Separation of functional West Nile virus replication complexes from intracellular membrane fragments.
Flaviviruses encode seven non-structural proteins for which functions have not yet been described. The identification of the viral and possible host proteins which may be involved in flavivirus replication has been impeded by the fact that the viral replication complexes are tightly associated with endoplasmic reticular membranes within infected cells and that in vitro polymerase activity is associated with large membrane fragments. To facilitate further study of flavivirus replication complexes, selected ultrapure detergents were analysed for their effect on West Nile virus (WNV) in vitro RNA-dependent RNA polymerase activity and for their ability to release functional replication complexes from partially purified intracellular BHK-21 membrane fragments. A few previous reports indicated that flavivirus in vitro polymerase activity was sensitive to detergent treatment. The present study indicates that WNV polymerase activity is variably inhibited depending on the concentration and identity of the detergent used. Of the five detergents (Tween 20, maltoside, octylglucoside, lubrol PX and sodium deoxycholate) tested, sodium deoxycholate was the most efficient at releasing functional viral replication complexes from intracellular membranes.
Grun
J B
JB
Wistar Institute, Philadelphia, Pennsylvania 19104.
Brinton
M A
MA
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000382:microbiology; D007425:Intracellular Membranes / Q000382:microbiology*; D008565:Membrane Proteins / Q000302:isolation & purification; D013501:Surface-Active Agents / Q000494:pharmacology*; D014764:Viral Proteins / Q000302:isolation & purification*; D014779:Virus Replication; D014902:West Nile virus
D003902:Detergents; D008565:Membrane Proteins; D013501:Surface-Active Agents; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases