Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing

Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been impl icated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg -Arg) in the P1 through P4 positions of this cleavage site. Multiple alanin e substitutions were made for these residues in order to investigate the su bstrate specificity and biological significance of this cleavage. Mutants w ere analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a trans processing ass ay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM producti on; (iii) an infectivity assay of full-length transcripts to determine plaq ue-forming ability; and (iv) analysis of proteins expressed from full-lengt h transcripts to assess processing in the context of the complete genome. M utants that exhibited severe defects in processing in vitro and in vivo wer e incapable of forming plaques. Mutants that contained two adjacent basic r esidues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infec tious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the p roteolytic activity of NS3, These data support a model of two coordinated c leavages, one that generates the carboxy terminus of C and another that gen erates the amino terminus of prM, A block in the viral protease-mediated cl eavage inhibits the production of prM by the signal peptidase, inhibits par ticle release, and eliminates plaque formation.