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>

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 Exploring the Conformational Dynamics of RNA Dependent RNA Polymerase of SARS-CoV-2 in the Presence of Various Nucleotide Analogues

2020 ◽  
Author(s):  
Shruti Koulgi ◽  
Vinod Jani ◽  
Mallikarjunachari Uppuladinne V N ◽  
Uddhavesh Sonavane ◽  
Rajendra Joshi
Keyword(s):  
Rna Polymerase ◽  
Conformational Dynamics ◽  
Principal Component ◽  
Inhibitory Effect ◽  
New Drugs ◽  
Strong Interactions ◽  
Binding Motif ◽  
Entry Site ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogues

<p>RNA dependent RNA polymerase (RdRP) from positive stranded RNA viruses has always been a hot target for designing of new drugs as it is responsible for viral replication. The major class of drugs that are targeted against RdRP are nucleotide analogues. An extensive docking and molecular dynamics study describing the role of natural nucleotides (NTPs) and its analogues in imparting an inhibitory effect on the RdRP has been presented here. RdRP simulations in its apo, NTP-bound and analogue-bound form have been performed for a cumulative time of 1.9 μs. The conformational flexibility of the RdRP molecule was explored using Principal Component Analysis (PCA) and Markov State Modeling (MSM) Analysis. PCA inferred the presence of correlated motions along the conserved motifs of the RdRP. The ligand binding motif F and template binding motif G showed motions that are negatively correlated with one another. LYS 551, ARG 553 and ARG 555 which are a part of the motif F appear to form strong interactions with the ligand molecules. ARG 836, a primer binding residue was observed to strongly bind to the nucleotide analogues. The MSM analysis helped to observe different conformational states explored by the RdRP. The ensemble docking of the ligands on the Markov states suggested the involvement of the above residues in ligand interactions. The Markov states obtained clearly demarcated the open and closed conformations. The closed states were observed to have more favorable docking of the ligands. MSM analysis predicted a probable inhibitory mechanism involving the closing of the template entry site by reduction in the distance between the flanking finger and thumb subdomain. </p>

scholarly journals Exploring the Conformational Dynamics of RNA Dependent RNA Polymerase of SARS-CoV-2 in the Presence of Various Nucleotide Analogues

2020 ◽  
Author(s):  
Shruti Koulgi ◽  
Vinod Jani ◽  
Mallikarjunachari Uppuladinne V N ◽  
Uddhavesh Sonavane ◽  
Rajendra Joshi
Keyword(s):  
Rna Polymerase ◽  
Conformational Dynamics ◽  
Principal Component ◽  
Inhibitory Effect ◽  
New Drugs ◽  
Strong Interactions ◽  
Binding Motif ◽  
Entry Site ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogues

<p>RNA dependent RNA polymerase (RdRP) from positive stranded RNA viruses has always been a hot target for designing of new drugs as it is responsible for viral replication. The major class of drugs that are targeted against RdRP are nucleotide analogues. An extensive docking and molecular dynamics study describing the role of natural nucleotides (NTPs) and its analogues in imparting an inhibitory effect on the RdRP has been presented here. RdRP simulations in its apo, NTP-bound and analogue-bound form have been performed for a cumulative time of 1.9 μs. The conformational flexibility of the RdRP molecule was explored using Principal Component Analysis (PCA) and Markov State Modeling (MSM) Analysis. PCA inferred the presence of correlated motions along the conserved motifs of the RdRP. The ligand binding motif F and template binding motif G showed motions that are negatively correlated with one another. LYS 551, ARG 553 and ARG 555 which are a part of the motif F appear to form strong interactions with the ligand molecules. ARG 836, a primer binding residue was observed to strongly bind to the nucleotide analogues. The MSM analysis helped to observe different conformational states explored by the RdRP. The ensemble docking of the ligands on the Markov states suggested the involvement of the above residues in ligand interactions. The Markov states obtained clearly demarcated the open and closed conformations. The closed states were observed to have more favorable docking of the ligands. MSM analysis predicted a probable inhibitory mechanism involving the closing of the template entry site by reduction in the distance between the flanking finger and thumb subdomain. </p>

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 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.

scholarly journals Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2272 ◽  
Author(s):  
Ming-Yue Ma ◽  
Xin-Ran Lan ◽  
Deng-Ke Niu
Keyword(s):  
Rna Polymerase ◽  
Tandem Duplication ◽  
Donor Site ◽  
Intron Gain ◽  
Rna Dependent Rna Polymerase ◽  
Gene Encoding ◽  
Spliceosomal Introns ◽  
Branch Site ◽  
Eukaryotic Gene ◽  
Genomic Duplication

The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In anRNA-dependent RNA polymerase(RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among otherSolanumplants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals.

scholarly journals Ivermectin as a promising RNA-dependent RNA polymerase inhibitor and a therapeutic drug against SARS-CoV2: Evidence from in silico studies

2020 ◽  
Author(s):  
Ananta Swargiary
Keyword(s):  
Rna Polymerase ◽  
3D Structure ◽  
Antiviral Drug ◽  
Strong Interactions ◽  
Therapeutic Drug ◽  
Binding Property ◽  
Binding Interaction ◽  
Rna Dependent Rna Polymerase ◽  
Potential Inhibitor ◽  
In Silico Studies

Abstract Purpose: COVID-19, caused by SARS-CoV2 virus is a contagious disease affecting millions of lives throughout the globe. Currently, there are no clinically approved drugs for SARS-CoV2 although some drugs are undergoing clinical trials. The present study investigates the binding property of ivermectin on four important drug targets, spike protein, RNA-dependent RNA polymerase, 3-chymotrypsin- and papain-like proteases of SARS-CoV2. Methods: The 3D structure of ivermectin along with known antiviral drug lopinavir, simeprevir and four nucleotides ATP, GTP, CTP, and UTP were downloaded from PubChem database. Crystal structures of proteins were downloaded from PDB database. PDB files were converted into pdbqt file using AutoDock tools. After proper processing and grid formation, docking was carried out in AutoDock vina. Furthermore, the co-crystallized RNA and its binding interactions with RdRp were studied using various visualization tools including Discovery studio.Results: Docking study showed that ivermectin is the best binding drug compared to lopinavir and simeprevir. The best binding interaction was found to be -9.7kcal/mol with RdRp suggesting potential inhibitor of the protein. Twenty-one amino acid residues of RdRp were found to interact with ivermectin including the catalytic residue Asp760. Furthermore, RNA-RdRp complex revealed that the catalytic active residues Ser759 and Asp760 of RdRp formed strong interactions with RNA chain. Binding of ivermectin in the active site of RdRp make clash with the nucleotides of RNA chain suggesting the possible inhibition of replication.Conclusions: The present study suggests ivermectin as a potential inhibitor of RdRp which may be crucial to combat the SARS-CoV2.

scholarly journals Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Philipp Schult ◽  
Maren Nattermann ◽  
Chris Lauber ◽  
Stefan Seitz ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Rna Dependent Rna Polymerase ◽  
Internal Initiation ◽  
Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

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