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. 2004 Mar 23;101(12):4240-5.
doi: 10.1073/pnas.0306446101. Epub 2004 Mar 9.

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Graham Simmons  1 , Jacqueline D Reeves, Andrew J Rennekamp, Sean M Amberg, Andrew J Piefer, Paul Bates
Affiliations

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Graham Simmons et al. Proc Natl Acad Sci U S A. .

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

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Figures

Fig. 1.
Fig. 1.
Expression and incorporation of SARS-CoV S into retroviral particles. (A) Detection of V5 epitope in transfected 293T cells. Lanes 1 and 4, mock transfected cells; lanes 2 and 3, cells transfected with pCDNA6 S/V5-His; lane 5, pCAGGS S/V5-His expressing cells. Lanes 3 and 4 were coinfected with Vaccinia expressing T7 pol. Markers represent kDa. (B) Surface expression of S. Shown are 293T cells transfected with empty vector (filled histogram) or pCAGGS S (open histogram) were analyzed, using human convalescent sera (1/200) and an anti-human Ig/FITC conjugate. (C) Release and cleavage of S/V5-His or Ebola GP/V5-His in transfected 293T cells. Lane 1, mock; lanes 2 (cell lysate) and 3 (supernatant), pCAGGS Ebola GPΔmuc/V5-His; lanes 4 (cell lysate) and 5 (supernatant), pCAGGS S/V5-His; lane 6, cell supernatant of cells cotransfected with pCAGGS S/V5-His and HIV gag/pol. (D) Reflotation of particles analyzed for V5 (Upper) or HIV p24 (Lower).
Fig. 2.
Fig. 2.
Optimization and neutralization of HIV(S) pseudovirions. (A) Titers of HIV(S) pseudovirions encoding β-gal on Vero E6 cells calculated as focus-forming units per ml (FFU/ml). Cells were incubated for 18 h at 37°C with 250 μl of diluted virus supernatants (alone), or with polybrene (poly) or DEAE-dextran (DEAE) at 3.2 μg/ml. Alternatively, cells were spin infected (Spin) with 500 μl of diluted virus supernatants or together with DEAE-dextran at 3.2μg/ml (Spin + DEAE). Values are means of quadruplet wells ± SD. (B) Lucencoding HIV(S) was preincubated with serial dilutions of 18-day convalescent patient serum (solid line, diamonds) or normal human serum (dotted line, squares). Results are presented as a percentage of no sera (3.2 × 105 relative light units) and represent the means of triplicate wells ± SD. HIV(VSV-G) was also preincubated with patient serum (dashed line, triangles). The experiment is representative of two experiments.
Fig. 3.
Fig. 3.
Effects of lysosomotropic agents on HIV(S) transduction. (A) Bafilomycin A inhibition of Vero E6 transduction by HIV(S) encoding β-gal (solid line, diamonds), HIV(VSV-G) (dashed line, triangles), or HIV(MLV-A env) (dotted line, squares). Results are expressed as a percentage of no drug and represent the means of triplicate wells ± SD. The experiment is representative of two experiments. (B) Inhibition of Vero E6 transduction by NH4Cl (shaded bars) or chloroquine (open bars). Results are a percentage of no drug control (filled bars) and are the means of quadruplet wells ± SD. (C)NH4Cl inhibition of 293T transduction by HIV(S) encoding β-gal (solid line, diamonds), HIV(VSV-G) (dashed line, triangles), or HIV(MLV-A env) (dotted line, squares). Results are expressed as a percentage of no NH4Cl control and represent the means of quadruplet wells ± SD. This experiment is representative of three experiments. (D) Pretreatment of pseudovirions with pH 7.5 (filled bars) or 5.0 (open bars). Results are presented as a percentage of the pH 7.5 results (6.9 × 103, 1.6 × 104, and 2.2 × 104 relative light units for S, HA, and VSV-G, respectively) and are the means of triplicate wells ± SD. This experiment is representative of two experiments.
Fig. 4.
Fig. 4.
Cell–cell fusion mediated by SARS-CoV S. S- and GFP-expressing 293T effector cells incubated with CMTMR-labeled Vero E6 target cells are shown. Effectors were either used directly (A and B) or pretreated with TPCK trypsin (C and D). After 1 h, cells were pulsed with pH 7.5 (A and C) or pH 5.0 (B and D) medium. Control cells expressing GFP and empty vector alone were pulsed with pH 5.0 (E). Other conditions for GFP and empty vector alone were all negative for fusion (data not shown). This experiment is representative of three experiments.
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