EXPERIMENTAL AND MOLECULAR PATHOLOGY 53, 152-159 (lm) Bovine Coronavirus Antigen in the Host Cell Plasmalemma H.R. PAYNE, 1,2J.S,1 W.G. HENK Departments of Veterinary Microbiology and Parasitology and Veterinary Anatomy and Fine Structure, Louisiana State University, Baton Rouge, Louisiana 70803 Received October 4, 1989, and in revised form July 19, 1990 Expression of bovine coronavirus (BCV) antigen in the plasmalemma of epithelioid hu- man rectal tumor (HRT-18) and fibroblastic bovine fetal spleen (BFS) cell lines was traced by immunofluorescence and immunoelectron microscopy facilitated by colloidal gold. Cy- toplasmic fluorescence was fast observed at 12 hr postinfection (h.p.i) in infected HRT-18 cultures. This fluorescence coincided with the appearance of cell surface antigen reacting with colloidal gold-labeled antibodies to BCV antigens. At 24 h.p.i the amount of viral antigens at the surface of HRT-18 had increased, although cytoplasmic fluorescence re- mained constant. Infected BFS cells but not HRT-18 cells formed polykaryons when incu- bated in the presence of trypsin. One viral antigen in the plasma membrane of BFS cells was thus identified as the S glycoprotein with a fusion domain. In contrast to HRT-18 cells, the overall amount of BCV antigens at the surface of BFS cells remained constant after the onset of fusion. Analysis of the labeling characteristics established that the gold- marked-sites represented de novo expression of BCV antigen in the plasma membrane of infected cells. o 1990 Academic press, 1. INTRODUCTION Bovine coronavirus (BCV) infections are associated with enteric disease of viral etiology in newborn calves (Mebus et al., 1973; Doughri et al., 1976). The enve- lope of BCV virions contains two types of morphologically and functionally dis- tinct spikes containing glycoproteins (Storz et al., 1981; King et al., 1985). The major envelope-associated glycoprotein S has a molecular mass of 185 kDa which is cleaved into Sl and S2 (100-l 10 kDa) while HE has 62 kDa in the reduced and 140 kDa in the nonreduced form. S carries the structural sites responsible for virus attachment and fusion of host cells (Collins et al., 1982; St. Cyr-Coats et al., 1988; Deregt et al., 1987). Hemagglutination of rodent red blood cells and acetylesterase activity is associated with HE of BCV (King et al., 1985; Vlasak et al., 1988; Cavanagh et al., 1990). The integral membrane glycoprotein M has a molecular mass of 24-26 kDa. The nucleocapsid protein N is phosphorylated (King and Brian, 1982; Lapps et al., 1987). The initial adaptation of wild-type strains of BCV to different cultured cells is difficult but most successful in the human rectal tumor (HRT-18) cell line which was derived from an adenocarcinoma (Tompkins et al., 1974; Laporte et al., 1979; St. Cyr-Coats and Storz, 1988). Monolayers of these polarized epithelioid cells resemble enterocytes, the type of cells targeted by BCV in natural infections (Doughri and Storz, 1977). Cultures of different bovine fetal cells support noncy- tocidal multiplication of the cell-adapted strain of BCV (Mebus et al., 1978). Bovine fetal spleen (BFS) cells are highly susceptible to BCV-induced cell fusion if trypsin is added to the medium of infected cultures. Cell fusion depends on To whom correspondence should be addressed at Case Western Reserve University, Department of Neurosciences, School of Medicine, 2040 Adelbert Road, Cleveland, OH 44106. 152 0014-4800190 $3.00 Copyright 0 I!990 by Academic Press. Inc. AU rights of reproduction in my form reserved. PLASMALEMMA ANTIGEN OF BCV-INFECTED CELLS 153 trypsin cleavage of the 185-kDa precursor to the lOO- to IlO-kDa Sl and S2 (St. Cyr-Coats et al., 1988; Storz et af., 1981;.Cavanagh et al., 1990). This phenom- enon implies that virally encoded macromolecules are expressed in the plasma membrane (White et al., 1983). Viral proteins in the host plasma membrane are not necessary for BCV maturation because coronaviral particles are enveloped by budding into intracellular compartments (Sturman and Holmes, 1982). Viral com- ponents at the cell surface contribute to intracellular spread of the infection by fusion with uninfected cells, and they serve as targets for immune surveillance. Immunoelectron and immunofluorescence microscopy were used in this investi- gation to analyze the expression of virus-specific antigen in the plasmalemma of cells infected with BCV. MATERIALS AND METHODS Monolayers of HRT-18 cells (Tompkins et al., 1974) were maintained in Dul- beccos modified Eagles medium (DMEM) buffered with 44 mM NaHCO, and supplemented with 5% fetal calf serum. The D2 strain of the BFS cell line was maintained in minimal essential medium (MEM) buffered with 25 mM N- 2-hydroxyethylpiperazine- N-Zethanesulfonic acid and supplemented with 10% fetal calf serum. Working stocks of the Mebus strain L9 of BCV (Mebus et al., 1978) with lo6 to 10 p.f.u. per milliliter were prepared in HRT-18 cells. Monolay