Design of recombinant protein-based SARS-CoV entry inhibitors targeting the heptad-repeat regions of the spike protein S2 domain

Entry of SARS-CoV into a target cell is initiated by binding of the S1 domain of spike protein to a receptor, followed by conformational changes of the spike protein S2 domain, resulting in the formation of a six-helix bundle by the heptad-repeat (HR1 and HR2) regions. Our previous studies have demonstrated that peptides derived from HR2 region could inhibit SARS-CoV entry. However, synthesis of these peptides is at high cost. In this study, we designed two recombinant proteins, one containing two HR1 and one HR2 peptides (denoted HR121), and the other consisting of two HR2 and one HR1 peptides (designated HR212). These two proteins could be easily purified with the low cost of production, exhibiting high stability and potent inhibitory activity on entry of the HIV/SARS pseudoviruses with IC(50) values of 4.13 and 0.95muM, respectively. These features suggest that HR121 and HR212 can serve as potent inhibitors of SARS-CoV entry.     

Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein

Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC(50) values of 0.14 and 1.19microM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.     

Following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a newly identified member of Family Coronaviridae. Coronavirus envelope spike protein S is a class I viral fusion protein which is characterized by the existence of two heptad repeat regions (HR1 and HR2) (forming a complex called fusion core). Here we report that by using in vitro bio-engineering techniques, SARS-CoV HR1 and HR2 bind to each other and form a typical 6-helix bundle. The HR2, either as a synthetic peptide or as a GST-fusion polypeptide, is a potent inhibitor of virus entry. The results do show that SARS-CoV follows the general fusion mechanism of class I viruses and this lays the ground for identification of virus fusion/entry inhibitors for this devastating emerging virus.     

Importance of SARS-CoV spike protein Trp-rich region in viral infectivity

SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.     

Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.     

Fusion core structure of the severe acute respiratory syndrome coronavirus (SARS-CoV): in search of potent SARS-CoV entry inhibitors

Severe acute respiratory coronavirus (SARS-CoV) spike (S) glycoprotein fusion core consists of a six-helix bundle with the three C-terminal heptad repeat (HR2) helices packed against a central coiled-coil of the other three N-terminal heptad repeat (HR1) helices. Each of the three peripheral HR2 helices shows prominent contacts with the hydrophobic surface of the central HR1 coiled-coil. The concerted protein-protein interactions among the HR helices are responsible for the fusion event that leads to the release of the SARS-CoV nucleocapsid into the target host-cell. In this investigation, we applied recombinant protein and synthetic peptide-based biophysical assays to characterize the biological activities of the HR helices. In a parallel experiment, we employed a HIV-luc/SARS pseudotyped virus entry inhibition assay to screen for potent inhibitory activities on HR peptides derived from the SARS-CoV S protein HR regions and a series of other small-molecule drugs. Three HR peptides and five small-molecule drugs were identified as potential inhibitors. ADS-J1, which has been used to interfere with the fusogenesis of HIV-1 onto CD4+ cells, demonstrated the highest HIV-luc/SARS pseudotyped virus-entry inhibition activity among the other small-molecule drugs. Molecular modeling analysis suggested that ADS-J1 may bind to the deep pocket of the hydrophobic groove on the surface of the central coiled-coil of SARS-CoV S HR protein and prevent the entrance of the SARS-CoV into the host cells.     

Why are HIV-1 fusion inhibitors not effective against SARS-CoV? Biophysical evaluation of molecular interactions

The envelope spike (S) glycoprotein of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) mediates the entry of the virus into target cells. Recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as HIV-1. As it happens with other viruses peptidic fusion inhibitors, SARS-CoV S protein HR2-derived peptides are potential therapeutic drugs against the virus. It is believed that HR2 peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the HR1 region. It is a matter of discussion if the HIV-1 gp41 HR2-derived peptide T20 (enfuvirtide) could be a possible SARS-CoV inhibitor given the similarities between the two viruses. We tested the possibility of interaction between both T20 (HIV-1 gp41 HR2-derived peptide) and T-1249 with S protein HR1- and HR2-derived peptides. Our biophysical data show a significant interaction between a SARS-CoV HR1-derived peptide and T20. However, the interaction is only moderate (K(B)=(1.1+/-0.3)x10(5) M(-1)). This finding shows that the reasoning behind the hypothesis that T20, already approved for clinical application in AIDS treatment, could inhibit the fusion of SARS-CoV with target cells is correct but the effect may not be strong enough for application.     

Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted

The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.     

A convenient cell fusion assay for the study of SARS-CoV entry and inhibition

SARS-CoV spike (S) protein-mediated cell fusion is important for the viral entry mechanism and identification of SARS-CoV entry inhibitors. In order to avoid the high risks involved in handling SARS-CoV and to facilitate the study of viral fusion mechanism, we established the cell lines: SR-COS7 cells that stably express both SARS-CoV S protein and red fluorescence protein, R-COS7 cells that stably express red fluorescence protein, and AG-COS7 cells that stably express both ACE2 and green fluorescence protein, respectively. When SR-COS7 cells or R-COS7 cells were cocultured with AG-COS7 cells, syncytia with yellow fluorescence were conveniently observed after 12 h in SR-COS7 cells plus AG-COS7 cells, but not in R-COS7 cells plus AG-COS7 cells. The cell-to-cell fusion efficiency was simply determined for quantitative analysis based on the number of syncytium detected by flow cytometry. Such new cell-to-cell fusion model was further assessed by the potent HR2 peptide inhibitor, which led to the obvious decrease of the cell-to-cell fusion efficiency. The successful fusion and inhibition of cell-based binding assay shows that it can be well used for the study of SARS-CoV entry and inhibition.     

Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome coronavirus

To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an alpha-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight S(L) form, while the wild type and all other mutants produced similar levels of both the S(L) form and the slower-migrating, higher-molecular-weight S(H) form. The wild-type S(L) form was processed to the S(H) form, whereas the S(L) form of the 913P mutant was inefficiently converted to the S(H) form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the S(H) form into the virions as effectively as did the wild-type S(H) form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.     

Template-based coiled-coil antigens elicit neutralizing antibodies to the SARS-coronavirus

The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.     

Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.     

Phosphatidylinositol 4-kinase IIIβ is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.     

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Lipid rafts often serve as an entry site for certain viruses. Here, we report that lipid rafts in Vero E6 cells are involved in the entry of severe acute respiratory syndrome coronavirus (SARS-CoV). Infectivity assay showed the integrity of lipid rafts was required for productive infection of pseudotyped SARS-CoV. Depletion of plasma membrane cholesterol with MβCD relocalized raft-resident marker caveolin-1 as well as SARS-CoV receptor ACE2 to a nonraft environment, but did not significantly change the surface expression of ACE2. MβCD-treatment inhibited infectivity of pseudotyped SARS-CoV by 90%. Biochemical fractionation and confocal imaging confirmed that ACE2 colocalized with raft-resident markers. Furthermore, an ectodomain of SARS-CoV S protein (S1188HA) could associate with lipid rafts after binding to its receptor, and colocalize with raft-resident marker ganglioside GM1. The binding of S1188HA was not affected by depleting plasma membrane cholesterol. Taken together, our results support that lipid rafts serve as an entry port for SARS-CoV.     

Characterization of the heptad repeat regions, HR1 and HR2, and design of a fusion core structure model of the spike protein from severe acute respiratory syndrome (SARS) coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902-952) and HR2 (1145-1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection.     

Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors

We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.     

ACE2在SARS发病中作用的研究进展

重症急性呼吸综合征（SARS）是由SARS冠状病毒（SARS-CoV）引起的一种急性传染病,在其序列被测出后几个月内人们就找到了SARS-CoV的受体血管紧张素转换酶2（ACE2）。因病毒受体与病毒入侵细胞密切相关,因而有必要深入研究ACE2与SARS-CoV之间的关系。本文总结了ACE2在各组织器官的分布及功能,分析了ACE2基因的变异与病毒进入及SARS疾病严重程度之间的关系、ACE2基因的表达水平与病毒进入及SARS疾病严重程度之间的关系。这些研究将为理解SARS-CoV与ACE2之间的相互作用及设计针对ACE2的抗SARS药物提供重要的理论依据。     

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

While severe acute respiratory syndrome coronavirus （SARS-CoV）~as initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that yirus entry may also involve endocytosis. We have found that SARS-CoV enters cells viapH- and receptor-dependent endocytosis. Treatment of cells with either SARS-COV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 （ACE2）, the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic path- way inhibitors and dominant-negative Epsl5 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.     

Identification of a New Region of SARS-CoV S Protein Critical for Viral Entry

Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) is initiated by specific interactions between the SARS-CoV spike (S) protein and its receptor ACE2. In this report, we screened a peptide library representing the SARS-CoV S protein sequence using a human immunodeficiency virus-based pseudotyping system to identify specific regions that affect viral entry. One of the 169 peptides screened, peptide 9626 (S residues 217-234), inhibited SARS-CoV S-mediated entry of the pseudotyped virions in 293T cells expressing a functional SARS-CoV receptor (human angiotensin-converting enzyme 2) in a dose-dependent manner (IC₅₀ ∼ 11 μM). Alanine scanning mutagenesis was performed to assess the roles of individual residues within this region of S, which was previously uncharacterized. The effects included significant reductions in expression (K223A), viral incorporation (L218A, I230A, and N232A), and reduced viral entry (L224A, L226A, I228A, T231A, and F233A). Taken together, these results reveal a new region of the S protein that is crucial for SARS-CoV entry.     

Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2

Replication of viruses in species other than their natural hosts is frequently limited by entry and postentry barriers. The coronavirus that causes severe acute respiratory syndrome (SARS-CoV) utilizes the receptor angiotensin-converting enzyme 2 (ACE2) to infect cells. Here we compare human, mouse, and rat ACE2 molecules for their ability to serve as receptors for SARS-CoV. We found that, compared to human ACE2, murine ACE2 less efficiently bound the S1 domain of SARS-CoV and supported less-efficient S protein-mediated infection. Rat ACE2 was even less efficient, at near background levels for both activities. Murine 3T3 cells expressing human ACE2 supported SARS-CoV replication, whereas replication was less than 10% as efficient in the same cells expressing murine ACE2. These data imply that a mouse transgenically expressing human ACE2 may be a useful animal model of SARS.