scholarly journals Fidelity of Nucleotide Incorporation by the RNA-Dependent RNA Polymerase from Poliovirus

2016 ◽  
pp. 293-323 ◽  
Author(s):  
C.E. Cameron ◽  
I.M. Moustafa ◽  
J.J. Arnold
Keyword(s):  
Rna Polymerase ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Incorporation

scholarly journals Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 326 ◽  
Author(s):  
Egor Tchesnokov ◽  
Joy Feng ◽  
Danielle Porter ◽  
Matthias Götte
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Virus Disease ◽  
Chain Termination ◽  
Mitochondrial Rna ◽  
Long Distance ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogue ◽  
Nucleotide Incorporation

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

scholarly journals Vaccine-derived Mutation in Motif D of Poliovirus RNA-dependent RNA Polymerase Lowers Nucleotide Incorporation Fidelity

2013 ◽  
Vol 288 (45) ◽  
pp. 32753-32765 ◽  
Author(s):  
Xinran Liu ◽  
Xiaorong Yang ◽  
Cheri A. Lee ◽  
Ibrahim M. Moustafa ◽  
Eric D. Smidansky ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Incorporation

scholarly journals RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

2020 ◽  
Author(s):  
Juan Aranda ◽  
Modesto Orozco
Keyword(s):  
Rna Polymerase ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Nitrile Group ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Incorporation ◽  
Mechanism Of Reaction ◽  
Combine Sequence ◽  
Kinetic Barriers ◽  
Inhibition Analysis

We combine sequence analysis, molecular dynamics and hybrid quantum mechanics/molecular mechanics simulations to obtain the first description of the mechanism of reaction of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and of the inhibition of the enzyme by Remdesivir. Despite its evolutionary youth, the enzyme is highly optimized to have good fidelity in nucleotide incorporation and a good catalytic efficiency. Our simulations strongly suggest that Remdesivir triphosphate (the active form of drug) is incorporated into the nascent RNA replacing ATP, leading to a duplex RNA which is structurally very similar to an unmodified one. We did not detect any reason to explain the inhibitory activity of Remdesivir at the active site. Displacement of the nascent Remdesivir-containing RNA duplex along the exit channel of the enzyme can occur without evident steric clashes which would justify delayed inhibition. However, after the incorporation of three more nucleotides we found a hydrated Serine which is placed in a perfect arrangement to react through a Pinner’s reaction with the nitrile group of Remdesivir. Kinetic barriers for crosslinking and polymerization are similar suggesting a competition between polymerization and inhibition. Analysis of SARS-CoV-2 mutational landscape and structural analysis of polymerases across different species support the proposed mechanism and suggest that virus has not explored yet resistance to Remdesivir inhibition.

Sign in / Sign up

Export Citation Format

Share Document