scholarly journals Conformational changes in human Norovirus polymerase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1595-C1595
Author(s):  
Kenneth Ng ◽  
Dmitry Zamyatkin ◽  
Hayeong Rho ◽  
Elesha Hoffarth ◽  
Gabriela Jurca ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Rna Binding ◽  
Divalent Metal ◽  
Rna Dependent Rna Polymerase ◽  
Human Norovirus ◽  
Conformational States ◽  
Crystal Forms ◽  
Divalent Metal Cations ◽  
Positive Strand Rna

Human Noroviruses (NV) belong in the Caliciviridae family and are a major cause of gastroenteritis outbreaks throughout the world. Crystal structures of the RNA-dependent RNA polymerase from the human Norovirus have been determined in over ten different crystal forms in the presence and absence of divalent metal cations, nucleoside triphosphates, inhibitors and primer-template duplex RNA. These structures show how the polymerase enzyme can adopt a range of conformations in which the thumb, fingers and palm domains change orientations depending on the step of the enzymatic cycle trapped in different crystal forms. We discuss how the evidence from crystallographic and biochemical experiments combine to better understand how viral RNA polymerase enzymes from human Norovirus and related positive-strand RNA viruses can adopt a range of conformational states to facilitate RNA binding, NTP binding, catalysis, RNA translocation and pyrophosphate release. The detailed structural and mechanistic understanding of these conformational changes is important for providing a sound basis for understanding viral replication in general, as well as for the design of novel inhibitors capable of trapping the enzyme in specific conformational states.

scholarly journals Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase

2001 ◽  
Vol 75 (6) ◽  
pp. 2818-2824 ◽  
Author(s):  
Cheng-Yen Huang ◽  
Yih-Leh Huang ◽  
Menghsiao Meng ◽  
Yau-Heiu Hsu ◽  
Ching-Hsiu Tsai
Keyword(s):  
Rna Polymerase ◽  
Untranslated Region ◽  
Rna Binding ◽  
Bovine Liver ◽  
Viral Rna ◽  
Mobility Shift ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Competition Analysis ◽  
Reading Frame

ABSTRACT The 3′ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555–565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that theEscherichia coli-expressed RNA-dependent RNA polymerase domain (Δ893) derived from BaMV open reading frame 1 could specifically bind to the 3′ UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3′ UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3′ UTR showed that Δ893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Δ893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.

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 Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Chen Li ◽  
Haiwei Wang ◽  
Jiabao Shi ◽  
Decheng Yang ◽  
Guohui Zhou ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Polymerase ◽  
Crucial Role ◽  
Virus Evolution ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Fidelity ◽  
Rdrp Region ◽  
Positive Strand Rna

ABSTRACTSenecavirus A (SVA) is a reemerging virus, and recent evidence has emphasized the importance of SVA recombinationin vivoon virus evolution. In this study, we report the development of an infectious cDNA clone for the SVA/HLJ/CHA/2016 strain. We used this strain to develop a reporter virus expressing enhanced green fluorescent protein (eGFP), which we then used to screen for a recombination-deficient SVA by an eGFP retention assay. Sequencing of the virus that retained the eGFP following passage allowed us to identify the nonsynonymous mutations (S460L alone and I212V-S460L in combination) in the RNA-dependent RNA polymerase (RdRp) region of the genome. We developed a Senecavirus-specific cell culture-based recombination assay, which we used to elucidate the role of RdRp in SVA recombination. Our results demonstrate that these two polymerase variants (S460L and I212/S460L) have reduced recombination capacity. These results indicate that the RdRp plays a central role in SVA replicative recombination. Notably, our results showed that the two recombination-deficient variants have higher replication fidelity than the wild type (WT) and display decreased ribavirin sensitivity compared to the WT. In addition, these two mutants exhibited significantly increased fitnessin vitrocompared to the WT. These results demonstrate that recombination and mutation rates are intimately linked. Our results have important implications for understanding the crucial role of the RdRp in virus recombination and fitness, especially in the molecular mechanisms of SVA evolution and pathogenicity.IMPORTANCERecent evidence has emphasized the importance of SVA recombination on virus evolutionin vivo. We describe the first assays to study Senecavirus A recombination. The results show that the RNA-dependent RNA polymerase plays a crucial role in recombination and that recombination can impact the fitness of SVA in cell culture. Further, SVA polymerase fidelity is closely related to recombination efficiency. The results provide key insights into the role of recombination in positive-strand RNA viruses.

