S Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Mediates Entry into Hepatoma Cell Lines and Is Targeted by Neutralizing Antibodies in Infected Patients

Abstract

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARSCoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T-and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replicationcompetent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera fromSARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.

Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

“…For SARS-CoV, it appears that neutralizing antibodies against S can provide some form of immunity against SARS-CoV infection (2,5,6,12,27). In this study, we raised rabbit polyclonal antibodies against five bacterially expressed S fragments, covering the entire ectodomain (aa 48 to 1192).…”

Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies

“…SARS-CoV infection leads to generation of potent neutralizing Abs (nAbs). Antibodies that neutralize the virus in vitro were also detected in SARS-CoV-infected patients (9)(10)(11)(12)(13)(14) and in mice (15), hamsters (16), and monkeys (17) infected with the virus. By passive transfer of immune serum before intranasal challenge, these antibodies also protected naïve animals from SARS-CoV infection in a mouse model of SARS-CoV replication (15).…”

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

“…Pseudotyping with retroviral and lentiviral vectors has been used to analyze the mechanism and the specificity of entry, which faithfully models infection of different cell types by virus (6,7,16,17). S-dependent entry occurs through pHsensitive endocytosis (6,7,16) and can be inhibited by antisera that protect against pulmonary viral replication in mice (8,18). To analyze the sensitivity of alternative SARS-CoV S glycoproteins to antibody neutralization, S sequences from alternative human isolates and two palm civet strains were synthesized as codon-modified cDNAs and inserted into expression vectors as described in ref.…”