Functional and antigenic domains of the dengue-2 virus nonstructural glycoprotein NS-1.
The gene coding for the nonstructural glycoprotein of dengue-2 virus was cloned, sequenced, and expressed in Escherichia coli. There was about 70% conservation at the amino acid level with dengue serotypes 1 and 4 suggesting an important common function for this protein. Conserved hydrophobic domains were found both before the amino-terminus and at the carboxy-terminus, consistent with transmembrane roles. Evidence for at least partial translocation of NS-1 through the inner membrane of E. coli was found. Also conserved were two signals for N-linked glycosylation located near the middle of NS-1. Various regions of NS-1 were tested for antigenicity with mouse and rabbit polyclonal and mouse monoclonal antibodies. The mouse polyclonal antibodies, made against a crude dengue-infected mouse brain immunogen, reacted most strongly with N-terminal regions of NS-1, whereas, the rabbit antiserum, made against purified NS-1 protein, reacted strongest with C-terminal regions. These findings suggest that immunogen presentation or species differences could be important. Although most of the monoclonals appeared to be unreactive in Western blots with expressed NS-1 proteins, two appeared to react strongly; the region from amino acid (a.a.) 273 to a.a. 346 was required for antibody binding. This region, located adjacent to the two conserved C-terminal hydrophobic domains, is highly charged and contains 5 of the 10 conserved cysteine residues of NS-1.
Putnak
J R
JR
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington D.C. 20307.
Charles
P C
PC
Padmanabhan
R
R
Irie
K
K
Hoke
C H
CH
Burke
D S
DS
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003716:Dengue Virus / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D017361:Viral Nonstructural Proteins
D000956:Antigens, Viral; D006023:Glycoproteins; D021382:Protein Sorting Signals; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins