Mutagenesis of the signal sequence of yellow fever virus prM protein: Enhancement of signalase cleavage in vitro is lethal for virus production

Protcolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an i nternal signal sequence. We have introduced at the COOH terminus of the yel low fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM pr otein from its dependence on prior cleavage in the cytoplasm of the C prote in mediated by the viral NS2B-3 protease, Infectivity assays with full-leng th YFV RNA transcripts showed that the VPQAQA mutation, which enhanced sign al peptidase cleavage in vitro, was lethal for infectious virus production, Revertants or second-site mutants were recovered from cells transfected wi th VPQAQA RNA. Analysis of these viruses revealed that single amino acid su bstitutions in different domains of the prM signal sequence could restore v iability, These variants had growth properties in vertebrate cells which di ffered only slightly from those of the parent virus, despite efficient sign al peptidase cleavage of prM in cell-free expression assays. However, the n eurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the bio logical properties of the mutant viruses. Factors other than the rate of pr oduction of prM are vitally controlled by regulated cleavages at this site.