title: Identification of two essential aspartates for polymerase activity in parainfluenza virus L protein by a minireplicon system expressing secretory luciferase.
abstract: Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.
authors.affiliation: Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohta
authors.affiliation: Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Yumine
authors.affiliation: Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Goto
authors.affiliation: Department of Microbiology, School of Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
authors.forename: Machiko
authors.initials: M
authors.lastname: Nishio
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D002478:Cells, Cultured; D017124:Conserved Sequence; D033342:Copepoda / Q000201:enzymology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D010223:Parainfluenza Virus 2, Human / Q000201:enzymology* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D014764:Viral Proteins; C069129:L protein, Human parainfluenza virus 2; D001224:Aspartic Acid; D008156:Luciferases
keywords: L protein; minireplicon system; parainfluenza virus