PROCESSING OF JAPANESE ENCEPHALITIS-VIRUS NONSTRUCTURAL PROTEINS - NS2B-NS3 COMPLEX AND HETEROLOGOUS PROTEASES

Processing of Japanese encephalitis (JE) virus nonstructural (NS) prot eins expressed by recombinant vaccinia viruses was analysed to charact erize the responsible viral protease. Analysis of the processing of po lyprotein NS2A-2B-3' containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergeni c junctions as well as at one or possibly two sites within NS2A, Cleav age at the alternate site(s) containing the cleavage sequence motif wi thin NS2A could possibly explain the production of the NS1' protein in JE virus-infected cells. Polyprotein NS2A-d2B-3' containing a large d eletion within NS2B was cleavage-defective, despite the presence of th e proposed NS3 protease domain. Cleavage of NS2A-d2B-3' was restored i f NS2B or NS2A-2B was supplied in trans, providing evidence that NS2B is strictly required for NS3 proteolytic activity. NS2B- or NS3-specif ic sera raised against the bacterial TrpE fusion protein co-precipitat ed NS2B and NS3 or NS3'from the lysate of JE virus or recombinant viru s-infected cells. Thus both protease components are associated as a co mplex, presumably representing the active JE virus protease. JE virus and the analogous dengue 4 (DEN-4) protease components were employed t o examine the activity of heterologous proteases. The defective cleava ge of JE virus NS2A-d2B-3' was complemented by heterologous DEN-4 NS2B , whereas the defective cleavage of DEN-4 NS2A-d2B-3' was not correcte d by heterologous JE virus NS2B. This suggests that the heterologous J E virus NS2B-DEN-4 NS3 protease is not active, despite the considerabl e sequence conservation of NS2B and NS3 between the two viruses. The c leavage activity was restored by replacement of the C-terminal 80 amin o acids of JE virus NS2B with the corresponding DEN-4 sequence, consis tent with the notion that the C-terminal region contains amino acid re sidues for interaction with DEN-4 NS3.