Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus.
Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-alpha synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly E1) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30- to 300-fold lower than that of the parental virus. The transcription of IFN-alpha genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic determinant resides in the N-terminal, exposed part of the molecule and provided further evidence for the direct role of M protein in the induction of IFN-alpha by transmissible gastroenteritis virus. The acronym VIP (viral interferogenic protein) is proposed as a designation for this particular class of proteins.
Laude
H
H
Laboratoire de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Gelfi
J
J
Lavenant
L
L
Charley
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D007369:Interferon Inducers; D016898:Interferon-alpha / Q000096:biosynthesis*; D007668:Kidney; D007700:Kinetics; D008214:Lymphocytes / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral / Q000235:genetics; D013552:Swine; D013737:Testis; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D007369:Interferon Inducers; D016898:Interferon-alpha; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D014763:Viral Matrix Proteins