scholarly journals Isolation of reconstructed functional ribonucleoprotein complexes of Machupo virus

2021 ◽  
Author(s):  
Jesse D. Pyle ◽  
Sean P. J. Whelan
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Severe Disease ◽  
Host Cells ◽  
Genomic Rna ◽  
Replication Machinery ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Ribonucleoprotein Complexes ◽  
Machupo Virus

Arenaviruses initiate infection by delivering a transcriptionally-competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (vRNA) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA, affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodisperse L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase – which copies the viral genome segments and synthesizes all four viral mRNAs – bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery, and thus advancing antiviral strategies targeting this essential complex.

scholarly journals Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Nidhi Kaushik ◽  
Chandru Subramani ◽  
Saumya Anang ◽  
Rajagopalan Muthumohan ◽  
Shalimar ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Hepatitis E Virus ◽  
Zinc Supplementation ◽  
Hepatitis E ◽  
Viral Rna ◽  
Health Concern ◽  
Healthy Individuals ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Salts

ABSTRACT Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

scholarly journals RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery

2020 ◽  
Vol 25 (10) ◽  
pp. 1141-1151 ◽  
Author(s):  
Wei Zhu ◽  
Catherine Z. Chen ◽  
Kirill Gorshkov ◽  
Miao Xu ◽  
Donald C. Lo ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Host Cells ◽  
Host Proteins ◽  
Rna Dependent Rna Polymerase ◽  
Global Health Issue ◽  
Repurposed Drugs ◽  
Viral Enzyme ◽  
Target Effects

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

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 The structure of a dimeric form of SARS-CoV-2 polymerase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Florian A. Jochheim ◽  
Dimitry Tegunov ◽  
Hauke S. Hillen ◽  
Jana Schmitzová ◽  
Goran Kokic ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Catalytic Subunit ◽  
Template Switching ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Dimeric Form

AbstractThe coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.

Potential Candidates against COVID-19 Targeting RNA-Dependent RNA Polymerase: A Comprehensive Review

2021 ◽  
Vol 22 ◽  
Author(s):  
Neetu Agrawal ◽  
Ahsas Goyal
Keyword(s):  
Rna Polymerase ◽  
In Silico ◽  
Host Cells ◽  
Rna Dependent Rna Polymerase ◽  
In Vivo Experiments ◽  
Contagious Nature ◽  
In Silico Studies ◽  
Viral Enzyme

: Due to the extremely contagious nature of SARS-COV-2, it presents a significant threat to humans worldwide. A plethora of studies are going on all over the world to discover the drug to fight SARS-COV-2. One of the most promising targets is RNA-dependent RNA polymerase (RdRp), responsible for viral RNA replication in host cells. Since RdRp is a viral enzyme with no host cell homologs, it allows the development of selective SARS-COV-2 RdRp inhibitors. A variety of studies used in silico approaches for virtual screening, molecular docking, and repurposing of already existing drugs and phytochemicals against SARS-COV-2 RdRp. This review focuses on collating compounds possessing the potential to inhibit SARS-COV-2 RdRp based on in silico studies to give medicinal chemists food for thought so that the existing drugs can be repurposed for the control and treatment of ongoing COVID-19 pandemic after performing in vitro and in vivo experiments.

scholarly journals Genetic Interactions among the West Nile Virus Methyltransferase, the RNA-Dependent RNA Polymerase, and the 5′ Stem-Loop of Genomic RNA

2008 ◽  
Vol 82 (14) ◽  
pp. 7047-7058 ◽  
Author(s):  
Bo Zhang ◽  
Hongping Dong ◽  
Yangsheng Zhou ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Genetic Interactions ◽  
Genome Length ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
West Nile ◽  
Stem Loop ◽  
Compensatory Mutations

ABSTRACT Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m7GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA.

scholarly journals Nodamura Virus RNA Replication in Saccharomyces cerevisiae: Heterologous Gene Expression Allows Replication-Dependent Colony Formation

2005 ◽  
Vol 79 (1) ◽  
pp. 495-502 ◽  
Author(s):  
B. Duane Price ◽  
Lance D. Eckerle ◽  
L. Andrew Ball ◽  
Kyle L. Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Heterologous Gene Expression ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Genomic Rnas ◽  
Nodamura Virus

ABSTRACT Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 104-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.

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 Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Hayden Thomas Pacl ◽  
Jennifer L Tipper ◽  
Ritesh R Sevalkar ◽  
Andrew Crouse ◽  
Camerron Crowder ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Severe Disease ◽  
Water Soluble ◽  
Glycol Succinate ◽  
Viral Inhibition ◽  
Current Standard ◽  
Rna Dependent Rna Polymerase ◽  
Recent Emergence ◽  
Derivatives Of

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits. Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and β-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp. In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection.

scholarly journals RNA-dependent RNA polymerase complex of Brome mosaic virus: analysis of the molecular structure with monoclonal antibodies

2002 ◽  
Vol 83 (11) ◽  
pp. 2879-2890 ◽  
Author(s):  
Koji Dohi ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
Tetsuro Okuno
Keyword(s):  
Monoclonal Antibodies ◽  
Rna Polymerase ◽  
Mosaic Virus ◽  
Membrane Structure ◽  
Brome Mosaic Virus ◽  
Host Cells ◽  
Intermediate Region ◽  
Rna Dependent Rna Polymerase ◽  
Cellular Factors ◽  
Membrane Bound

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

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