scholarly journals Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

2017 ◽  
Vol 115 (2) ◽  
pp. E162-E171 ◽  
Author(s):  
François Ferron ◽  
Lorenzo Subissi ◽  
Ana Theresa Silveira De Morais ◽  
Nhung Thi Tuyet Le ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Mutation Rates ◽  
Bifunctional Enzyme ◽  
Antiviral Compound ◽  
Mismatch Correction ◽  
Limited Efficacy

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

scholarly journals Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus

2005 ◽  
Vol 79 (15) ◽  
pp. 9777-9785 ◽  
Author(s):  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Dual Function ◽  
Long Distance ◽  
Replication Assay ◽  
Rna Interaction ◽  
Stimulation Of

ABSTRACT Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

scholarly journals Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

2014 ◽  
Vol 89 (1) ◽  
pp. 275-286 ◽  
Author(s):  
Grace Campagnola ◽  
Seth McDonald ◽  
Stéphanie Beaucourt ◽  
Marco Vignuzzi ◽  
Olve B. Peersen
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Mutation Rates ◽  
Rapid Adaptation ◽  
Replication Fidelity ◽  
Virus Growth ◽  
Polymerase Structure ◽  
Positive Strand Rna

ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

scholarly journals Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

2001 ◽  
Vol 75 (22) ◽  
pp. 11088-11095 ◽  
Author(s):  
Hongyan Yang ◽  
Eugene V. Makeyev ◽  
Dennis H. Bamford
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Regulatory Mechanism ◽  
Biochemical Properties ◽  
Double Stranded Rna ◽  
The Family ◽  
Dsrna Viruses ◽  
Template Specificity

ABSTRACT The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription inCystoviridae is controlled by the template specificity of the polymerase subunit.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Plasmodium vivax and Drug Resistance

2021 ◽  
Author(s):  
Puji Budi Setia Asih ◽  
Din Syafruddin
Keyword(s):  
Drug Resistance ◽  
Molecular Basis ◽  
Indirect Evidence ◽  
Antimalarial Drugs ◽  
In Vivo Studies ◽  
In Vitro Cultivation ◽  
Radical Cure ◽  
The Status

Resistance to antimalarial drugs is a threat to global efforts to eliminate malaria by 2030. Currently, treatment for vivax malaria uses chloroquine or ACT for uncomplicated P. vivax whereas primaquine is given to eliminate latent liver stage infections (a method known as radical cure). Studies on P. vivax resistance to antimalarials and the molecular basis of resistance lags far behind the P. falciparum as in vitro cultivation of the P. vivax has not yet been established. Therefore, data on the P. vivax resistance to any antimalarial drugs are generated through in vivo studies or through monitoring of antimalarial treatments in mixed species infection. Indirect evidence through drug selective pressure on the parasites genome, as evidenced by the presence of the molecular marker(s) for drug resistance in areas where P. falciparum and P. vivax are distributed in sympatry may reflect, although require validation, the status of P. vivax resistance. This review focuses on the currently available data that may represent the state-of-the art of the P. vivax resistance status to antimalarial to anticipate the challenge for malaria elimination by 2030.

scholarly journals Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

2020 ◽  
Author(s):  
Florian Geiger ◽  
Guido Papa ◽  
William E. Arter ◽  
Julia Acker ◽  
Kadi L. Saar ◽  
...  
Keyword(s):  
Phase Separation ◽  
Unique Feature ◽  
Rna Viruses ◽  
Therapeutic Approaches ◽  
Subcellular Organelles ◽  
Selective Enrichment ◽  
Novel Therapeutic ◽  
Liquid Liquid Phase Separation

AbstractRNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

scholarly journals Thioredoxin 2 Negatively Regulates Innate Immunity to RNA Viruses by Disrupting the Assembly of the Virus-Induced Signaling Adaptor Complex

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Dan Li ◽  
Wenping Yang ◽  
Yi Ru ◽  
Jingjing Ren ◽  
Xiangtao Liu ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Viruses ◽  
Critical Role ◽  
Type I Interferons ◽  
Type I ◽  
Host Proteins ◽  
Innate Antiviral Response ◽  
Thioredoxin 2

ABSTRACT The virus-induced signaling adaptor (VISA) complex plays a critical role in the innate immune response to RNA viruses. However, the mechanism of VISA complex formation remains unclear. Here, we demonstrate that thioredoxin 2 (TRX2) interacts with VISA at mitochondria both in vivo and in vitro. Knockdown and knockout of TRX2 enhanced the formation of the VISA-associated complex, as well as virus-triggered activation of interferon regulatory factor 3 (IRF3) and transcription of the interferon beta 1 (IFNB1) gene. TRX2 inhibits the formation of VISA aggregates by repressing reactive oxygen species (ROS) production, thereby disrupting the assembly of the VISA complex. Furthermore, our data suggest that the C93 residue of TRX2 is essential for inhibition of VISA aggregation, whereas the C283 residue of VISA is required for VISA aggregation. Collectively, these findings uncover a novel mechanism of TRX2 that negatively regulates VISA complex formation. IMPORTANCE The VISA-associated complex plays pivotal roles in inducing type I interferons (IFNs) and eliciting the innate antiviral response. Many host proteins are identified as VISA-associated-complex proteins, but how VISA complex formation is regulated by host proteins remains enigmatic. We identified the TRX2 protein as an important regulator of VISA complex formation. Knockout of TRX2 increases virus- or poly(I·C)-triggered induction of type I IFNs at the VISA level. Mechanistically, TRX2 inhibits the production of ROS at its C93 site, which impairs VISA aggregates at its C283 site, and subsequently impedes the assembly of the VISA complex. Our findings suggest that TRX2 plays an important role in the regulation of VISA complex assembly.

scholarly journals Polyamine Depletion Abrogates Enterovirus Cellular Attachment

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Thomas M. Kicmal ◽  
Patrick M. Tate ◽  
Courtney N. Dial ◽  
Jeremy J. Esin ◽  
Bryan C. Mounce
Keyword(s):  
Rna Viruses ◽  
Severe Disease ◽  
Coxsackievirus B3 ◽  
Human Pathogens ◽  
Polyamine Biosynthesis ◽  
Virus Attachment ◽  
Significant Public Health ◽  
Approved Drugs

ABSTRACT Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells treated with FDA-approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate nucleic acid packaging, transcription, and translation, but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to the depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential proviral role for polyamines using a forward screen. Passaging CVB3 in polyamine-depleted cells generated a mutation in capsid protein VP3 at residue 234. We show that this mutation confers resistance to polyamine depletion. Through attachment assays, we demonstrate that polyamine depletion limits CVB3 attachment to susceptible cells, which is rescued by incubating virus with polyamines. Furthermore, the capsid mutant rescues this inhibition in polyamine-depleted cells. More divergent viruses also exhibited reduced attachment to polyamine-depleted cells, suggesting that polyamines may facilitate attachment of diverse RNA viruses. These studies inform additional mechanisms of action for polyamine-depleting pharmaceuticals, with implications for potential antiviral therapies. IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease. These viruses rely on polyamines, small positively charged molecules, for robust replication, and polyamine depletion limits infection in vitro and in vivo. The mechanisms by which polyamines enhance enteroviral replication are unknown. Here, we describe how Coxsackievirus B3 (CVB3) utilizes polyamines to attach to susceptible cells and initiate infection. Using a forward genetic screen, we identified a mutation in a receptor-binding amino acid that promotes infection of polyamine-depleted cells. These data suggest that pharmacologically inhibiting polyamine biosynthesis may combat virus infection by preventing virus attachment to susceptible cells.

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