Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.
The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318 -510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318 -490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC 50 of less than 10 nM, whereas the IC 50 of the S1 domain was ϳ50 nM. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.A distinct coronavirus (SARS-CoV) 1 has been identified as the etiological agent of SARS, an acute pulmonary syndrome characterized by an atypical pneumonia that results in progressive respiratory failure and death in close to 10% of infected individuals (1-4). SARS-CoV does not belong to any of the three previously defined genetic and serological coronavirus groups; the SARS-CoV S protein, a surface glycoprotein that mediates coronavirus entry into receptor-bearing cells, is also distinct from those of other coronaviruses (5, 6). Reflecting this difference, SARS-CoV does not utilize any previously identified coronavirus receptors to infect cells. Rather, as we have recently demonstrated, angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for this coronavirus (7).The S proteins of some coronaviruses, for example, that of mouse hepatitis virus (MHV), can be cleaved into two subunits (S1 and S2) (8, 9). The S proteins of other coronaviruses, such as those of human coronavirus 229E (HCoV-229E) and SARSCoV, are not cleaved by the virus-producing cell (10). Nonetheless, S1 and S2 domains of these latter S proteins can be identified through their homology with the S1 and S2 subunits of cleaved coronavirus S proteins. The S1 domain of all characterized coronaviruses, including that of SARS-CoV, mediates an initial high affinity interaction with a cellular receptor (11-13).Independently folded receptor-binding domains of two coronaviruses have been described. The first 330 amino acids of the 769-residue S1 subunit of the MHV S protein is sufficient to bind carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), the cellular receptor for MHV (13)(14)(15). A very different region of the S1 domain of HCoV-229E, between residues 407 and 547, is sufficient to associate with the cellular receptor for this coronavirus, aminopeptidase N (APN, CD13) (11, 12, 16). Here we show that a 193-amino acid fragment of the SARS-CoV S protein, residues 318 -510, binds the SARSCoV receptor ACE2 and blocks S protein-mediated infection more efficiently than does the full-length S1 domain. This region in...
Legionella pneumophila, the causative agent of Legionnaires' pneumonia, replicates within alveolar macrophages by preventing phagosome-lysosome fusion. Here, a large number of mutants called dot (defective for organelle trafficking) that were unable to replicate intracellularly because of an inability of the bacteria to alter the endocytic pathway of macrophages were isolated. The dot virulence genes encoded a large putative membrane complex that functioned as a secretion system that was able to transfer plasmid DNA from one cell to another.
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