Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein.
Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.
Homann
H E
HE
Abteilung für Virusforschung, Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Willenbrink
W
W
Buchholz
C J
CJ
Neubert
W J
WJ
D000818:Animals; D002642:Chick Embryo; D002872:Chromosome Deletion; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005814:Genes, Viral; D007700:Kinetics; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D011485:Protein Binding; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014709:Vero Cells; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases