title: Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo.
abstract: Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.
authors.affiliation: Friedrich-Loeffler-Institut, Institute of Molecular Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Sindy
authors.initials: S
authors.lastname: Böttcher
authors.forename: Christina
authors.initials: C
authors.lastname: Maresch
authors.forename: Harald
authors.initials: H
authors.lastname: Granzow
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.forename: Jens P
authors.initials: JP
authors.lastname: Teifke
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003545:Cysteine / Q000235:genetics*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000821:virology*; D012694:Serine / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure; D014779:Virus Replication / Q000235:genetics*
chemical_list: D015678:Viral Structural Proteins; D012694:Serine; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases; D003545:Cysteine