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Functional and antigenic analysis of the F glycoprotein of human parainfluenza type 3 viruses
7 SYNTHESIS, PROCESSING AND MATURATION OF THE RESPIRATORY SYNCYTIAL VIRUS SURFACE GLYCOPROTEIN, G
GAIL W WERTZ AND DREW LICHTENSTEIN, Dept. of Microbiology, University Birmingham, Ala 35294 and Dept. of Biochemistry,
University of of Wisconsin,
Alabama Medical School, Madison, Wi 53706, USA
The surface glycoprotein, G of human respiratory syncytial (RS) virus is structurally distinct from known paramyxoviral transmembrane proteins. The amino acid sequence is rich in serine and threonine residues (30%), the potential acceptor sites for O-linked carbohydrate chains and it has been estimated that as much as 50-60X: of the mol.wt. of the protein may be contributed by a an extensively glycosylated protein. An internal, combination of N and O-linked sugars , yielding N-terminal hydrophobic region (residues 38-66) has been proposed as the signal/anchor for the G protein which would result in the N-terminus being orientated in the cytoplasm. Two approaches have been used to probe the structure of the G protein. A mutant cell line with an absolute but reversible defect in protein 0-glycosylation has been used to examine the pathway of maturation, processing and surface expression of the G protein and the role of 0 and N-linked In separate experiments, site specific mutations were introduced sugars in these processes. The effects of these into cDNA copies of the G gene within the proposed transmembrane domain. transport and surface expression of the protein from recombinant vaccinia mutations on synthesis, virus expression vectors have been measured.
8 FUNCTIONAL AND ANTIGENIC PARAINFLUENZA TYPE 3 VIRUSES. EVELINE L. Bethesda, Md.
TIERNEY
AND
ANALYSIS
KATHLEEN
OF
L.
THE
VAN
F
WYKE
GLYCOPROTEIN
COELINGH.
OF
HUMAN
NIH,
Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 non-neutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) protein of human parainfluenza type 3 virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which are neutralization epitopes. Competitive-binding assays indicated that the 14 neutralization epitopes are organized into 3 distinct antigenic sites and the 6 non-neutralization epitopes form 4 sites. Neutralization MAbs differ in the efficiency with which they inhibit fusion activity of PIV3, suggesting that neutralization by F-specific antibodies does not necessarily require inhibition of fusion activity. The degree of antigenic variation in the neutralization epitopes of the F protein of clinical isolates was examined with binding and neutralization tests. These data indicated that PIV3 frequently develops mutations that produce altered F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies. Sequence analysis of the F genes of these naturally-occurring strains and of laboratoryselected antigenic variants will be used to identify residues involved in F glycoprotein neutralization epitopes.
4
GAIL W WERTZ AND DREW LICHTENSTEIN, Dept. of Microbiology, University Birmingham, Ala 35294 and Dept. of Biochemistry,
University of of Wisconsin,
Alabama Medical School, Madison, Wi 53706, USA
The surface glycoprotein, G of human respiratory syncytial (RS) virus is structurally distinct from known paramyxoviral transmembrane proteins. The amino acid sequence is rich in serine and threonine residues (30%), the potential acceptor sites for O-linked carbohydrate chains and it has been estimated that as much as 50-60X: of the mol.wt. of the protein may be contributed by a an extensively glycosylated protein. An internal, combination of N and O-linked sugars , yielding N-terminal hydrophobic region (residues 38-66) has been proposed as the signal/anchor for the G protein which would result in the N-terminus being orientated in the cytoplasm. Two approaches have been used to probe the structure of the G protein. A mutant cell line with an absolute but reversible defect in protein 0-glycosylation has been used to examine the pathway of maturation, processing and surface expression of the G protein and the role of 0 and N-linked In separate experiments, site specific mutations were introduced sugars in these processes. The effects of these into cDNA copies of the G gene within the proposed transmembrane domain. transport and surface expression of the protein from recombinant vaccinia mutations on synthesis, virus expression vectors have been measured.
8 FUNCTIONAL AND ANTIGENIC PARAINFLUENZA TYPE 3 VIRUSES. EVELINE L. Bethesda, Md.
TIERNEY
AND
ANALYSIS
KATHLEEN
OF
L.
THE
VAN
F
WYKE
GLYCOPROTEIN
COELINGH.
OF
HUMAN
NIH,
Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 non-neutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) protein of human parainfluenza type 3 virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which are neutralization epitopes. Competitive-binding assays indicated that the 14 neutralization epitopes are organized into 3 distinct antigenic sites and the 6 non-neutralization epitopes form 4 sites. Neutralization MAbs differ in the efficiency with which they inhibit fusion activity of PIV3, suggesting that neutralization by F-specific antibodies does not necessarily require inhibition of fusion activity. The degree of antigenic variation in the neutralization epitopes of the F protein of clinical isolates was examined with binding and neutralization tests. These data indicated that PIV3 frequently develops mutations that produce altered F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies. Sequence analysis of the F genes of these naturally-occurring strains and of laboratoryselected antigenic variants will be used to identify residues involved in F glycoprotein neutralization epitopes.
4