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.

Download full-text

Full-text

Available from: Amiya Banerjee, Mar 03, 2014
0 Followers
 · 
71 Views
 · 
20 Downloads
  • Source
    • "The HR motif [64] at position 1217 which has been shown to be necessary for the PRNT ase activity of the L protein, at the step of enzyme-pRNA intermediate formation [61], was found to be conserved in all the CHPV isolates as in other rhabdoviruses and the equally important R in the vicinity of the HR motif at position 1211 was also conserved in the isolates. The same was also conserved in the other rhabdoviruses studied here. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Chandipura virus (CHPV) belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003-2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003-2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV.
    PLoS ONE 01/2012; 7(1):e30315. DOI:10.1371/journal.pone.0030315 · 3.23 Impact Factor
  • Source
    • "Mapping of the putative PRNTase domain followed eventually by X-ray crystallographic analyses of its three-dimensional structures would certainly provide deeper insight into the mode of the unique capping reaction. In contrast to abrogation of the VSV L PRNTase and RdRp activities upon mutations in the HR and GDN motifs, respectively, these mutations did not abolish the GTPase activity (Ogino et al., 2010). These results suggest that the GTPase active site is located separately from the PRNTase and RdRp active sites. "
    [Show abstract] [Hide abstract]
    ABSTRACT: mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5'-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.
    Virus Research 09/2011; 162(1-2):100-9. DOI:10.1016/j.virusres.2011.09.012 · 2.83 Impact Factor
  • Source
    • "In addition, it was shown that mRNA capping reaction of Chandipura virus, another member within the family Rhabdoviridae, utilizes the same mechanism (Ogino and Banerjee, 2010). Whether this unconventional mechanism is conserved in other families of NNS RNA viruses is not known. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.
    Virology 10/2010; 408(2):241-52. DOI:10.1016/j.virol.2010.09.017 · 3.28 Impact Factor
Show more