scholarly journals Concentration of 2’C-methyladenosine triphosphate byLeishmania guyanensisenables specific inhibition ofLeishmaniaRNA virus 1 via its RNA polymerase

2018 ◽  
Author(s):  
John I. Robinson ◽  
Stephen M. Beverley
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Protozoan Parasite ◽  
Cesium Chloride ◽  
Rdrp Activity ◽  
Cellular Mechanisms ◽  
The Impact ◽  
Leishmania Guyanensis ◽  
Good Agreement

AbstractLeishmaniais a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virusLeishmaniaRNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2’-C-methyladenosine (2CMA) potently inhibited LRV1 inLeishmania guyanensis(Lgy) andL. braziliensis, leading to viral eradication at concentrations above 10 µM. Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation and inhibition of the viral RNA dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMATP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50s ranging from 150 to 910 µM, depending on the CsCl density of the virion (empty, ssRNA- and dsRNA-containing).Lgyparasites incubatedin vitrowith 10 µM 2CMA accumulated 2CMA-TP to 410 µM, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

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

2021 ◽  
Vol 478 (13) ◽  
pp. 2425-2443 ◽  
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.

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

2002 ◽  
Vol 76 (4) ◽  
pp. 1707-1717 ◽  
Author(s):  
K. S. Rajendran ◽  
J. Pogany ◽  
P. D Nagy
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
De Novo ◽  
Rna Virus ◽  
In Vitro Assays ◽  
Turnip Crinkle Virus ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

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.

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.

A Mechanistic Motor-Clutch Model That Explains Cell Shape Dynamics to Cyclic Stretch

2022 ◽  
Author(s):  
Benjamin W. Scandling ◽  
Jia Gou ◽  
Jessica Thomas ◽  
Jacqueline Xuan ◽  
Chuan Xue ◽  
...  
Keyword(s):  
Computational Model ◽  
Computational Models ◽  
The Body ◽  
Cyclic Stretch ◽  
Substrate Stiffness ◽  
Cell Alignment ◽  
Cellular Mechanisms ◽  
Actin Bundles ◽  
The Impact

Many cells in the body experience cyclic mechanical loading, which can impact cellular processes and morphology. In vitro studies often report that cells reorient in response to cyclic stretch of their substrate. To explore cellular mechanisms involved in this reorientation, a computational model was developed by utilizing the previous computational models of the actin-myosin-integrin motor-clutch system developed by others. The computational model predicts that under most conditions, actin bundles align perpendicular to the direction of applied cyclic stretch, but under specific conditions, such as low substrate stiffness, actin bundles align parallel to the direction of stretch. The model also predicts that stretch frequency impacts the rate of reorientation, and that proper myosin function is critical in the reorientation response. These computational predictions are consistent with reports from the literature and new experimental results presented here. The model suggests that the impact of different stretching conditions (stretch type, amplitude, frequency, substrate stiffness, etc.) on the direction of cell alignment can largely be understood by considering their impact on cell-substrate detachment events, specifically whether detachment occurs during stretching or relaxing of the substrate.

scholarly journals Genome Characterization, Prevalence, and Transmission Mode of a Novel Picornavirus Associated with the Threespine Stickleback Fish (Gasterosteus aculeatus)

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Megan A. Hahn ◽  
Nolwenn M. Dheilly
Keyword(s):  
Gasterosteus Aculeatus ◽  
Rna Virus ◽  
Wide Distribution ◽  
Model System ◽  
Threespine Stickleback ◽  
Tissue Samples ◽  
Content Type ◽  
Threespine Sticklebacks ◽  
The Impact

ABSTRACT The complete genome sequence of an RNA virus was assembled from RNA sequencing of virus particles purified from threespine stickleback intestine tissue samples. This new virus is most closely related to the Eel picornavirus and can be assigned to the genus Potamipivirus in the family Picornaviridae. Its unique genetic properties are enough to establish a new species, dubbed the Threespine Stickleback picornavirus (TSPV). Due to their broad geographic distribution throughout the Northern Hemisphere and parallel adaptation to freshwater, threespine sticklebacks have become a model in evolutionary ecology. Further analysis using diagnostic PCRs revealed that TSPV is highly prevalent in both anadromous and freshwater populations of threespine sticklebacks, infects almost all fish tissues, and is transmitted vertically to offspring obtained from in vitro fertilization in laboratory settings. Finally, TSPV was found in Sequence Reads Archives of transcriptome of Gasterosteus aculeatus, further demonstrating its wide distribution and unsought prevalence in samples. It is thus necessary to test the impact of TSPV on the biology of threespine sticklebacks, as this widespread virus could interfere with the behavioral, physiological, or immunological studies that employ this fish as a model system. IMPORTANCE The threespine stickleback species complex is an important model system in ecological and evolutionary studies because of the large number of isolated divergent populations that are experimentally tractable. For similar reasons, its coevolution with the cestode parasite Schistocephalus solidus, its interaction with gut microbes, and the evolution of its immune system are of growing interest. Herein we describe the discovery of an RNA virus that infects both freshwater and anadromous populations of sticklebacks. We show that the virus is transmitted vertically in laboratory settings and found it in Sequence Reads Archives, suggesting that experiments using sticklebacks were conducted in the presence of the virus. This discovery can serve as a reminder that the presence of viruses in wild-caught animals is possible, even when animals appear healthy. Regarding threespine sticklebacks, the impact of Threespine Stickleback picornavirus (TSPV) on the fish biology should be investigated further to ensure that it does not interfere with experimental results.

