scholarly journals Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase

2020 ◽  
Vol 65 (1) ◽  
pp. e01508-20
Author(s):  
Gaofei Lu ◽  
Xi Zhang ◽  
Weinan Zheng ◽  
Jialei Sun ◽  
Lan Hua ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antiviral Treatment ◽  
Screening Method ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Vitro Assay ◽  
Assay Conditions ◽  
Incorporation Efficiency

ABSTRACTNucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

2020 ◽  
Author(s):  
Gaofei Lu ◽  
Xi Zhang ◽  
Weinan Zheng ◽  
Jialei Sun ◽  
Lan Hua ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mechanism Of Action ◽  
Antiviral Treatment ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Vitro Assay ◽  
Nucleotide Analog ◽  
Incorporation Efficiency

AbstractNucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2’-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2’-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

Incorporation Efficiency and Inhibition Mechanism of 2’-Substituted Nucleotide Analogs against SARS-CoV-2 RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Congmin Yuan ◽  
Eshani C. Goonetilleke ◽  
Ilona Christy Unarta ◽  
Xuhui Huang
Keyword(s):  
Rna Polymerase ◽  
Inhibition Mechanism ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Common Group ◽  
Incorporation Efficiency

The ongoing pandemic caused by SARS-CoV-2 emphasizes the need for effective therapeutics. Inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by nucleotide analogs provides a promising antiviral strategy. One common group...

scholarly journals Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

2020 ◽  
Author(s):  
Jack PK Bravo ◽  
Tyler L Dangerfield ◽  
David W Taylor ◽  
Kenneth A Johnson
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rational Design ◽  
Antiviral Drugs ◽  
Chain Termination ◽  
Nucleoside Analog ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

scholarly journals Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution

2007 ◽  
Vol 81 (9) ◽  
pp. 4753-4765 ◽  
Author(s):  
Thai Leong Yap ◽  
Ting Xu ◽  
Yen-Liang Chen ◽  
Helene Malet ◽  
Marie-Pierre Egloff ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Catalytic Domain ◽  
Crystallographic Structure ◽  
Transferase Activity ◽  
Human Pathogens ◽  
Rna Dependent Rna Polymerase ◽  
Vitro Assay ◽  
Positive Strand Rna

ABSTRACT Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.

scholarly journals RNA ligands activate the Machupo virus polymerase and guide promoter usage

2019 ◽  
Vol 116 (21) ◽  
pp. 10518-10524 ◽  
Author(s):  
Jesse D. Pyle ◽  
Sean P. J. Whelan
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Rna Virus ◽  
Binding Pocket ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Vitro Assay ◽  
Guanine Residue ◽  
Rna Ligands

Segmented negative-sense (SNS) RNA viruses initiate infection by delivering into cells a suite of genomic RNA segments, each sheathed by the viral nucleocapsid protein and bound by the RNA-dependent RNA-polymerase (RdRP). For the orthomyxovirus influenza and the bunyavirus La Crosse, the 5′ end of the genomic RNA binds as a hook-like structure proximal to the active site of the RdRP. Using an in vitro assay for the RNA-dependent RNA-polymerase (RdRP) of the arenavirus Machupo (MACV), we demonstrate that the 5′ genomic and antigenomic RNAs of both small and large genome segments stimulate activity in a promoter-specific manner. Functional probing of the activating RNAs identifies intramolecular base-pairing between positions +1 and +7 and a pseudotemplated 5′ terminal guanine residue as key for activation. Binding of structured 5′ RNAs is a conserved feature of all SNS RNA virus polymerases, implying that promoter-specific RdRP activation extends beyond the arenaviruses. The 5′ RNAs and the RNA binding pocket itself represent targets for therapeutic intervention.

scholarly journals Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Shuli Mao ◽  
Michael Hager ◽  
Bharat P. Gurale ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Mitochondrial Rna ◽  
Mitochondrial Toxicity ◽  
Simple Method ◽  
Vitro Assay ◽  
Human Mitochondrial Dna ◽  
Toxic Compound ◽  
Incorporation Efficiency

ABSTRACT There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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