scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase in complex with 2-thiouridine or ribavirin

Virology ◽  
2012 ◽  
Vol 426 (2) ◽  
pp. 143-151 ◽  
Author(s):  
Intekhab Alam ◽  
Ji-Hye Lee ◽  
Ki Joon Cho ◽  
Kang Rok Han ◽  
Jai Myung Yang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

scholarly journals Structural basis for VPg-induced formation of RNA-dependent RNA polymerase multimeric complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1601-C1601
Author(s):  
Ji-Hye Lee ◽  
Yeon Bin Chung ◽  
Jong Hyeon Seok ◽  
Kang Rok Han ◽  
Sella Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Helical Structure ◽  
Science Research ◽  
Binding Mode ◽  
Structural Basis ◽  
Murine Norovirus ◽  
Virion Protein ◽  
Electron Microscopic ◽  
Rna Dependent Rna Polymerase

Norovirus is the leading cause of epidemic acute, nonbacterial gastroenteritis, and adopts de novo and VPg (Virion protein genome linked)-primed RNA synthesis by RNA-dependent RNA polymerase (RdRp). To understand the interaction between RdRp and VPg in replication of murine norovirus-1 (MNV-1), we determined the crystal structure of MNV-1 RdRp-VPg(1-73)-RNA complex. VPg was bound to the base of the palm domain and the tip of the fingers domain of RdRp simultaneously, but RNA template could not be modeled. The binding affinity constants (Kd) for RdRp-VPg was 3.7411.57 nM and VPg(1-73) showed approximately 90-fold less affinity than that of full-length VPg. In addition to this multiple binding mode, VPg enhanced the interactions of RdRp hexamers, leading to the formation of high-order multimers or tubular fibrils with significantly increased polymerase activity, confirmed by electron microscopic and biochemical studies. Our data indicated that MNV-1 VPg with helical structure was bound to RdRp at multiple sites and induces RdRp multimerization in viral replication. The multimers of RdRp-VPg-RNA can provide a mechanistic understanding of viral polymerase multimeric arrays and a new tool for development of antivirals to control norovirus outbreaks. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs (A085119 K.H.K), Basic Science Research Program through the National Research Foundation (NRF-2013R1A1A2064940, L.J-H), Korea University Grant (L.J-H), and the BK21 plus program of the Ministry of Education, Korea.

scholarly journals PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases

FEBS Letters ◽  
2014 ◽  
Vol 588 (9) ◽  
pp. 1720-1725 ◽  
Author(s):  
Romina Croci ◽  
Delia Tarantino ◽  
Mario Milani ◽  
Margherita Pezzullo ◽  
Jacques Rohayem ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase

scholarly journals Crystal Structures of Active and Inactive Conformations of a Caliciviral RNA-dependent RNA Polymerase

2001 ◽  
Vol 277 (2) ◽  
pp. 1381-1387 ◽  
Author(s):  
Kenneth K. S. Ng ◽  
Maia M. Cherney ◽  
Ana López Vázquez ◽  
Ángeles Machı́n ◽  
José M. Martı́n Alonso ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Rna Dependent Rna Polymerase

scholarly journals Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg

2010 ◽  
Vol 91 (7) ◽  
pp. 1713-1722 ◽  
Author(s):  
K. R. Han ◽  
Y. Choi ◽  
B. S. Min ◽  
H. Jeong ◽  
D. Cheon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Polymerase Activity ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase Activity

scholarly journals Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

2012 ◽  
Vol 86 (18) ◽  
pp. 10138-10149 ◽  
Author(s):  
Chennareddy V. Subba-Reddy ◽  
Muhammad Amir Yunus ◽  
Ian G. Goodfellow ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Murine Norovirus ◽  
Structural Protein ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Concentration Dependent Manner

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRpsin vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR).Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.

scholarly journals Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics

2015 ◽  
Vol 89 (15) ◽  
pp. 7722-7734 ◽  
Author(s):  
Huidan Zhang ◽  
Shelley K. Cockrell ◽  
Abimbola O. Kolawole ◽  
Assaf Rotem ◽  
Adrian W. R. Serohijos ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Phylogenetic Analyses ◽  
Low Frequency ◽  
Template Switching ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase ◽  
Infectious Gastroenteritis

ABSTRACTHuman noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture orin vitrosystems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants followingin vitroandin vivocoinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.IMPORTANCERNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

scholarly journals The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis

2014 ◽  
Vol 89 (2) ◽  
pp. 1218-1229 ◽  
Author(s):  
Muhammad Amir Yunus ◽  
Xiaoyan Lin ◽  
Dalan Bailey ◽  
Ioannis Karakasiliotis ◽  
Yasmin Chaudhry ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Subgenomic Rna ◽  
Stem Loop Structure ◽  
Loop Sequence

ABSTRACTAll members of theCaliciviridaefamily of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3′ of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally,in vitrobiochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter.IMPORTANCENoroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells andin vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase.

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