Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3

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.

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Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309107020416 ◽

2007 ◽

Vol 63 (6) ◽

pp. 495-498 ◽

Cited By ~ 7

Author(s):

Ilham Jabafi ◽

Barbara Selisko ◽

Bruno Coutard ◽

Armando M. De Palma ◽

Johan Neyts ◽

...

Keyword(s):

Rna Polymerase ◽

Coxsackievirus B3 ◽

Rna Dependent Rna Polymerase

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Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using φ6 RNA-dependent RNA polymerase

Journal of General Virology ◽

10.1099/vir.0.011338-0 ◽

2009 ◽

Vol 90 (10) ◽

pp. 2468-2473 ◽

Cited By ~ 10

Author(s):

Michaela Nygårdas ◽

Tytti Vuorinen ◽

Antti P. Aalto ◽

Dennis H. Bamford ◽

Veijo Hukkanen

Keyword(s):

Rna Polymerase ◽

Genomic Sequence ◽

Coxsackievirus B3 ◽

Human Enterovirus ◽

Rna Dependent Rna Polymerase ◽

Coxsackievirus B4 ◽

Novel Approach ◽

Experimental Therapies ◽

Coxsackievirus A9 ◽

Interfering Rna

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage φ6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837–7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the φ6 RdRP (φ6–siRNAs) was considerably more effective than single-site siRNAs. The φ6–siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

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Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

10.1101/2021.07.12.452099 ◽

2021 ◽

Author(s):

Moises Ernesto Romero ◽

Chunhong Long ◽

Daniel La Rocco ◽

Anusha Mysore Keerthi ◽

Dajun Xu ◽

...

Keyword(s):

Free Energy ◽

Rna Polymerase ◽

Active Site ◽

Energy Barrier ◽

Umbrella Sampling ◽

Dynamics Simulation ◽

Free Energy Barrier ◽

Base Stacking ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Analogue

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with the viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can bind comparatively stabilized to the viral RdRp active site, as it primarily forms base stacking with the template Uracil nucleotide (at +1), which is under freely fluctuations and supports a low free energy barrier of the RDV-TP insertion (~ 1.5 kcal/mol). In comparison, the corresponding natural substrate ATP binds to the RdRp active site in Watson-Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~ 2.6 kcal/mol), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be particularly stabilized by motif B-N691, S682, and motif F-K500 with the sugar, base, and the template backbone, respectively. Although the RDV-TP insertion can be hindered by motif-F R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif-A D623, respectively.

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Vitamin B12 may inhibit RNA-dependent-RNA polymerase activity of nsp12 from the COVID-19 Virus

10.35543/osf.io/p48fa ◽

2020 ◽

Cited By ~ 1

Author(s):

deepak t nair ◽

naveen narayanan

Keyword(s):

Rna Polymerase ◽

Vitamin B12 ◽

Active Site ◽

Protein A ◽

Homology Model ◽

Polymerase Activity ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Rna Polymerase Activity ◽

Approved Drugs

COVID-19 is the causative agent for the ongoing pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that Vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

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Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019276118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2019276118

Author(s):

Vibhor Mishra ◽

Jasleen Singh ◽

Feng Wang ◽

Yixiang Zhang ◽

Akihito Fukudome ◽

...

Keyword(s):

Rna Polymerase ◽

Active Site ◽

Rna Dependent Rna Polymerase ◽

Dna Viruses ◽

Selfish Genetic Elements ◽

Pol Iv ◽

Nuclear Rna ◽

Tightly Coupled ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV’s 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3′ ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

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Targeting SARS-CoV-2 RNA-dependent RNA polymerase: An in silico drug repurposing for COVID-19

F1000Research ◽

10.12688/f1000research.26359.1 ◽

2020 ◽

Vol 9 ◽

pp. 1166

Author(s):

Krishnaprasad Baby ◽

Swastika Maity ◽

Chetan H. Mehta ◽

Akhil Suresh ◽

Usha Y. Nayak ◽

...

Keyword(s):

Rna Polymerase ◽

Active Site ◽

In Silico ◽

Drug Repurposing ◽

Drug Binding ◽

Rna Dependent Rna Polymerase ◽

Target Proteins ◽

Dynamics Simulations ◽

Preclinical And Clinical Studies ◽

And Control

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), took more lives than combined epidemics of SARS, MERS, H1N1, and Ebola. Currently, the prevention and control of spread are the goals in COVID-19 management as there are no specific drugs to cure or vaccines available for prevention. Hence, the drug repurposing was explored by many research groups, and many target proteins have been examined. The major protease (Mpro), and RNA-dependent RNA polymerase (RdRp) are two target proteins in SARS-CoV-2 that have been validated and extensively studied for drug development in COVID-19. The RdRp shares a high degree of homology between those of two previously known coronaviruses, SARS-CoV and MERS-CoV. Methods: In this study, the FDA approved library of drugs were docked against the active site of RdRp using Schrodinger's computer-aided drug discovery tools for in silico drug-repurposing. Results: We have shortlisted 14 drugs from the Standard Precision docking and interaction-wise study of drug-binding with the active site on the enzyme. These drugs are antibiotics, NSAIDs, hypolipidemic, coagulant, thrombolytic, and anti-allergics. In molecular dynamics simulations, pitavastatin, ridogrel and rosoxacin displayed superior binding with the active site through ARG555 and divalent magnesium. Conclusion: Pitavastatin, ridogrel and rosoxacin can be further optimized in preclinical and clinical studies to determine their possible role in COVID-19 treatment.

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In Silico Identification of a Potent Arsenic Based Approved Drug Darinaparsin against SARS-CoV-2: Inhibitor of RNA dependent RNA polymerase (RdRp) and Necessary Proteases

10.26434/chemrxiv.12200495 ◽

2020 ◽

Cited By ~ 1

Author(s):

Trinath Chowdhury ◽

Gourisankar Roymahapatra ◽

Santi M Mandal

Keyword(s):

Rna Polymerase ◽

Active Site ◽

In Silico ◽

Significant Interaction ◽

Rna Dependent Rna Polymerase ◽

Docking Analysis ◽

Replicase Protein ◽

Arsenical Compounds ◽

Viral Replicase ◽

In Silico Identification

The work demonstrate screening of several arsenical compounds against RdRp of coronavirus. The study implies out of all arsenical compounds, darinaparsin shows its most effective results based on <i>in silico</i> docking analysis. This study also confirmed the significant interaction between the active site of viral replicase protein, endoribonuclease protein and different proteases with darinaparsin.

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