COTRANSLATIONAL MEMBRANE INSERTION OF THE SERINE PROTEINASE PRECURSORNS2B-NS3(PRO) OF DENGUE VIRUS TYPE-2 IS REQUIRED FOR EFFICIENT IN-VITRO PROCESSING AND IS MEDIATED THROUGH THE HYDROPHOBIC REGIONS OF NS2B

Polyprotein processing of dengue virus type 2, a positive strand RNA v irus, is carried out by the host signal peptidase and a novel two-comp onent viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum, Using an in vitro processing system, we examined the cis and trans cleavages of the 2B/3 and 4B/5 sites by NS 2B/NS3(Pro), respectively, Lysates of BHK-21 cells coexpressing NS2B a nd NS3(Pro) mediated trans cleavage of the 4B/5 site in vitro, and the protease activity was associated with the membrane fraction, To study the role of membranes in the protease activity of NS2B/NS3(Pro), labe led precursors, NS2B-NS3(Pro) and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed u sing a coupled in vitro transcription/translation system (TnT), The re sults showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site i n a dose-dependent manner, NS2B synthesized in the presence of membran es also facilitated trans cleavage of the 2B/3 site in the mutant prec ursor, The cleavage products, NS2B and NS3(Pro), were membrane-associa ted, Furthermore, this membrane requirement was dictated by the hydrop hobic regions of NS2B, Deletion of hydrophobic regions of NS2B, leavin g only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site in vitro in the absence of microsomal membranes.