. Naqi. 1995. A novel variant of

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avian infectious bronchitis virus resulting from recombination among three
This work was jointly funded by a State Key Program for Basic different strains. Arch. Virol. 140:259–271.
Research Grant (2005CB523004) from the Chinese Ministry of Sci- 23. Kan, B., M. Wang, H. Jing, H. Xu, X. Jiang, M. Yan, W. Liang, H. Zheng, K.
ence and Technology, a special fund from the president of the Chinese Wan, Q. Liu, B. Cui, Y. Xu, E. Zhang, H. Wang, J. Ye, G. Li, M. Li, Z. Cui,
Academy of Sciences (no. 1009), the Knowledge Innovation Program X. Qi, K. Chen, L. Du, K. Gao, Y. T. Zhao, X.-Z. Zou, Y.-J. Feng, Y.-F. Gao,
Key Project of the Chinese Academy of Sciences (KSCX1-YW-R-07) R. Hai, D. Yu, Y. Guan, and J. Xu. 2005. Molecular evolution analysis and
to Z. Shi, the Sixth Framework Program “EPISARS” of the European geographic investigation of severe acute respiratory syndrome coronavirus-
Commission, a National Nature Science Foundation of China for Cre- like virus in palm civets at an animal market and on farms. J. Virol. 79:
ative Research group grant (30421004) to H. Deng, and the Australian 11892–11900.
24. Krueger, D. K., S. M. Kelly, D. N. Lewicki, R. Ruffolo, and T. M. Gallagher.
Biosecurity CRC for Emerging Infectious Diseases (project 1.026RE) 2001. Variations in disparate regions of the murine coronavirus spike protein
to L.-F. Wang. impact the initiation of membrane fusion. J. Virol. 75:2792–2802.
25. Ksiazek, T. G., D. Erdman, C. S. Goldsmith, S. R. Zaki, T. Peret, S. Emery,
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