Normal goat serum IgG was purchased from R & D Systems, and normal rabbit serum IgG was purchased from Bethyl Laboratories (Montgomery, TX)

Normal goat serum IgG was purchased from R & D Systems, and normal rabbit serum IgG was purchased from Bethyl Laboratories (Montgomery, TX). showed that CD209L is usually expressed in human lung in type II alveolar cells and endothelial cells, both potential Liquidambaric lactone targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of access, and HIV and hepatitis Liquidambaric lactone C computer virus can bind to CD209L on cell membranes but do not use it to mediate computer virus access. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in computer virus contamination and pathogenesis. Severe acute respiratory syndrome (SARS) is usually caused by a novel coronavirus called SARS-CoV (1C3). The computer virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of 10% that ranges from 0% in children and 50% in persons over 65 (2). Coronaviruses bind to their glycoprotein receptors by the 200-kDa spike glycoprotein, S, around the viral envelope. Identification of computer virus receptors can provide insight into mechanisms of computer virus access, tissue tropism, pathogenesis, and host range. Several types of receptors were previously recognized for coronavirus S glycoproteins. The receptors for the murine coronavirus mouse hepatitis computer virus are murine carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) and related murine glycoproteins in the carcinoembryonic antigen family in the Ig superfamily (4). The receptors for human coronavirus 229E (HCoV-229E), transmissible gastroenteritis computer virus of swine, and feline coronavirus in genetic group 1 are aminopeptidase N (APN) glycoproteins (5C8). Angiotensin-converting enzyme 2 (ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9, 10). Because the Vero line of rhesus monkey kidney cells is usually highly susceptible to contamination with SARS-CoV, Li (9) used a codon-optimized soluble SARS-CoV spike glycoprotein (amino acids 12C672) fused to the Fc domain name of human IgG1 to immunoprecipitate a putative receptor glycoprotein from Vero cell membranes and recognized the simian ACE2 protein by mass spectrometry. They showed that expression of recombinant human ACE2 greatly enhanced the susceptibility of human 293T cells to contamination by SARS-CoV. Wang (10) independently demonstrated that human ACE2 is usually a receptor for SARS-CoV by transducing HeLa cells with a retrovirus library of cDNAs from Vero E6 cells and by using flow cytometry to select transduced cells that bound to purified soluble SARS-CoV S glycoprotein (amino acids 14C502) with a 6-histidine tag. They found that the simian cDNA in these cells, which encoded triosephosphate isomerase, enhanced expression of human ACE2 by inserting into the HeLa cell genome immediately upstream of the ACE2 ORF. Murine NIH 3T3 cells expressing recombinant human ACE2, but not those expressing recombinant triosephosphate isomerase, were susceptible to contamination by HIV pseudovirus expressing SARS-CoV S protein. In this statement, we describe the discovery of an additional receptor for SARS-CoV, CD209L (also Rabbit Polyclonal to SLC9A6 called L-SIGN, DC-SIGNR, and DC-SIGN2) (11). Our strategy for identifying a SARS-CoV receptor was to transduce a human lung cDNA library carried by a retroviral vector into Chinese hamster ovary (CHO) cells and use flow cytometry to select transduced CHO cells that bound soluble, codon-optimized, c-myc-tagged SARS-CoV S590 glycoprotein (amino acids 1C590) expressed in 293T cells. The SARS-CoV S590-binding cells were then challenged with infectious SARS-CoV, and contamination was exhibited by detection of subgenomic viral RNA synthesis and immunofluorescence with antiviral antibody. The finding that CD209L is usually expressed in human type II alveolar cells and lung endothelial cells that may be targets for SARS-CoV (12, 13) suggests that ACE2 and CD209L may both participate in SARS-CoV access. Materials and Methods Virus. The Urbani strain of SARS-CoV was kindly provided by W. Bellini and T. Ksiazek (Centers for Disease Control and Prevention, Atlanta) (1) and propagated in Vero E6 cells (from American Type Culture Collection) in a Biosafety Level 3 laboratory. The inocula utilized for these experiments were the 24- to 28-h supernatant media from the second passage of computer virus in our laboratories, centrifuged at 1,000 rpm in a Beckman Coulter Allegra 64R refrigerated centrifuge with a Beckman Coulter GH3.8 rotor in Aerosolve aerosol-resistant canisters, aliquoted, flash frozen, and stored at -80C. Cells were inoculated with Liquidambaric lactone computer virus at a multiplicity of contamination (moi) of 0.01. Computer virus was adsorbed for 1 h at 37C in serum-free DMEM, and the inoculum was removed and replaced with DMEM, 10% FBS, and 2% penicillin, streptomycin, and fungisone.