title: Polymerase activity of in vitro mutated rabies virus L protein.
abstract: The large (L) protein of nonsegmented negative-strand RNA viruses is the multifunctional catalytic component of the viral ribonucleoprotein (RNP) complex. To address the role of conserved rabies virus (RV) L protein sequences predicted to be involved in RNA polymerase activity, a reverse genetics approach was applied that allows intracellular reconstitution of transcriptionally active RV RNPs from plasmid-encoded proteins. Artificial RV model genomes encoding bacterial chloramphenicol acetyltransferase or firefly luciferase was used to determine the polymerase activity of a series of 23 RV L proteins mutated in the highly conserved C motif of the proposed polymerase module. All constructs with mutations of the GDN core sequence of motif C, which is proposed to be a variant of the catalytical XDD residues of RNA polymerase and reverse transcriptases, failed to express the reporter genes. In addition, the identity of the upstream residues AQ was crucial for maintenance of polymerase activity. Several conservative and nonconservative mutations introduced into the three amino acids QVL located downstream of the GDN core resulted in reduced polymerase activities and expression of luciferase in the range 0.4 to 92% compared to the parental L protein.
authors.affiliation: Federal Research Centre for Virus Diseases of Animals, Tübingn, Germany.
authors.forename: M J
authors.initials: MJ
authors.lastname: Schnell
authors.forename: K K
authors.initials: KK
authors.lastname: Conzelmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000378:metabolism; D011820:Rabies virus / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C097625:L protein, Rabies virus; D012321:DNA-Directed RNA Polymerases