Journal of General Virology (1992), 73, 2591-2600. Printed in Great Britain 2591 Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus Dongwan Yoo, 1. Frank L. Graham, 3 Ludvik Prevec, 3 Michael D. Parker, l~ Maria Benk6,1 Tim Zamb ~ and Lorne A. Babiuk 1,2 Veterinary Infectious Disease Organization and 2Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO and 3Departments of Biology and Pathology, McMaster University, Hamilton, Ontario, Canada LSS 4K1 The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombi- nation between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [aH]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypep- tide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haem- agglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recom- binant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo. Introduction Coronaviruses contain a large, positive-sense ssRNA of approximately 30 kb in length that is associated with nucleocapsid (N) protein (55K) in a helical ribonucleo- protein complex (MacNaughton et al., 1978). In addition to the N protein, two membrane glycoproteins are found in virions: the spike (S) protein (180K) and the integral membrane (M) protein (23K). The S glycoprotein forms surface peplomers and functions in virus attachment (Collins et al., 1982) and cell fusion (Sturman et al., 1985; de Groots et al., 1989; Yoo et al., 1991), whereas the M protein determines the site of virus maturation (Tooze et al., 1984; Rottier & Rose, 1987). A third membrane glycoprotein, haemagglutinin-esterase (HE; 65K), is found only in certain species of coronaviruses, such as bovine coronavirus (BCV) (King & Brian, 1982; King et t Present address: VirologyAnnex, USAMRIID, Fort Detrick, Frederick, Maryland21701-5000, U.S.A. al., 1985; Deregt et al., 1987), haemagglutinating encephalomyelitis virus (Callebaut & Pensaert, 1980) and human coronavirus OC43 (Hogue & Brian, 1986). In strains of mouse hepatitis virus (MHV), the HE gene is present but is expressed in only a few isolates (Makino & Lai, 1989; Shieh et al., 1989). It appears that the HE gene in MHV-JHM is a pseudogene which is not essential for virus replication (Yokomori et al., 1991). Moreover, the HE gene does not exist at all in avian infectious bronchitis virus (Stern & Sefton, 1982; Boursnell et al., 1987) or porcine transmissible gastroenteritis virus (Garwes & Reynolds, 1981). In contrast, the HE protein of BCV is a major membrane-associated glycoprotein and is essential for virus replication (Vlasak et al., 1988b). The BCV HE has been reported to exhibit significant sequence homology with the haemagglutinin of type C human influenza virus (Nakada et al., 1984; Luytjes et al., 1988; Parker et al., 1989, 1990b), having known haemagglutination and acetylesterase functions 0001-0928 © 1992SGM