Description of a procedure which allows isolation of viral nonstructural proteins from BHK vertebrate cells infected with the West Nile flavivirus in a state which allows their direct chemical characterization.
We have developed a procedure which allows isolation of virus-coded nonstructural (NS) proteins from BHK cells infected with the West Nile (WN) flavivirus in a state of purity which allows their chemical characterization. A crude membrane fraction proved to be a suitable starting material. Incubation of crude membranes in buffer containing 1.2 M guanidine hydrochloride (GH) allows isolation of weakly washed membranes. Incubation of weakly washed membranes in the presence of either 5 M GH or 8 M urea allows the isolation of stringently washed membranes, as well as a soluble protein-containing wash which is called the differential wash. Stringently washed membranes contain proteins with apparent molecular weights of 14, 19, 23, 29, and 50 kDa as predominant constituents. These proteins are of sufficient purity after SDS-PAGE to allow amino-terminal sequence determination. Together with the genome RNA sequence these analyses show that these molecules represent the virus-coded proteins NS 2b, NS 2a, pre M, NS 4b, and E, respectively. Similar analysis of the proteins present in the differential wash shows that the proteins NS 5, NS 3, and NS 1 are major constituents of this material. The carboxy-terminal sequences of NS 5 and NS 1 have also been determined.
Wengler
G
G
Institut für Virologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Wengler
G
G
Nowak
T
T
Castle
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000096:biosynthesis / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney; D007700:Kinetics; D008566:Membranes / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014461:Ultracentrifugation; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D017361:Viral Nonstructural Proteins; D014902:West Nile virus / Q000235:genetics* / Q000378:metabolism
D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins