In brief, the column was first washed with 10 column volumes of the binding buffer (20?mM sodium phosphate, pH 7.0) at a flow rate of 5?ml/min. (SARS) emerged as a deadly global threat (Lee et al., 2003, Poutanen et al., 2003, Tsang et al., 2003). The pathogen was identified as severe acute respiratory syndrome coronavirus (SARS-CoV) (Drosten et al., 2003, Ksiazek et al., 2003, Marra et al., 2003, Rota et al., 2003), which is an enveloped, single-strand plus-sense RNA computer virus. Spike (S), nucleocapsid (N), membrane (M) and envelope (E) are its major structural proteins (Drosten et al., 2003, Marra et al., 2003, Rota et al., 2003). Ametantrone Like other coronaviruses, SARS-CoV entry is mediated by the S protein (Hofmann et al., 2004, Inoue et al., in press, Simmons et al., 2004, Yang et al., 2004). The S protein consists of 1255 amino acids that forms common petal-shaped spikes on the surface of SARS-CoV (Ksiazek et al., 2003). There is no direct evidence that this S protein of SARS-CoV is usually processed proteolytically into the S1 receptor-binding subunit and the S2 membrane fusion subunit, but the two subunits can be predicted by sequence alignment with other coronavirus S proteins (Rota et al., 2003, Spiga et al., 2003). Angiotensin Ametantrone converting enzyme 2 (ACE2) has been demonstrated to be a functional receptor for SARS-CoV in vitro and in vivo (Kuba et al., 2005, Li et al., 2003) by binding to the receptor-binding domain name (RBD, amino acids 319C510) of the S protein (Chakraborti et al., 2005, Wong et al., 2004). Additionally, there are 23 potential N-linked glycosylation sites in the SARS-CoV S protein (Rota et al., 2003), and two are in the RBD. Usually, ligand binding induces endocytosis of the receptors. Our previous study demonstrated that this binding of the S protein to endogenous ACE2 in mice resulted in down-regulation of ACE2 surface expression (Kuba et Ametantrone al., 2005), implying ACE2 internalization. Therefore, we would like to explore whether RBD, the minimal receptor-binding domain name around the S protein, could induce endocytosis of the receptor. To test this hypothesis, we used the recombinant RBD spike protein as a defined model system, which avoided possible effects of other fragments around the S protein. We constructed a new vector using a human codon-optimized RBD DNA sequence, and created a stable RBD-Fc-expressing cell line. The RBD spike protein could then be secreted into culture medium and easily purified by Protein A affinity chromatography. The flow cytometry assay and immunostaining experiments exhibited the endocytosis of the RBD spike protein by susceptible cells together with ACE2. At the same time, the removal of N-glycans from the RBD spike protein could still induce ACE2 internalization. To our knowledge, this is the first report showing that this receptor-binding domain name of SARS-CoV alone can trigger the endocytosis of susceptible cells. 2.?Materials and methods 2.1. Construction of the recombinant plasmid The amino acids 319C510 of the SARS-CoV spike protein are mapped as the minimal ACE2-binding domain name (RBD) (Chakraborti et al., 2005, Wong et al., 2004). The cDNA fragment encoding the RBD was amplified by PCR using a plasmid, PUC18-S as the template, which contains the human codon-optimized SARS-CoV (Urbani strain) spike protein (GenBank accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAP13441″,”term_id”:”30027620″,”term_text”:”AAP13441″AAP13441) coding sequence synthesized by Generay Inc., and the primers (forward: 5-GGCGCTAGCCATCACCAACCTGTGCCCC-3, made up of NheI recognition site; reverse: 5-CGCGGATCCGTCACGGTGGCGGGGGCGTTC-3, made up of BamHI recognition site). The PCR product Cxcr4 was digested with NheI and BamHI, and then cloned in-frame downstream of the leader peptide of human CD5 Ametantrone antigen (CD5L), and upstream of the Fc portion of human IgG1 (Fc) in the Peak13 expression vector (provided by B. Seed, Harvard Ametantrone Medical School, Boston, MA), which was also digested by NheI and BamHI. The resulting recombinant plasmid was named Peak13-RBD-Fc. 2.2. Cell cultures VeroE6 cells (African green monkey kidney cell line), HEK293 cells (human embryo kidney cell line) and a HEK293 cell line stably expressing RBD-Fc (RBD-Fc-293) or human ACE2-GFP (ACE2-GFP-293) were maintained in Dulbecco’s altered Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100?U/ml penicillin and 100?g/ml streptomycin at 37?C with 5% CO2. 2.3. Establishment of a.