scholarly journals Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

2016 ◽  
Vol 92 ◽  
pp. 26-30 ◽  
Author(s):  
Deeba Amraiz ◽  
Najam-us-Sahar Sadaf Zaidi ◽  
Munazza Fatima
Keyword(s):  
Rna Polymerase ◽  
Drug Testing ◽  
Antiviral Drug ◽  
Rna Dependent Rna Polymerase

scholarly journals A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability

2019 ◽  
Vol 48 (3) ◽  
pp. 1392-1405 ◽  
Author(s):  
Wei Shi ◽  
Han-Qing Ye ◽  
Cheng-Lin Deng ◽  
Rui Li ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Vaccine Development ◽  
Antiviral Drug ◽  
Enterovirus 71 ◽  
Binding Pocket ◽  
Genome Replication ◽  
Elongation Complex ◽  
Rna Dependent Rna Polymerase

Abstract The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5′-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.

scholarly journals A dynamic regulatory interface on SARS-CoV-2 RNA polymerase

2020 ◽  
Author(s):  
Wei Shi ◽  
Ming Chen ◽  
Yang Yang ◽  
Wei Zhou ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Center ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Genome Replication ◽  
Specific Interactions ◽  
Global Public Health ◽  
Rna Dependent Rna Polymerase ◽  
Viral Genome Replication

AbstractThe RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is the core machinery responsible for the viral genome replication and transcription and also a major antiviral target. Here we report the cryo-electron microscopy structure of a post-translocated SARS-CoV-2 RdRp core complex, comprising one nsp12, one separate nsp8(I) monomer, one nsp7-nsp8(II) subcomplex and a replicating RNA substrate. Compared with the recently reported SARS-CoV-2 RdRp complexes, the nsp8(I)/nsp7 interface in this RdRp complex shifts away from the nsp12 polymerase. Further functional characterizations suggest that specific interactions between the nsp8(I) and nsp7, together with the rearrangement of nsp8(I)/nsp7 interface, ensure the efficient and processive RNA synthesis by the RdRp complex. Our findings provide a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nsp12 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RdRp for antiviral design.Author summarySince it was first discovered and reported in late 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by highly contagious SARS-CoV-2 virus is wreaking havoc around the world. Currently, no highly effective and specific antiviral drug is available for clinical treatment. Therefore, the threat of COVID-19 transmission necessitates the discovery of more effective antiviral strategies. Viral RNA-dependent RNA polymerase (RdRp) is an important antiviral drug target. Here, our cryo-EM structure of a SARS-CoV-2 RdRp/RNA replicating complex reveals a previously uncharacterized overall shift of the cofactor nsp8(I)/nsp7 interface, leading to its rearrangement. Through in vitro functional test, we found that the specific interactions on the interface are important to the efficient RNA polymerase activity of SARS-CoV-2 RdRp. These observations let us to suggest this interface as a potential new drug intervention site, outside of the canonical polymerase active center, in RdRp for antiviral design. Our findings would provide new insights into regulatory mechanism of this novel SARS-CoV-2 RdRp, contribute to the design of antiviral drugs against SARS-CoV-2, and benefit the global public health.

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.

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

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.

scholarly journals Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

1999 ◽  
Vol 73 (1) ◽  
pp. 198-204 ◽  
Author(s):  
Scott Stevenson Stawicki ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Dna And Rna ◽  
In The Wild ◽  
Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

scholarly journals Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

Virology ◽  
2012 ◽  
Vol 427 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Greta A. Van Slyke ◽  
Alexander T. Ciota ◽  
Graham G. Willsey ◽  
Joachim Jaeger ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Point Mutations ◽  
Viral Fitness ◽  
Dependent Manner ◽  
The West ◽  
Rna Dependent Rna Polymerase ◽  
West Nile
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