title: Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production.
abstract: Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence 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 biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.
authors.affiliation: Division of Immunology and Cell Biology, John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Lee
authors.forename: C E
authors.initials: CE
authors.lastname: Stocks
authors.forename: S M
authors.initials: SM
authors.lastname: Amberg
authors.forename: C M
authors.initials: CM
authors.lastname: Rice
authors.forename: M
authors.initials: M
authors.lastname: Lobigs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers; D016679:Genome, Viral; D006868:Hydrolysis; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009420:Nervous System / Q000821:virology; D021382:Protein Sorting Signals / Q000235:genetics*; D011995:Recombination, Genetic; D012697:Serine Endopeptidases / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics*; D014774:Virulence; D014779:Virus Replication / Q000235:genetics; D015005:Yellow fever virus / Q000472:pathogenicity / Q000502:physiology*
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; D012697:Serine Endopeptidases; C026515:type I signal peptidase