Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.
As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features.
Thompson
S D
SD
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Laver
W G
WG
Murti
K G
KG
Portner
A
A
D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D015707:HN Protein; D006385:Hemagglutination Inhibition Tests; D006386:Hemagglutination Tests; D006389:Hemagglutinins, Viral / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D008854:Microscopy, Electron; D010222:Parainfluenza Virus 1, Human; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification / Q000648:ultrastructure
D015707:HN Protein; D006389:Hemagglutinins, Viral; D014759:Viral Envelope Proteins