MUTAGENESIS OF THE YELLOW-FEVER VIRUS NS2B 3 CLEAVAGE SITE - DETERMINANTS OF CLEAVAGE SITE-SPECIFICITY AND EFFECTS ON POLYPROTEIN PROCESSING AND VIRAL REPLICATION/

The determinants of cleavage site specificity of the yellow fever viru s (YF) NS3 proteinase for its 2B/3 cleavage site have been studied by using site-directed mutagenesis. Mutations at residues within the GARR down arrow S sequence were tested for effects on cia cleavage of an N S2B-3(181) polyprotein during cell-free translation, At the P1 positio n, only the conservative substitution R-->K exhibited Significant leve ls of cleavage. Conservative and nonconservative substitutions were to lerated at the P1' and P2 positions, resulting in intermediate levels of cleavage. Substitutions at the P3 and P4 positions had no effects o n cleavage efficiency in the cell-free assay. Processing at other diba sic sites was studied by using transient expression of a sig2A-5(356) polyprotein. Cleavage at the 2B/3 site was not required for processing at downstream sites. However, increased accumulation of high-molecula r-weight viral polyproteins was generally observed for mutations which reduced cleavage efficiency at the 2B/3 site. Several mutations were also tested for their effects on viral replication. Virus was not reco vered from substitutions which blocked or substantially reduced cleava ge in the cell-free assay,suggesting that efficient cleavage at the 2B /3 site is required for flavivirus replication.