A Novel Cell-based Binding Assay System Reconstituting Interaction Between SARS-CoV S Protein and Its Cellular Receptor

Overview

Journal J Virol Methods

Specialty Microbiology

Date 2004 Dec 8

PMID 15582697

Citations 18

Authors

Chih-Fong Chou

Shuo Shen

Yee-Joo Tan

Burtram C Fielding

Timothy H P Tan

Jianlin Fu

Qiurong Xu

Seng Gee Lim

Wanjin Hong

Affiliations

Soon will be listed here.

Abstract

Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1M NaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.

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