Remdesivir-Bound and Ligand-Free Simulations Reveal the Probable Mechanism of Inhibiting the RNA Dependent RNA Polymerase of SARS-CoV-2

2020 ◽  
Author(s):  
Shruti Koulgi ◽  
Vinod Jani ◽  
Mallikarjunachari Uppuladinne V N ◽  
Uddhavesh Sonavane ◽  
Rajendra Joshi
Keyword(s):  
Rna Polymerase ◽  
Principal Component ◽  
Experimental Information ◽  
Strong Interactions ◽  
Entry Site ◽  
Rna Dependent Rna Polymerase ◽  
Gene Encoding ◽  
Strong Inhibitor ◽  
Ensemble Docking ◽  
Positive Strand Rna

<p>The efforts towards developing a potential drug against the current global pandemic, COVID-19, has increased in the past few months. Drug development strategies to target the RNA dependent RNA polymerase (RdRP) are being tried worldwide. The gene encoding this protein, is known to be conserved amongst positive strand RNA viruses. This enables an avenue to repurpose the drugs designed against earlier reported inhibitors of RdRP. One such strong inhibitor is remdesivir which has been used against EBOLA infections. The binding of remdesivir to RdRP of SARS-CoV-2 has been studied using the classical molecular dynamics and ensemble docking approach. A comparative study of the simulations of RdRP in the apo and remdesivir-bound form revealed blocking of the template entry site in the presence of remdesivir. The conformation changes leading to this event were captured through principal component analysis. The conformational and thermodynamic parameters supported the experimental information available on the involvement of crucial arginine, serine and aspartate residues belonging to the conserved motifs in RdRP functioning. The catalytic site comprising of SER 759, ASP 760, and ASP 761 (SDD) was observed to form strong contacts with remdesivir. The significantly strong interactions of these residues with remdesivir may infer the latter’s binding similar to the normal nucleotides thereby remaining unidentified by the exonuclease activity of RdRP. The ensemble docking of remdesivir too, comprehended the involvement of similar residues in interaction with the inhibitor. This information on crucial interactions between conserved residues of RdRP with remdesivir through <i>in-silico</i> approaches may be useful in designing inhibitors.<b></b></p>

scholarly journals Functional Analysis of RNA Binding by the Hepatitis C Virus RNA-dependent RNA Polymerase

2005 ◽  
Vol 280 (45) ◽  
pp. 38011-38019 ◽  
Author(s):  
Young-Chan Kim ◽  
William K. Russell ◽  
C. T. Ranjith-Kumar ◽  
Michael Thomson ◽  
David H. Russell ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Binding ◽  
Hepatitis C Virus Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

scholarly journals Analysis of tombusvirus revertants to identify essential amino acid residues within RNA-dependent RNA polymerase motifs

2005 ◽  
Vol 86 (3) ◽  
pp. 823-826 ◽  
Author(s):  
K. Boonrod ◽  
S. Chotewutmontri ◽  
D. Galetzka ◽  
G. Krczal
Keyword(s):  
Rna Polymerase ◽  
Nicotiana Benthamiana ◽  
Essential Amino Acid ◽  
Selection Pressure ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Rna Dependent Rna Polymerase ◽  
Viral Rdrp ◽  
In Trans ◽  
Residue Position

The RNA-dependent RNA polymerase (RdRp) of Tomato bushy stunt virus (TBSV) contains an arginine- and proline-rich (RPR) motif. This motif functions as an RNA-binding domain and is essential for tombusvirus replication. A mutant carrying three arginine substitutions in this motif rendered the virus unable to replicate in Nicotiana benthamiana plants and protoplasts. When the replicase function was provided in trans, by expressing the TBSV RdRp in N. benthamiana plants, an infectious variant could be isolated. Sequence analysis showed that only the substituted glycine residue (position 216) had reverted to arginine; all other substitutions remained unchanged. This finding suggested that strong selection pressure is active to maintain necessary sequences of the viral RdRp and that the analysis of revertants may help to identify essential viral functions.

