NS2B-3 PROTEINASE-MEDIATED PROCESSING IN THE YELLOW-FEVER VIRUS STRUCTURAL REGION - IN-VITRO AND IN-VIVO STUDIES

Several of the cleavages required to generate the mature nonstructural proteins from the flaviviral polyprotein are known to be mediated by a complex consisting of NS2B and a serine proteinase domain located in the N-terminal one-third of NS3. These cleavages typically occur afte r two basic residues followed by a short side chain residue. Cleavage at a similar dibasic site in the structural region is believed to prod uce the C terminus of the virion capsid protein. To study this cleavag e, we developed a cell-free trans cleavage assay for yellow fever viru s (YF)-specific proteolytic activity by using a substrate spanning the C protein dibasic site. Cleavage at the predicted site was observed w hen the substrate was incubated with detergent-solubilized lysates fro m YF-infected BHK cells. NS2B and NS3 proteinase domain were the only YF-specific proteolytic activity was membrane associated and that acti vity could be detected only after detergent solubilization. Previous c ell-free studies led to a hypothesis that processing in the C-prM regi on involves (i) translation of C followed by translocation and core gl ycosylation of prM by using an internal signal sequence, (ii) signalas e cleavage to produce a membrane-anchored form of the C protein (anchC ) and the N terminus of prM, and (iii) NS2B-3-mediated cleavage at the anchC dibasic site to produce the C terminus of the virion C protein. However, the results of in vivo transient-expression studies do not s upport this temporal cleavage order. Rather, expression of a YF polypr otein extending from C through the N-terminal one-third of NS3 reveale d that C-prM processing, but not translation, was dependent on an acti ve NS2B-3 proteinase. This suggests that signalase-mediated cleavage i n the lumen of the endoplasmic reticulum may be dependent on prior cle avage at the anchC dibasic site. Possible pathways for processing in t he C-prM region are outlined and discussed.