Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins

Abstract

Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.

FIG. 1.

Binding of TGEV and the mutant m10 to BBM. BBM were isolated from the small intestine of a 3-day-old piglet and either mock treated (−) or treated with VCNA (+). Following electrophoretic separation under nonreducing conditions, the proteins were transferred to nitrocellulose. The immobilized proteins were incubated with purified virus, and bound virus was detected by an enzyme-linked immunoassay. Lanes 5 and 6, Western blot with the anti-pAPN antibody. On the left the positions of molecular mass markers are indicated.

FIG. 2.

Detection of major BBM glycoproteins. BBM from a 3-day-old piglet were either mock treated (−) or treated with VCNA (+). After electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride membrane. Carbohydrate residues (lanes 1 and 2) and sialic acid residues (lanes 3 and 4) were detected by metaperiodate oxidation.

FIG. 3.

Binding of TGEV and the mutant m10 to cryosections of the small intestine from a 3-day-old piglet. The cryosections were incubated with purified virions and with monoclonal antibody 6A.C3 (for detection of bound virus). The sections were examined by immunofluorescence microscopy (B, D, F, and H). The same parts of the sections are shown in phase contrast on the left (A, C, E, and G). The binding of TGEV is shown in an overview (B; arrows) and at a higher magnification (D; arrows). The binding of TGEV after VCNA treatment of the section (F) and the binding of the mutant m10 to the mock-treated intestine (H) are shown in an overview.