Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell-cell fusion

Abstract

Hybridoma cell lines producing monoclonal antibodies to the JHM strain of mouse hepatitis virus-4 (MHV-4) were established. By indirect immunofluorescence and immune precipitation, monoclonal antibodies of three viral polypeptide specificities were characterized. Monoclonal antibodies to nucleocapsid reacted in the cytoplasm of infected cells and precipitated the 60,OOOd nucleocapsid polypeptide (VP-4) of MHV-4. Other monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and were found to precipitate the 170,OOOd viral glycoprotein (GP-1). A third set of monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and precipitated the 25,OOOd viral glycoprotein (GP-5) and its precursor VP-6 (23,OOOd). Anti-GP-1 alone had direct neutralizing activity for MHV-4 virus, while in the presence of complement both anti-GP-1 and anti-GP-5 neutralized virus. Only anti-GP-1 had the ability to inhibit the spread of infection due to fusion in L241 cells. Thus, the viral glycoprotein GP-1 likely contains both the attachment and fusion activities of MHV-4.

Host Genetic Control of Mouse Hepatitis Virus Type 4 (JHM Strain) Replication. I. Restriction of Virus Amplification and Spread in Macrophages from Resistant Mice

“…Cultured cells on glass coverslips were fixed with 4~ paraformaldehyde and 0.5% glutaraldehyde in 0.1 M-phosphate buffer pH 7-3, washed in phosphate-buffered saline (PBS), and incubated with either polyvalent mouse antibody to mouse hepatitis virus (provided by the Resource Branch of the National Cancer Institute, Bethesda, Md., U.S.A.) or monoclonal antibodies to MHV-4 (Collins et al, 1982) for 30 min. The coverslips were washed three times in PBS and incubated with the F(ab)~ fragment of rabbit antibody to mouse IgG, conjugated with fluorescein isothiocyanate or peroxidase (Cappel Laboratories, Cochranville, Pa., U.S.A.) for 30 min, as reported by Knobler et al (1981).…”

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

“…The peplomeric E2 glycoprotein is often cleaved post-translationally by host cell proteases into two 85K to 100K subunits (Sturman et al, 1985;Cavanagh et al, 1986b). The latter also is known to be responsible for virus attachment and cell membrane fusion (Collins et al, 1982), and elicits the production of virus-neutralizing antibodies (Fleming et al, 1983;Laude et al, 1986;Niesters et al, 1987; which may be protective (Buchmeier et al, 1984;Wege et al, 1984;Cavanagh et al, 1986a). An additional glycoprotein of 130K to 140K, a disulphide-linked dimer of 65K subunits, is associated with the haemagglutinin (E3) of viruses belonging to the subgroup of haemagglutinating mammalian coronaviruses (King et al, 1985 ;Hogue & Brian, 1986: Sugiyama et al, I986).…”

Antigenic Structure of Transmissible Gastroenteritis Virus. II. Domains in the Peplomer Glycoprotein

“…The virions contain three functional classes of polypeptides: the nucleoprotein and two glycosylated proteins, namely a small transmembrane protein (El) and a large complex protein (called E2) which forms the characteristic surface projections. These peplomers have been shown to be responsible for attachment to cells, induction of neutralizing antibodies and cell fusion (Garwes et al, 1978/79;Hasony & Macnaughton, 1981 ;Holmes et al, 1981;Schmidt & Kenny, 1982;Collins et al, 1982;Vautherot et al, 1984). Because these properties are essential in determining the virulence and tissue tropism of coronaviruses, studies have been recently undertaken to localize the biologically active sites of the protein with the help of monoclonal antibodies (MAbs).…”