Structural basis for active site closure by the poliovirus 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.

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

Journal of General Virology ◽

10.1099/vir.0.031104-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1607-1616 ◽

Cited By ~ 28

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.

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Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

Journal of Virology ◽

10.1128/jvi.00324-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 5

Author(s):

Weiwei Li ◽

Baixing Wu ◽

Wibowo Adian Soca ◽

Lei An

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Economic Losses ◽

Rna Dependent Rna Polymerase ◽

Content Type ◽

Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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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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Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

10.1101/2020.10.26.355099 ◽

2020 ◽

Author(s):

Pramod R. Bhatt ◽

Alain Scaiola ◽

Gary Loughran ◽

Marc Leibundgut ◽

Annika Kratzel ◽

...

Keyword(s):

Rna Polymerase ◽

Viral Proteins ◽

Viral Rna ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Ribosomal Frameshifting ◽

Infected Cells ◽

Rna Genome ◽

Exit Tunnel ◽

Viral Polyprotein

AbstractProgrammed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

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Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19

10.21203/rs.3.rs-99513/v1 ◽

2020 ◽

Author(s):

H. Eric Xu ◽

Wanchao Yin ◽

Xiaodong Luan ◽

Zhihai Li ◽

Leike Zhang ◽

...

Keyword(s):

Active Site ◽

Binding Sites ◽

Potent Inhibitor ◽

Structural Basis ◽

Rna Dependent Rna Polymerase ◽

Structural Mechanism ◽

Template Strand ◽

Viral Rdrp ◽

Biochemical Assays ◽

Catalytic Active Site

Abstract The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 Å cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19.

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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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