title: Analysis of serine codon conservation reveals diverse phenotypic constraints on hepatitis C virus glycoprotein evolution.
abstract: Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes.
authors.affiliation: Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, a joint venture between Medical School Hannover and Helmholtz Centre for Infection Research, Hannover, Germany.
authors.forename: Richard J P
authors.initials: RJ
authors.lastname: Brown
authors.forename: George
authors.initials: G
authors.lastname: Koutsoudakis
authors.forename: Richard A
authors.initials: RA
authors.lastname: Urbanowicz
authors.forename: Deeman
authors.initials: D
authors.lastname: Mirza
authors.forename: Corinne
authors.initials: C
authors.lastname: Ginkel
authors.forename: Nina
authors.initials: N
authors.lastname: Riebesehl
authors.forename: Noémie
authors.initials: N
authors.lastname: Calland
authors.forename: Anna
authors.initials: A
authors.lastname: Albecka
authors.forename: Louisa
authors.initials: L
authors.lastname: Price
authors.forename: Natalia
authors.initials: N
authors.lastname: Hudson
authors.forename: Véronique
authors.initials: V
authors.lastname: Descamps
authors.forename: Matthijs
authors.initials: M
authors.lastname: Backx
authors.forename: C Patrick
authors.initials: CP
authors.lastname: McClure
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.forename: Eve-Isabelle
authors.initials: EI
authors.lastname: Pecheur
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.forename: Sofia
authors.initials: S
authors.lastname: Perez-del-Pulgar
authors.forename: Xavier
authors.initials: X
authors.lastname: Forns
authors.forename: Eike
authors.initials: E
authors.lastname: Steinmann
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tarr
authors.forename: Thomas
authors.initials: T
authors.lastname: Pietschmann
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Ball
mesh_terms: D003062:Codon; D019143:Evolution, Molecular; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry*; D010641:Phenotype; D010802:Phylogeny; D012694:Serine / Q000737:chemistry*
chemical_list: D003062:Codon; D006023:Glycoproteins; D012694:Serine