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. 2006 Jun 20;350(1):15-25.
doi: 10.1016/j.virol.2006.01.029. Epub 2006 Feb 28.

Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor

Dong P Han  1 , Adam Penn-Nicholson, Michael W Cho
Affiliations

Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor

Dong P Han et al. Virology. .

Abstract

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus, SARS-CoV. Virus entry into cells is mediated through interactions between spike (S) glycoprotein and angiotensin-converting enzyme 2 (ACE2). Alanine scanning mutagenesis analysis was performed to identify determinants on ACE2 critical for SARS-CoV infection. Results indicated that charged amino acids between residues 22 and 57 were important, K26 and D30, in particular. Peptides representing various regions of ACE2 critical for virus infection were chemically synthesized and evaluated for antiviral activity. Two peptides (a.a. 22-44 and 22-57) exhibited a modest antiviral activity with IC50 of about 50 microM and 6 microM, respectively. One peptide comprised of two discontinuous segments of ACE2 (a.a. 22-44 and 351-357) artificially linked together by glycine, exhibited a potent antiviral activity with IC50 of about 0.1 microM. This novel peptide is a promising candidate as a therapeutic agent against this deadly emerging pathogen.

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Figures

Fig. 1
Fig. 1
Structure of ACE2. (A) Sequence alignment of human ACE2 and two homologous domains of human ACE. Charged amino acids are indicated in red or blue (negative and positive, respectively). K31 and Y41 are highlighted in yellow. (B) A crystal structure of ACE2 without the collectrin domain at the C-terminal end. Locations of amino acids shown to be important for binding S glycoprotein are indicated (Li et al., 2005b). α-Helices 1 and 2 are highlighted in yellow. Amino acid D615 at the C-terminus is shown as a reference. (C) Close-up view of α-helices 1 and 2. Charged amino acids are shown in red and blue. Polar and hydrophobic residues are shown in green and white, respectively. White dashed line represents an imaginary line connecting residues known to be important for binding S protein.
Fig. 2
Fig. 2
Characterization of mutant ACE2 proteins. (A) Infectivity analyses of the wild type and mutant ACE2 (60-min adsorption). (B) Kinetic analyses of SARS pseudovirus entry using the wild type or D30A mutant ACE2. Pseudoviruses were adsorbed to cells for various times as indicated. (C) Infectivity analyses of the wild type and mutant ACE2 (20-min adsorption). (D) Western blot analyses of ACE2 protein expression. (E) Comparison of cell surface expression of the wild type and D30A mutant ACE2 by flow cytometry. Cells transfected with pcDNA were used as a negative control.
Fig. 3
Fig. 3
Characterization of mutant ACE2 proteins. (A) Analyses of SARS pseudovirus infectivity as a function of ACE2 amount. HeLa cells were transfected with indicated amounts of plasmids encoding either wild type or D30A mutant ACE2. The total amount of DNA transfected remained constant (1 μg) using pcDNA as filler DNA. (B) Western blot analyses of ACE2 expression in cells transfected with indicated amounts of plasmids expressing either wild type or D30A mutant proteins. (C) Infectivity analyses of the wild type and mutant ACE2 (0.25 μg plasmid, 60-min adsorption). (D) Infectivity analyses of the wild type and mutant ACE2 (0.25 μg plasmid, 40-min adsorption).
Fig. 4
Fig. 4
Inhibitory effects of ACE2-derived peptides on SARS pseudovirus infection. (A) A crystal structure of an ACE2 peptide fragment (residues 22–57) as it appears in an intact protein. Five peptides derived from this fragment are shown (P1–P5). Amino acid residues are color-coded as in Fig. 1. (B) A crystal structure of ACE2 peptide fragments (residues 22–44 and 351–357). The primary sequence of peptide P6 is shown. Backbone tracing of a potential conformation of the two fragments connected by glycine is shown in the inset. (C) Inhibition of SARS pseudovirus infection as a function of concentration of six different peptides derived from ACE2. HeLa cells transfected with a plasmid expressing the wild type ACE2 were used. (D) VSV-G pseudovirus infection was not inhibited even at 100 μM.
Fig. 5
Fig. 5
Structural analyses of interactions between ACE2 and RBD. (A) A bird's eye view of a crystal structure of the RBD of S protein (lime) bound to ACE2 (gray) (Li et al., 2005a). The RBM portion of the RBD is colored pink. The surface of amino acids that actually make contacts with ACE2 is shown in purple. The first two α-helices of ACE2 are shown in yellow as a reference. ACE2 residues that make contacts with RBM are shown in green. Charged residues shown to be important for pseudovirus infection in this study are shown in either red or blue. (B) A top view of the co-crystal structure. Residues shown to be important for infection but have not shown to make contacts with the RBD are indicated. (C) A side view of the co-crystal structure. The position of D454 residue of S protein is shown in orange. (D) A potential binding site of the P6 peptide on the RBD. Peptide fragments 22–44 and 351–357 are shown in yellow and cyan, respectively. The RBM is shown in pink, while the rest of the RBD is shown in lime.
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