Immunoglobulin Fc binding activity is associated with the mouse hepatitis virus E2 peplomer protein

Abstract

Antigenic variation among murine coronaviruses is associated primarily with the surface peplomer protein E2 (180,000 Da). E2 is responsible for attachment of the virus to the host cell, MHV-induced cell fusion, and eliciting neutralizing antibody. We report here the molecular mimicry between E2 and Fc gamma receptor (Fc gamma R). Molecular mimicry between E2 and Fc gamma R may allow the escape of virus-infected cells from destruction by immunological mechanisms. Rabbit IgG, monoclonal rat IgG1 and IgG2b, monoclonal mouse IgG2a and IgG2b, and the rat anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-infected cells a polypeptide with a molecular mass identical to that immunoprecipitated by anti-E2 antibodies. F(ab')2 fragments of rabbit IgG did not immunoprecipitate any proteins from MHV-infected cells. All of these antibodies did not immunoprecipitate any proteins from uninfected cells. The anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-, MHV-3-, or MHV-A59-infected L-2 cells and 17CL-1 cells, or MHV-JHM-infected cultures of neonatal BALB/c brain cells, a protein with a molecular weight identical to that of MHV-JHM E2. The anti-Fc gamma R monoclonal antibody did not immunoprecipitate any proteins from uninfected cells. Furthermore, the 2.4G2 monoclonal antibody (mab), unrelated rat and mouse monoclonal antibodies, and a goat antiserum against E2, but not normal goat serum, immunoprecipitated a 75,000- to 77,000-Da molecule from uninfected WEHI-3 cells, a Fc gamma R bearing cell line. Several lines of evidence demonstrated that the protein immunoprecipitated by the anti-Fc gamma R mab from MHV-JHM-infected cells is the E2 glycoprotein: (1) Partial proteolytic maps obtained by Staphylococcus aureus V-8 protease treatment of the 180,000-Da proteins immunoprecipitated by the anti Fc gamma R mab and the anti-E2 mab were identical. (2) Sequential immunoprecipitation experiments from MHV-JHM-infected cells revealed that the same polypeptide chain was recognized by the anti-E2 mab and by the anti-Fc gamma R mab 2.4G2, (3) Actinomycin D did not influence the induction and expression of the 180,000-Da polypeptide chain that was immunoprecipitated by the anti-Fc gamma R mab, demonstrating that this protein is of viral origin.

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

“…Transfected 293T cells and infected DBT cells were radiolabeled with 400 Ci/ml [ 35 S]-methionine and cysteine for 6-7 h post transfection or 8 h post infection until 95-100% of the monolayer was involved in syncytia, respectively. Cytoplasmic extracts of infected and control cells were prepared in 250 l of lysing buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40, 0.2 TIU/ml aprotinin) on ice as described previously (Oleszak et al, 1990) and stored at −80 • C. Twenty microliter of protein G agarose beads (Calbiochem) were incubated with secondary antibody (40 g of goat anti-mouse IgG or 120 g of anti-rat IgG, respectively) for 1 h on ice, than washed twice with PBS and incubated with primary antibodies (A2.1 and A2.3 or 2.4G2, respectively) for an additional hour. Unbound antibodies were washed away with PBS and the protein G beads-antibody complexes were resuspended in MRIP buffer (10 mM phosphate, pH 7.4, 500 mM NaCI, 0.25% NP40, 0.2 TIU/ml aprotinin, 1 mM PMSF) as described before (Oleszak et al, 1990).…”

“…Cytoplasmic extracts of infected and control cells were prepared in 250 l of lysing buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40, 0.2 TIU/ml aprotinin) on ice as described previously (Oleszak et al, 1990) and stored at −80 • C. Twenty microliter of protein G agarose beads (Calbiochem) were incubated with secondary antibody (40 g of goat anti-mouse IgG or 120 g of anti-rat IgG, respectively) for 1 h on ice, than washed twice with PBS and incubated with primary antibodies (A2.1 and A2.3 or 2.4G2, respectively) for an additional hour. Unbound antibodies were washed away with PBS and the protein G beads-antibody complexes were resuspended in MRIP buffer (10 mM phosphate, pH 7.4, 500 mM NaCI, 0.25% NP40, 0.2 TIU/ml aprotinin, 1 mM PMSF) as described before (Oleszak et al, 1990). Cell lysates in a volume of 50 l [for 2.4G2 binding assays 150 l of the lysates were concentrated into final volume of 50 l with a Microcon YM-100 centrifugal concentrator (Millipore)] were added to antibody-protein G coated beads and the mixture incubated on ice for 1 h. The immunocomplexes were collected by centrifugation and washed five times with MRIP buffer.…”

Identification and Expression of Human Cytomegalovirus Transcription Units Coding for Two Distinct Fcγ Receptor Homologs

“…Coronaviruses (52) and certain herpesviruses, such as herpes simplex virus type I (HSV-1) (3, 53), HSV-2 (10), varicella zoster virus (51), and murine cytomegalovirus (MCMV) (63), produce molecules with Fc␥R activity. The HSV-encoded vFc␥R is formed by a protein complex composed of two type I transmembrane glycoproteins, gE and gI, which are part of the virion structure (32).…”

Microheterogeneity of S-glycoprotein of mouse hepatitis virus temperature-sensitive mutants

“…Cells were propagated in Dulbecco's Modified Eagle Medium (DMEM, Gibco Laboratories, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS) and a 100 units/ml of penicillin G and 100 micrograms/ml of streptomycin. wt MHV-JHM employed in these studies has been described previously (Oleszak and Leibowitz, 1990a). Five independent RNA + ts mutants of MHV-JHM, namely ts104, tsl10, tsl15 Bond et al, 1979), ts8 and ts15 (Haspel et al, 1978), have been employed in this study and their characteristics are summarized in Table 1.…”