scholarly journals Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention

2014 ◽  
Vol 42 (14) ◽  
pp. 9399-9409 ◽  
Author(s):  
Abdelhakim Ahmed-Belkacem ◽  
Jean-François Guichou ◽  
Rozenn Brillet ◽  
Nazim Ahnou ◽  
Eva Hernandez ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Binding ◽  
Hepatitis C Virus Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

scholarly journals The 3′ end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Takashi Shimoike ◽  
Tsuyoshi Hayashi ◽  
Tomoichiro Oka ◽  
Masamichi Muramatsu
Keyword(s):  
Rna Polymerase ◽  
Genome Sequence ◽  
Rna Synthesis ◽  
Loop Structure ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Human Norovirus ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Positive Sense

Norovirus genome is a single-stranded positive-strand RNA. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp synthesized the complementary positive-sense RNA from the 100 nt template corresponding to the 3′ end region of the viral antisense genome sequence, but that RdRp did not synthesize the antisense genomic RNA from the 100 nt template, corresponding to the 5′ end region of the positive-sense genome sequence. The 31 nt region at the 3′ end of the RNA antisense template was then predicted to form the stem-loop structure. Its deletion resulted in the loss of complementary RNA synthesis by RdRp. The connection of the 31 nt to the 3′ end of the positive-sense RNA template allowed to be recognized by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that RdRp bound to the antisense RNA specifically, but the 31 nt deletion at the 3′ end lost the binding to RdRp. Therefore, combining this observation with further deletion and mutation analysis, we concluded that the predicted stem-loop structure in the 31 nt and region close to the antisense viral genomic stem sequences are important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.

Remdesivir-Bound and Ligand-Free Simulations Reveal the Probable Mechanism of Inhibiting the RNA Dependent RNA Polymerase of SARS-CoV-2

2020 ◽  
Author(s):  
Shruti Koulgi ◽  
Vinod Jani ◽  
Mallikarjunachari Uppuladinne V N ◽  
Uddhavesh Sonavane ◽  
Rajendra Joshi
Keyword(s):  
Rna Polymerase ◽  
Principal Component ◽  
Experimental Information ◽  
Strong Interactions ◽  
Entry Site ◽  
Rna Dependent Rna Polymerase ◽  
Gene Encoding ◽  
Strong Inhibitor ◽  
Ensemble Docking ◽  
Positive Strand Rna

<p>The efforts towards developing a potential drug against the current global pandemic, COVID-19, has increased in the past few months. Drug development strategies to target the RNA dependent RNA polymerase (RdRP) are being tried worldwide. The gene encoding this protein, is known to be conserved amongst positive strand RNA viruses. This enables an avenue to repurpose the drugs designed against earlier reported inhibitors of RdRP. One such strong inhibitor is remdesivir which has been used against EBOLA infections. The binding of remdesivir to RdRP of SARS-CoV-2 has been studied using the classical molecular dynamics and ensemble docking approach. A comparative study of the simulations of RdRP in the apo and remdesivir-bound form revealed blocking of the template entry site in the presence of remdesivir. The conformation changes leading to this event were captured through principal component analysis. The conformational and thermodynamic parameters supported the experimental information available on the involvement of crucial arginine, serine and aspartate residues belonging to the conserved motifs in RdRP functioning. The catalytic site comprising of SER 759, ASP 760, and ASP 761 (SDD) was observed to form strong contacts with remdesivir. The significantly strong interactions of these residues with remdesivir may infer the latter’s binding similar to the normal nucleotides thereby remaining unidentified by the exonuclease activity of RdRP. The ensemble docking of remdesivir too, comprehended the involvement of similar residues in interaction with the inhibitor. This information on crucial interactions between conserved residues of RdRP with remdesivir through <i>in-silico</i> approaches may be useful in designing inhibitors.<b></b></p>

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