Epitope mapping of human respiratory syncytial virus 22K transcription antitermination factor: role of N-terminal sequences in protein folding.
The reactivity of a panel of 12 monoclonal antibodies raised against the human respiratory syncytial virus 22 kDa (22K) protein was tested by Western blotting with a set of 22K deletion mutants. The results obtained identified sequences in the C-terminal half of the 22K polypeptide required for integrity of most antibody epitopes, except for epitope 112, which was lost in mutants with short N-terminal deletions. This antibody, in contrast to the others, failed to immunoprecipitate the native 22K protein, indicating that the N terminus of this protein is buried in the native molecule and exposed only under the denaturing conditions of Western blotting. In addition, N-terminal deletions that abolished reactivity with monoclonal antibody 112 also inhibited phosphorylation of the 22K protein previously identified at Ser-58 and Ser-61, suggesting that the N terminus is important in regulating the 22K protein phosphorylation status, most likely as a result of its requirement for protein folding.
García-Barreno
Blanca
B
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
Steel
John
J
MRC Virology Unit, Institute for Virology, Church Street, Glasgow G11 5JR, UK.
Payá
Monica
M
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
Martínez-Sobrido
Luis
L
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
Delgado
Teresa
T
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
Yeo
Robert P
RP
The Centre for Infectious Diseases, Wolfson Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, UK.
Melero
José A
JA
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D018604:Epitope Mapping; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D006801:Humans; D009154:Mutation; D017510:Protein Folding; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D014764:Viral Proteins