Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants

ABSTRACT Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.

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Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

10.1101/2021.04.07.438807 ◽

2021 ◽

Author(s):

Agustina P. Bertolin ◽

Florian Weissmann ◽

Jingkun Zeng ◽

Viktor Posse ◽

Jennifer C. Milligan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Virus ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Etiologic Agent ◽

Host Cells ◽

Rdrp Activity ◽

Chemical Library ◽

Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

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Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

Biochemical Journal ◽

10.1042/bcj20210200 ◽

2021 ◽

Vol 478 (13) ◽

pp. 2425-2443 ◽

Cited By ~ 1

Author(s):

Agustina P. Bertolin ◽

Florian Weissmann ◽

Jingkun Zeng ◽

Viktor Posse ◽

Jennifer C. Milligan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Virus ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Etiologic Agent ◽

Host Cells ◽

Rdrp Activity ◽

Chemical Library ◽

Rna Dependent Rna Polymerase

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication–transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

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Identification and Characterization of the Escherichia coli-Expressed RNA-Dependent RNA Polymerase of Bamboo Mosaic Virus

Journal of Virology ◽

10.1128/jvi.72.12.10093-10099.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10093-10099 ◽

Cited By ~ 59

Author(s):

Yi-Ija Li ◽

Yueh-Mei Cheng ◽

Yih-Leh Huang ◽

Ching-Hsiu Tsai ◽

Yau-Heiu Hsu ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Mosaic Virus ◽

Viral Protein ◽

Polymerase Activity ◽

Reaction Products ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Rna Fragments

ABSTRACT Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. The open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5′ end of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in Escherichia coli BL21(DE3) cells with thioredoxin, hexahistidine, and S · Tag fused consecutively at its amino terminus, and the fusion protein was purified by metal affinity chromatography. Several RNA fragments, prepared by in vitro transcription, were tested as substrates for the RNA-dependent RNA polymerase (RdRp) activity. Among them, the expressed fusion enzyme was able to generate a 32P-labeled RNA product when 3′-end RNA fragments of the positive strand or negative strand of BaMV were included in the assay mixture. Dot hybridization assay revealed that the reaction products are complementary to their RNA substrates. Taken together, the evidence suggests that the 155-kDa protein encoded by ORF1 of BaMV has an RdRp activity and should be involved in the replication of BaMV. Mutational analyses demonstrate the importance of the GDD motif in the polymerase activity, and deletion studies suggest that the polymerase activity resides in the carboxyl terminus of the 155-kDa viral protein.

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De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters

Journal of Virology ◽

10.1128/jvi.77.19.10730.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10730-10730 ◽

Cited By ~ 1

Author(s):

Masako Nomaguchi ◽

Matt Ackermann ◽

Changsuek Yon ◽

Shihyun You ◽

R. Padmanabhan

Keyword(s):

Dengue Virus ◽

Rna Polymerase ◽

De Novo ◽

De Novo Synthesis ◽

Rna Dependent Rna Polymerase ◽

Negative Strand ◽

Virus Rna

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01967-16 ◽

2016 ◽

Vol 61 (3) ◽

Cited By ~ 24

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.

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

Viruses ◽

10.3390/v13091738 ◽

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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De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.74.2.851-863.2000 ◽

2000 ◽

Vol 74 (2) ◽

pp. 851-863 ◽

Cited By ~ 217

Author(s):

Guangxiang Luo ◽

Robert K. Hamatake ◽

Danielle M. Mathis ◽

Jason Racela ◽

Karen L. Rigat ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Oligonucleotide Primer ◽

Transferase Activity ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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