PROCESSING OF THE INTRACELLULAR FORM OF THE WEST NILE VIRUS CAPSID PROTEIN BY THE VIRAL NS2B-NS3 PROTEASE - AN IN-VITRO STUDY

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus poly protein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracel lular form of the viral capsid protein (C-int) retaining the prM signa l sequence at its carboxy terminus. This hydrophobic anchor is subsequ ently removed by the viral protease, resulting in formation of the mat ure viral capsid protein found in virions (C-vir). We hate prepared in vitro expression cassettes coding for both forms of the capsid protei n, for the prM protein, for the C-prM precursor; and for the viral pro tease components of West Nile flavivirus and characterized their trans lation products. Using C-int and C-vir translation products as molecul ar markers, we have observed processing of the intracellular farm of t he West Nile capsid protein by the viral protease in vitro both upon c otranslation of the C-prM precursor and the viral protease-encoding ca ssette and by incubation of C prM translation products with a detergen t-solubilized extract of cells infected with a recombinant vaccinia vi rus expressing the active viral protease. The cleavage of C-int by the viral protease at the predicted dibasic site was verified by introduc tion of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.