MUTAGENESIS OF THE YELLOW-FEVER VIRUS NS2B PROTEIN - EFFECTS ON PROTEOLYTIC PROCESSING, NS2B-NS3 COMPLEX-FORMATION, AND VIRAL REPLICATION

To study the role of specific regions of the yellow fever virus NS2B p rotein in proteolytic processing and association with the NS3 proteina se domain, a series of mutations were created in the hydrophobic regio ns and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cle avage at the 2B/3 cleavage site and on processing at other consensus c leavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression i n BHK cells. Association between NS2B and the NS3 proteinase domain an d the effects of mutations on complex formation were investigated by n ondenaturing immunoprecipitation of these proteins expressed in infect ed cells, by cell-free translation, or by recombinant vaccinia viruses . Mutations within the hydrophobic regions had subtle effects on prote olytic processing, whereas mutations within the conserved domain drama tically reduced cleavage efficiency or abolished all cleavages. The co nserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eli minate both association of these two proteins and trans-cleavage activ ity. In addition, mutations which either eliminated proteolytic proces sing or had no apparent effect on processing were found to abolish rec overy of infectious virus following RNA transfection. These results su ggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose stru ctural integrity may not be essential for proteolytic processing may h ave additional functions during viral replication.