Downloaded from www.microbiologyresearch.org by IP: 23.22.250.46 On: Sat, 13 Feb 2016 11:36:33 Journal of General Virology (1990), 71, 1313-1323. Printed in Great Britain 1313 Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S Bernard Deimas, Denis Rasschaert, Murielle Godet, Jacqueline Gelfi and Hubert Laude* Laboratoire de Virologie et d'Immunologie Mol~culaires, Institut National de la Recherche Agronomique, Centre de Recherches de Jouy-en-Josas, 78350 Jouy-en-Josas, France Four major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were con- structed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutral- ization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites. Introduction Transmissible gastroenteritis virus (TGEV) causes an acute and usually fatal enteric disease in newborn piglets. Together with the murine hepatitis virus (MHV) and infectious bronchitis virus (IBV), TGEV is one of the most well studied coronaviruses, a family of enveloped positive-stranded RNA viruses (for review, see Spaan et al., 1988). The organization of the TGEV genome has been established (Rasschaert et al., 1987) as well as the sequence of the genes encoding the structural and most of the non-structural proteins (Kapke & Brian, 1986; Laude et al., 1987; Rasschaert & Laude, 1987; Rasschaert et al., 1987; Jacobs et al., 1987). TGEV virions contain three proteins, a nucleocapsid protein and two envelope glycoproteins, M (29K) and S (220K) (Garwes & Pocock, 1975; Laude et al., 1986). The spike protein S is 1431 residues long, highly glycosylated, with a membrane- anchoring domain near its carboxy-terminus and a globular domain assumed to correspond to its amino- terminal half (Rasschaert &Laude, 1987). Competition studies using monoclonal antibodies (MAbs) has led to the prediction of at least four main antigenic sites (Delmas et al., 1986; Garwes et al., 1987; Correa et al., 1988). Major antigenic sites, designated A, B, C and D, have been delineated using the library of MAbs established in our laboratory. Most of the epitopes critical for neutral- ization were found to be clustered in the immunodomin- ant A and B sites, but the other two sites contained subsidiary neutralization epitopes. The A, B and D sites have been found to be highly conserved among TGEV strains, whereas antigenic variation was observed on site C epitopes (Laude et al., 1986; Delmas et al., 1986). In order to gain information on the antigenic structure of S at the molecular level, but also to investigate possible vital functions of distinct epitopes, we have undertaken a systematic mapping of the antibody-binding regions. The present study reports the localization of four antigenic sites on the primary structure of S by combined approaches including production of antigenically active fragments of the protein by bacterial expression or proteolytic treatment, and analysis of epitope mutants. Methods Virusesand MAbs. The high-passagePurdue- 115strain of TGEVwas propagated in the pig kidney cell line PD5 as reported (Laude et al., 1986). Infected monolayers were maintained in Dulbecco'smodified Eagle's medium (DMEM) buffered with 25 mM-PIPESpH 6-8, and 0000-9404 © 1990SGM