UP-REGULATION OF SIGNALASE PROCESSING AND INDUCTION OF PRM-E SECRETION BY THE FLAVIVIRUS NS2B-NS3 PROTEASE - ROLES OF PROTEASE COMPONENTS

Recently, we have shown that the ability of the flavivirus NS2B-NS3 pr otease complex to promote efficient signalase processing of the C-prM precursor, as well as secretion of prM and E, does not appear to depen d strictly on cleavage of the precursor at its Lys-Arg-Gly dibasic sit e by the protease, We suggested that the association of the protease w ith the precursor via NS2B may be sufficient by itself for the above e ffects, To study the proposed association in more detail, we have deve loped an assay in which processing at the C-prM dibasic cleavage site is abolished by Lys --> Gly conversion, We constructed deletion mutant s and chimeras of the West Nile (WN) flavivirus NS2B protein and expre ssed them in the context of {5'-C --> NS3(243)} containing either wild -type C-prM or its cleavage site mutant. All NS2B variants were able t o form active protease complexes, Deletion of the carboxy-terminal clu ster of hydrophobic amino acids in NS2B had no apparent effect on the formation of prM and prM-E secretion for the cassettes containing eith er wild-type or mutated C-prM precursor, Deletion of the amino-termina l hydrophobic cluster in NS2B did not affect prM-E secretion for the c assettes with mild-type C-prM but abrogated prM(-)E secretion for the cassettes with the mutated dibasic cleavage site in C-prM, Similarly, the NS2B-NS3(178) protease of Japanese encephalitis (JE) virus, when s ubstituted for the WN virus NS2B-NS3(243) protease, was able to promot e prM-E secretion for the cassette with the wild-type C-prM precursor but not with the mutated one, Replacement of the deleted amino termina l hydrophobic cluster in the WN virus NS2B protein with an analogous J E virus sequence restored the ability of the protease to promote prM-E secretion, On the basis of these observations, roles of individual pr otease components in upregulation of C-prM signalase processing are di scussed.