The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specified by dengue virus.
cDNA of dengue virus type 2 encoding the glycoprotein NS1 was transiently expressed in COS cells using an SV40-based vector. To test the importance of selected regions of the protein in dimer formation, constructs were prepared which encoded amino acid changes at specific locations. Six of the 12 Cys residues in the NS1 protein were individually changed to Ala. Two amino acids in each of three hydrophilic and two hydrophobic areas of the protein were also changed to Ala. The ability of these 11 mutant proteins to form dimers, the sensitivity of the dimers to dissociation by heat, and the secretion of the mutant proteins from transfected cells were investigated by immunoblotting, immunoprecipitation, and indirect immunofluorescence. The results demonstrated that the carboxy terminal end of the protein is important in dimer formation. In particular, the substitution of Ala for any one of the last three Cys residues, which are located within the carboxy terminal forty amino acids of the protein, prevented dimer formation. Only the proteins which formed dimers were detected at the cell surface and in the extracellular medium. No monomers were secreted.
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
M J
MJ
Pryor
P J
PJ
Wright
D000409:Alanine / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C079340:NS1 protein, Dengue virus type 2; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D003545:Cysteine; D000409:Alanine