title: Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus.
abstract: In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.
authors.affiliation: Sir Henry Wellcome Laboratories, Division of Infection, Inflammation and Immunity, School of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom.
authors.forename: Morgan R
authors.initials: MR
authors.lastname: Herod
authors.affiliation: Medical Research Council-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, Scotland, United Kingdom.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Jones
authors.affiliation: Medical Research Council-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, Scotland, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: McLauchlan
authors.affiliation: Sir Henry Wellcome Laboratories, Division of Infection, Inflammation and Immunity, School of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom. Electronic address: cjm@soton.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: McCormick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D011498:Protein Precursors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080107:NS4B protein, flavivirus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
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