The sensitivity of cell-associated dengue virus proteins to trypsin and the detection of trypsin-resistant fragments of the nonstructural glycoprotein NS1.
Extracts of Vero cells infected with dengue virus type 2 were digested by trypsin in the presence and absence of detergents. The experiments were designed to test the models proposed for flavivirus translation in which the glycoproteins prM, E, and NS1 are inserted into the endoplasmic reticulum of the cell, whereas certain other nonstructural proteins are not. Viral polypeptides were detected by the use of radiolabel, by immunoprecipitation, or by immunoblotting. The results obtained for NS3 and NS5 were as predicted by the models, with membranes providing no protection against digestion by trypsin. Similarly, the results obtained for prM and E were consistent with the models, with membranes protecting against proteolysis. Some molecules of NS1 were protected, while others were sensitive to proteolysis; novel trypsin-resistant fragments of 69,000, 60,000, and 50,000 Mr (all heat-labile), and of 37,000 and 24,000 Mr were detected following treatment of cell extracts with various combinations of trypsin, detergent, and reducing agent. Preliminary experiments suggested that these tryptic fragments are potentially useful in mapping the antigenic epitopes of NS1.
Cauchi
M R
MR
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Henchal
E A
EA
Wright
P J
PJ
D000818:Animals; D015153:Blotting, Western; D002213:Capsid / Q000302:isolation & purification / Q000378:metabolism*; D003716:Dengue Virus / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism*; D014709:Vero Cells; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D014357:Trypsin