title: A replication defect of pseudorabies virus induced by targeted α-helix distortion in the syntaxin-like bundle of glycoprotein H (V275P) is corrected by an adjacent compensatory mutation (V271A).
abstract: Glycoprotein gH is essential for herpesvirus-induced membrane fusion during entry and cell-to-cell spread. Structural analyses of gH homologues revealed a conserved syntaxin-like bundle motif composed of three α-helices. Previous studies showed that targeted disruption of any of these helices strongly impaired maturation, cell surface expression and fusion activity of pseudorabies virus gH, as well as formation and spread of infectious virus. After passaging of one corresponding mutant (pPrV-gH-V275P) these replication defects were widely corrected by an adjacent spontaneous amino acid substitution (V271A). Although the doubly mutated gH was still non-functional in fusion assays, its targeted reinsertion into the cloned virus genome (pPrV-gH-V275P-V271A) led to a 200-fold increase in plaque sizes and 10,000-fold higher virus titres, compared with pPrV-gH-V275P. Thus, our results demonstrate that structural requirements for gH function in in vitro assays and virus replication are different, and that minor amounts of mature gH in virions are sufficient for productive replication.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Sebastian W
authors.initials: SW
authors.lastname: Böhm
authors.affiliation: Institut Pasteur, Unité de Virologie Structurale, Département de Virologie and CNRS Unité de Recherche Associée 3015, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.
authors.forename: Marija
authors.initials: M
authors.lastname: Backovic
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.affiliation: Institut Pasteur, Unité de Virologie Structurale, Département de Virologie and CNRS Unité de Recherche Associée 3015, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.
authors.forename: Felix A
authors.initials: FA
authors.lastname: Rey
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Fuchs
mesh_terms: D000818:Animals; D002460:Cell Line; D011558:Herpesvirus 1, Suid / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011557:Pseudorabies / Q000821:virology*; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013553:Swine Diseases / Q000821:virology*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
chemical_list: D050765:Qa-SNARE Proteins; D014759:Viral Envelope Proteins