Article

The HR motif in the RNA-dependent RNA polymerase L protein of Chandipura virus is required for unconventional mRNA-capping activity.

Department of Molecular Genetics, Section of Virology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Journal of General Virology (Impact Factor: 3.53). 05/2010; 91(Pt 5):1311-4. DOI: 10.1099/vir.0.019307-0
Source: PubMed

ABSTRACT Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA:GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5'-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme-pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme-pRNA intermediate formation.

0 Bookmarks
 · 
63 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: This article supports several aspects of the arguments by Hofmann, Asmundson, and Beck (2013--this issue) about the scientific basis of cognitive-behavioral therapy (CBT), for example, that CBT has a strong evidence base, and that studies of the mechanisms of change are warranted. This response discusses growth within the broad field of CBT, as well as the diverse research methods that are needed to explore both clinical efficacy and treatment mechanism questions. It is suggested that the field of CBT may be approaching a shift in emphasis from cognitive to metacognitive assessment and interventions. The article concludes with a statement of general support for further development of the field of CBT.
    Behavior therapy 06/2013; 44(2):224-7. · 2.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Ebola virus (EBOV) RNA-dependent RNA polymerase (RdRp) complex consists of the catalytic subunit of the polymerase, L, and its cofactor VP35. Using immunofluorescence analysis and coimmunoprecipitation assays, we mapped the VP35 binding site on L. A core binding domain spanning amino acids 280-370 of L was sufficient to mediate weak interaction with VP35, while the entire N-terminus up to amino acid 380 was required for strong VP35-L binding. Interestingly, the VP35 binding site overlaps with an N-terminal L homo-oligomerization domain in a non-competitive manner. N-terminal L deletion mutants containing the VP35 binding site were able to efficiently block EBOV replication and transcription in a minigenome system suggesting the VP35 binding site on L as a potential target for the development of antivirals.
    Virology 04/2013; · 3.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The multifunctional RNA-dependent RNA polymerase L protein of vesicular stomatitis virus catalyzes unconventional pre-mRNA capping via the covalent enzyme-pRNA intermediate formation, which requires the histidine-arginine (HR) motif in the polyribonucleotidyltransferase domain. Here, the effects of cap-defective mutations in the HR motif on transcription were analyzed using an in vitro reconstituted transcription system. The wild-type L protein synthesized the leader RNA from the 3'-end of the genome followed by 5'-capped and 3'-polyadenylated mRNAs from internal genes by a stop-start transcription mechanism. Cap-defective mutants efficiently produced the leader RNA, but displayed aberrant stop-start transcription using cryptic termination and initiation signals within the first gene, resulting in sequential generation of ∼40-nucleotide transcripts with 5'-ATP from a correct mRNA-start site followed by a 28-nucleotide transcript and long 3'-polyadenylated transcript initiated with non-canonical GTP from atypical start sites. Frequent transcription termination and re-initiation within the first gene significantly attenuated the production of downstream mRNAs. Consistent with the inability of these mutants in in vitro mRNA synthesis and capping, these mutations were lethal to virus replication in cultured cells. These findings indicate that viral mRNA capping is required for accurate stop-start transcription as well as mRNA stability and translation and, therefore, for virus replication in host cells.
    Nucleic Acids Research 10/2014; · 8.81 Impact Factor

Full-text (2 Sources)

Download
7 Downloads
Available from
Jun 4, 2014