title: The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.
abstract: The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. minako.ogino@case.edu.
authors.forename: Minako
authors.initials: M
authors.lastname: Ogino
authors.affiliation: Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. naotoito@gifu-u.ac.jp.|The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. naotoito@gifu-u.ac.jp.
authors.forename: Naoto
authors.initials: N
authors.lastname: Ito
authors.affiliation: Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. sugiyama@gifu-u.ac.jp.|The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. sugiyama@gifu-u.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sugiyama
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. tomoaki.ogino@case.edu.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ogino
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011820:Rabies virus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D006153:Guanosine Diphosphate; C097625:L protein, Rabies virus; D012321:DNA-Directed RNA Polymerases; D011117:Polyribonucleotide Nucleotidyltransferase; D020558:GTP Phosphohydrolases
keywords: GDP polyribonucleotidyltransferase; L protein; mRNA capping; rabies virus