scholarly journals A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

2003 ◽  
Vol 100 (12) ◽  
pp. 7289-7294 ◽  
Author(s):  
J. K. Pfeiffer ◽  
K. Kirkegaard
Keyword(s):  
Rna Polymerase ◽  
Single Mutation ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs

scholarly journals Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

2020 ◽  
Author(s):  
Jialei Sun
Keyword(s):  
Rna Polymerase ◽  
Polymerase Activity ◽  
Inhibitory Effects ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Dependent Inhibition ◽  
Catalytic Metal ◽  
Dose Dependent Inhibition ◽  
Dose Dependent ◽  
Low Activity

AbstractThe outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+ or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+ promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.

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 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.

scholarly journals Resistance Profile of a Hepatitis C Virus RNA-Dependent RNA Polymerase Benzothiadiazine Inhibitor

2003 ◽  
Vol 47 (11) ◽  
pp. 3525-3530 ◽  
Author(s):  
Tammy T. Nguyen ◽  
Adam T. Gates ◽  
Lester L. Gutshall ◽  
Victor K. Johnston ◽  
Baohua Gu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Single Point ◽  
Point Mutations ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Single Mutation ◽  
Rna Dependent Rna Polymerase ◽  
Resistance Profile

ABSTRACT Recently, a benzo-1,2,4-thiadiazine antiviral agent (C21H21N3O4S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 μM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

scholarly journals A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase

2021 ◽  
Author(s):  
Hui Wu ◽  
Ting Wei ◽  
Rui Cheng ◽  
Fengtao Huang ◽  
Xuelin Lu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Complex Structure ◽  
T7 Rna Polymerase ◽  
Prototype Model ◽  
Single Mutation ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop

ABSTRACTTranscription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) was known to response to two types of termination signals, while the mechanism underlying such termination especially the specific elements of the polymerase involved in is still unclear, due to the lack of a termination complex structure. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation S43Y that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNA polymerase (RdRp) of T7 RNAP without affecting the major DNA-dependent RNA polymerase (DdRp) activity of the enzyme, indicating the relationship between transcription termination and RdRp activity, and suggesting a model in which the stem-loop terminator induces the RdRp activity which competes with the ongoing DdRp activity to cause transcription termination. The T7 RNAP S43Y mutant as an enzymatic reagent for in vitro transcription reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

scholarly journals Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

2000 ◽  
Vol 74 (22) ◽  
pp. 10312-10322 ◽  
Author(s):  
Min-Ju Kim ◽  
Weidong Zhong ◽  
Zhi Hong ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Direct Synthesis ◽  
Amino Group ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Diarrhea Virus ◽  
Base Analog ◽  
Nascent Rna

ABSTRACT The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3′ end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1–7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2′-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2′-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2′-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.

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