ABSTRACT
Comparison of the haemagglutinins (HA) of the pathogenic avian influenza viruses A/FPV/Dutch/27 (H7N7) and A/FPV/Rostock/34 (H7N1) revealed 94.7% nucleotide and 93.8% amino acid sequence homologies. Six of the seven N-glycosidic oligosaccharides of the Rostock HA are at the same positions as the six carbohydrates of the Dutch strain. The additional oligosaccharide side chain of the Rostock strain, which is of the complex type, is attached to asparagine149 in antigenic epitope B. The accessibility of this antigenic epitope has been analysed by using rabbit antisera raised against synthetic peptides comprising amino acids 143-162. The carbohydrates of the HA of the Rostock strain have been modified (i) to truncated cores by expression in insect cells using a baculovirus vector, (ii) to oligomannosidic side chains by growth in the presence of the trimming inhibitor methyldeoxynojirimycin and (iii) to a single N-acetylglucosamine residue by removal of the oligomannosidic sugar with endo-beta-N-acetylglucosaminidase H. Neither the authentic nor the modified oligosaccharides allowed antibody binding, as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Reactivity was observed, however, after complete removal of the carbohydrate from HA of the Rostock strain by digestion with peptide-N-glycosidase F. HA of the Dutch strain was reactive without prior peptide-N-glycosidase F treatment. These results demonstrate that a single N-acetyl-glucosamine at asparagine149 is sufficient to prevent recognition of the peptide epitope.
Subject(s)
Hemagglutinins, Viral/chemistry , Influenza A virus/chemistry , Influenza A virus/immunology , Oligosaccharides/chemistry , Oligosaccharides/immunology , Amino Acid Sequence , Antigens, Viral/chemistry , Epitopes/chemistry , Hemagglutinins, Viral/immunology , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
Comparative nucleotide sequence analyses of the genome of Sendai virus (strain Z) and two host range mutants, ts-f1 and F1-R, previously described revealed that the ts defect of ts-f1 can be attributed to two nucleotide exchanges in the NP gene. These exchanges lead to a single amino acid substitution. A single base pair change was found in both the P and L genes of F1-R, but not of ts-f1. Both host range mutants have the two same exchanges in the M gene. These additional mutations are discussed concerning their significance in the pantropic properties of the host range mutants.
Subject(s)
DNA-Directed RNA Polymerases , Nucleoproteins , Parainfluenza Virus 1, Human/genetics , RNA, Viral/genetics , Viral Proteins/genetics , Animals , Base Sequence , HN Protein/genetics , Molecular Sequence Data , Mutation , Nucleocapsid Proteins , Phosphoproteins/genetics , Viral Core Proteins/genetics , Viral Matrix Proteins/geneticsABSTRACT
The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.
Subject(s)
Newcastle disease virus/genetics , Viral Fusion Proteins/genetics , Amino Acid Sequence , Binding Sites , Molecular Sequence Data , Mutation , Newcastle disease virus/classification , Newcastle disease virus/metabolism , Peptide Hydrolases/metabolism , Sequence Homology, Nucleic Acid , Species Specificity , Viral Fusion Proteins/metabolismABSTRACT
A variant (F1-R) was isolated from a temperature-sensitive host range mutant (ts-f1) of Sendai virus. F1-R was no longer temperature-sensitive but it retained the host range phenotype. Unlike wild-type virus, F1-R and ts-f1 undergo multiple cycles of replication in several cell lines in the absence of trypsin. This was attributed to proteolytic activation of the fusion (F) glycoprotein of the host range mutants, in cell nonpermissive to wild-type virus. In mice infected intranasally the variant F1-R caused a generalized infection. This was shown by immunohistology and with infectious virus being recovered from several organs whereas infection with wild-type virus was restricted to the lung. These observations indicate that the pantropic property of F1-R is the result of proteolytic activation of the virus by ubiquitous proteases. Nucleotide sequence analyses revealed that ts-f1 and F1-R differed from the wild-type virus by mutations at the region of the cleavage site of F and at the glycosylation site of the F2 subunit. The findings indicated that these mutations are responsible for the increased cleavability of the F protein of ts-f1 and F1-R and therefore are important determinants for the pantropism of F1-R.
