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 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.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals Complementary Mutations in the N and L Proteins for Restoration of Viral RNA Synthesis

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Weike Li ◽  
Ryan H. Gumpper ◽  
Yusuf Uddin ◽  
Ingeborg Schmidt-Krey ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Rna Synthesis ◽  
Large Subunit ◽  
Viral Rna ◽  
Structural Motif ◽  
General Mechanism ◽  
Rna Dependent Rna Polymerase ◽  
N Protein ◽  
Rna Genome

ABSTRACTDuring viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif—Glu169, Phe171, and Leu174—to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCEDuring viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

scholarly journals Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World

1999 ◽  
Vol 73 (8) ◽  
pp. 6424-6429 ◽  
Author(s):  
Robert W. Siegel ◽  
Laurent Bellon ◽  
Leonid Beigelman ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Competition Assay ◽  
Rna Dependent Rna Polymerase ◽  
Dna Molecule ◽  
Mechanism Of Catalysis ◽  
Insight Into

ABSTRACT All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.

scholarly journals De NovoRNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

2016 ◽  
Vol 36 (8) ◽  
pp. 1248-1259 ◽  
Author(s):  
Yoshiko Maida ◽  
Mami Yasukawa ◽  
Kenkichi Masutomi
Keyword(s):  
Rna Polymerase ◽  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Reverse Transcriptase ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Human Carcinoma ◽  
Content Type ◽  
Secondary Sirnas

RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such asCaenorhabditis elegansandNeurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAsde novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However,de novosynthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5′-triphosphorylated ends, which indicatesde novosynthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.

scholarly journals The Imidazopyrrolopyridine Analogue AG110 Is a Novel, Highly Selective Inhibitor of Pestiviruses That Targets the Viral RNA-Dependent RNA Polymerase at a Hot Spot for Inhibition of Viral Replication

2007 ◽  
Vol 81 (20) ◽  
pp. 11046-11053 ◽  
Author(s):  
Jan Paeshuyse ◽  
Jean-Michel Chezal ◽  
Matheus Froeyen ◽  
Pieter Leyssen ◽  
Hélène Dutartre ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Hot Spot ◽  
Resistance Mutation ◽  
Drug Resistance Mutation ◽  
Drug Binding ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Diarrhea Virus

ABSTRACT Ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 ± 0.5 μM, 5 ± 1 μM, and 2.3 ± 0.3 μM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 Å away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

scholarly journals Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

2006 ◽  
Vol 81 (4) ◽  
pp. 1858-1871 ◽  
Author(s):  
Stephen W. B. Fullerton ◽  
Martina Blaschke ◽  
Bruno Coutard ◽  
Julia Gebhardt ◽  
Alexander Gorbalenya ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Functional Characterization ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Rna Dependent Rna Polymerase ◽  
Key Enzyme

ABSTRACT Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

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