Expression of cDNA encoding the Sendai virus hemagglutinin-neuraminidase gene: characterization of wild-type and mutant gene products.
Cloned cDNA encoding the Sendai virus (SV) hemagglutinin-neuraminidase (HN) envelope glycoprotein was expressed in cultured cells in two ways: (I) infection with HN-expressing recombinant vaccinia virus, or (II) transfection with a plasmid with T7 promoter and termination sequences flanking the HN gene, with intracellular T7 RNA polymerase supplied by coinfection with recombinant vaccinia virus that expresses the enzyme. The HN expressed was indistinguishable from the authentic SV protein in antigenicity, cell surface location, and formation of oligomeric structures. In addition, HN expressed from cDNA functioned normally in both hemadsorption and neuraminidase activities. The usefulness of cDNA expression for analyzing HN structure and function was evaluated by mutating the HN cDNA and observing the consequences for HN protein activity. Since previous work indicated that the lysine residue at position 461 is important for the neuraminidase activity of HN, we used site-directed mutation to produce HN protein with this lysine residue changed to glutamic acid. The mutated HN had neuraminidase activity with significantly increased thermal stability, indicating that residue 461 may be essential to the protein's conformation.
Takahashi
T
T
Department of Virology & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318.
Ryan
K W
KW
Portner
A
A
D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D005814:Genes, Viral; D015707:HN Protein / Q000235:genetics* / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D010222:Parainfluenza Virus 1, Human / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D000954:Antigens, Surface; D015707:HN Protein; D015678:Viral Structural Proteins; D004247:DNA; D009439:Neuraminidase