Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin

Abstract

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 organizations 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.

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.

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 molecular masses (in kDa) of prestained protein markers (Fermentas, Canada) are given on the left.

FIG. 3.

Detection of ACE2 expression by immunofluorescence confocal microscopy. Cells were incubated with goat anti-huACE2 antibody, followed by probing with FITC-conjugated donkey anti-goat IgG. The top three rows (from the top down) show HeLa cells expressing huACE2, pcACE2, and RpACE2. The bottom row shows HeLa cells as a negative control. The columns (from left to right) show staining of expressed ACE2 (green fluorescence of FITC), staining of cell nuclei (blue fluorescence of Hoechst 33258), and the merged double-stained image.

FIG. 4.

Determination of ACE2 activity. The protease activities from different ACE2-expressing cell membrane fractions were determined using the ACE2-specific substrate QFS (see Materials and Methods). The liberated fluorescence (in relative fluorescence units [RFU]) determined at 320 to 420 nm in the absence (A) and presence (B) of EDTA is shown. HeLa cells with no exogenous ACE2 gene and PBS buffer were used as negative controls. The error bars indicate standard deviations.

FIG. 5.

Analysis of S protein expression and incorporation into pseudovirus. (A) Western blot analysis. S proteins expressed in transfected 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 reporter luciferase activity. Cell lysates were prepared 48 h p.i. from HeLa-huACE2 (A), HeLa-pcACE2 (B), and HeLa-RpACE2 (C), and luciferase activity was determined as described in Materials and Methods. The error bars indicate standard deviations.

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.