scholarly journals Network feedback regulates motor output across a range of modulatory neuron activity

2016 ◽  
Vol 115 (6) ◽  
pp. 3249-3263 ◽  
Author(s):  
Robert M. Spencer ◽  
Dawn M. Blitz
Keyword(s):  
Neuron Activity ◽  
Projection Neurons ◽  
Activity Levels ◽  
Cellular Mechanisms ◽  
Physiological Range ◽  
Network Activation ◽  
Network Output ◽  
The Impact

Modulatory projection neurons alter network neuron synaptic and intrinsic properties to elicit multiple different outputs. Sensory and other inputs elicit a range of modulatory neuron activity that is further shaped by network feedback, yet little is known regarding how the impact of network feedback on modulatory neurons regulates network output across a physiological range of modulatory neuron activity. Identified network neurons, a fully described connectome, and a well-characterized, identified modulatory projection neuron enabled us to address this issue in the crab ( Cancer borealis) stomatogastric nervous system. The modulatory neuron modulatory commissural neuron 1 (MCN1) activates and modulates two networks that generate rhythms via different cellular mechanisms and at distinct frequencies. MCN1 is activated at rates of 5–35 Hz in vivo and in vitro. Additionally, network feedback elicits MCN1 activity time-locked to motor activity. We asked how network activation, rhythm speed, and neuron activity levels are regulated by the presence or absence of network feedback across a physiological range of MCN1 activity rates. There were both similarities and differences in responses of the two networks to MCN1 activity. Many parameters in both networks were sensitive to network feedback effects on MCN1 activity. However, for most parameters, MCN1 activity rate did not determine the extent to which network output was altered by the addition of network feedback. These data demonstrate that the influence of network feedback on modulatory neuron activity is an important determinant of network output and feedback can be effective in shaping network output regardless of the extent of network modulation.

scholarly journals Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases

2015 ◽  
Vol 59 (12) ◽  
pp. 7504-7516 ◽  
Author(s):  
Zhinan Jin ◽  
Kathryn Tucker ◽  
Xiaoyan Lin ◽  
C. Cheng Kao ◽  
Ken Shaw ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mechanism Of Action ◽  
Rna Virus ◽  
Nucleoside Analogs ◽  
Effective Vaccine ◽  
Mitochondrial Rna ◽  
Biochemical Evaluation ◽  
Ribonucleoside Triphosphate ◽  
Human And Mouse

ABSTRACTNorovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replicationin vitroand in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

scholarly journals Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Purification and Enzymatic Analysis of the RNA-Dependent RNA Polymerase 3Dpol

2001 ◽  
Vol 75 (22) ◽  
pp. 10969-10978 ◽  
Author(s):  
Kinga Gerber ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Biochemical Properties ◽  
Human Rhinovirus ◽  
Hydroxyl Group ◽  
Genetic Studies ◽  
Vitro Assay ◽  
Enzyme Preparations ◽  
Synthetic Reactions

ABSTRACT The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathioneS-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3Dpol. Using in vitro assay systems previously described for poliovirus RNA polymerase 3Dpol(J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677–3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280–284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3Dpol is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn2+, and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)15 primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5′ end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

scholarly journals Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6

1998 ◽  
Vol 72 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Anja O. Paatero ◽  
Leonard Mindich ◽  
Dennis H. Bamford
Keyword(s):  
Rna Polymerase ◽  
Mutational Analysis ◽  
Rna Virus ◽  
Nucleoside Triphosphate ◽  
Particle Assembly ◽  
Polymerase Complex ◽  
Translocation Event ◽  
Shell Forming ◽  
Nucleoside Triphosphatase

ABSTRACT Bacteriophage φ6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.

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