S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients - PubMed (original) (raw)

S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients

Heike Hofmann et al. J Virol. 2004 Jun.

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 SARS-CoV 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 replication-competent 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 from SARS 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.

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Figures

FIG. 1.

FIG. 1.

Transient expression of the SARS-CoV S protein. (A) Western blot analysis of SARS-CoV S expression. 293T cells were transfected either with the empty eukaryotic expression vector pCAGGS (lane 1) or the SARS-CoV S expression constructs pcDNA3-S (lane 2) or pCAGGS-S (lane 3). After lysis in RIPA buffer, the extracts were separated by SDS-10% polyacrylamide gel electrophoresis and detected in Western blot analyses with a polyclonal rabbit serum directed against SARS-CoV particles. Numbers at left are molecular masses in kilodaltons. (B) Surface expression of SARS-CoV S protein. 293T cells were transfected with the empty expression plasmid pCAGGS or the pCAGGS-S construct. Thereafter, cells were prepared for FACS analysis and incubated with the polyclonal rabbit serum in order to detect the S protein. Results of one experiment representative of three are shown.

FIG. 2.

FIG. 2.

Tropism and infectivity of SARS-S-bearing lentiviral pseudotypes. (A) Cellular tropism of SARS-CoV S pseudotypes. SARS-CoV S-mediated infection of a panel of common cell lines was assessed. For infection, 3 ng of p24-normalized pseudotypes carrying either the pantropic VSV G as positive control, no GP (pcDNA3) as negative control, or the SARS-CoV S GP was added to the cells followed by a 2-h centrifugation at 800 × g in order to concentrate the infectious material on the cells. After 72 h, cells were lysed and luciferase activity was determined in the cell extracts. Results of a representative experiment performed in quadruplicate are shown. Comparable results were obtained in at least three different experiments with independent virus stocks. (B) Relative infectivity of pseudotypes bearing VSV G, MLV GP, and SARS-CoV S. Huh-7 cells were infected with culture supernatants containing 1 ng of viral antigen, and luciferase activity was measured 3 days after infection. Results of a representative experiment carried out in quadruplicate are shown; comparable results were obtained in an independent experiment. (C) Efficiency of transduction of Huh-7 cells with p24-normalized GFP reporter viruses. Huh-7 cells were spin infected for 2 h with 25 ng of pseudotypes bearing VSV G or SARS-CoV S or control pseudotypes lacking an envelope protein (pcDNA3). After 72 h, cells were fixed and analyzed by immunofluorescence. Results of a representative experiment carried out in quadruplicate are shown. The results were confirmed in two independent experiments.

FIG. 3.

FIG. 3.

Infection of hepatoma cells by replication-competent SARS-CoV. Huh-7 cells were mock infected (a) or infected with SARS-CoV (Frankfurt strain) (b and c) and stained with human anti SARS-CoV antiserum 24 h after infection. The cells were analyzed with a confocal laser scanning microscope (Zeiss LSM 510) at magnifications of ×10 (a and b) and ×63 (c).

FIG. 4.

FIG. 4.

Influence of inhibitors of acidification on SARS-CoV infection. Target cells were preincubated for 30 min with the indicated concentrations of ammonium chloride. Subsequently the cells were infected with pseudotypes carrying VSV G, MLV GP, or SARS-CoV S protein, which had been normalized for comparable luciferase activity upon infection of target cells in the absence of inhibitor. After 6 h, virus and inhibitor were removed from the cells, and fresh medium was added. Luciferase activity was determined in cell extracts after 72 h. The averages of three independent experiments performed in quadruplicate are shown. Error bars indicate standard errors of the means.

FIG. 5.

FIG. 5.

Neutralization of SARS-CoV S-mediated infection by polyclonal rabbit serum raised against purified SARS-CoV. Pseudotypes bearing VSV G, MLV GP, or SARS-CoV S protein, which had been normalized to equal luciferase activity upon infection of target cells in the absence of immune serum, were incubated for 30 min with the indicated dilutions of the polyclonal rabbit serum. Thereafter, the virus-antibody mixture was added to Huh-7 cells for 7 h followed by complete removal of the culture medium. Luciferase activity was determined in cell extracts after 72 h. Results of a representative experiment carried out in triplicate are shown. Similar results were obtained in two independent experiments.

FIG. 6.

FIG. 6.

Inhibition of SARS-CoV S-mediated infection by sera from SARS patients. (A) Detection of cell-surface-expressed SARS-CoV S protein by SARS patient serum. The SARS-CoV S protein was transiently expressed in 293T cells as described in the legend to Fig. 1. The cells were stained with serum from a convalescent SARS patient, and staining was analyzed by FACS. Results of a representative experiment are shown; similar results were obtained in an independent experiment. (B) Neutralization of S-bearing pseudotypes by sera from SARS patients. Pseudotypes bearing the VSV G or SARS-CoV S protein, which had been normalized to equal luciferase activity upon infection of target cells in the absence of serum, were preincubated for 30 min with serial dilutions of sera from convalescent SARS patients or control serum from a healthy donor. The virus-antibody mixture was incubated with Huh-7 cells for 7 h followed by complete removal of the culture medium. After 72 h, luciferase activity was determined in cell extracts. Each experiment was performed in triplicate; results of one out of two experiments are shown.

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