Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication

2021 ◽  
Author(s):  
Sari Mattila ◽  
Pirjo Merilahti ◽  
Sarah Wazir ◽  
Tania Quirin ◽  
Mirko M. Maksimainen ◽  
...  
Keyword(s):  
Semliki Forest Virus ◽  
Rna Viruses ◽  
Virus Production ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Cells ◽  
Febrile Disease ◽  
Replication Stage ◽  
Replication Systems ◽  
Positive Strand Rna

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human MacroD1 protein inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression. The inhibitor was similarly active in alphavirus trans -replication systems, indicating that it targets the viral RNA replication stage.

scholarly journals Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2540
Author(s):  
Nicole Doyle ◽  
Jennifer Simpson ◽  
Philippa C. Hawes ◽  
Helena J. Maier
Keyword(s):  
Life Cycle ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Rna ◽  
Host Cells ◽  
Infectious Bronchitis ◽  
Welfare Issue ◽  
Membrane Bound ◽  
Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

scholarly journals Picornavirus RNA Recombination Counteracts Error Catastrophe

2019 ◽  
Vol 93 (14) ◽  
Author(s):  
Brian J. Kempf ◽  
Colleen L. Watkins ◽  
Olve B. Peersen ◽  
David J. Barton
Keyword(s):  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Common Mechanism ◽  
Negative Consequences ◽  
Rna Recombination ◽  
Viral Polymerase ◽  
Advantages And Disadvantages ◽  
Error Catastrophe ◽  
Positive Strand Rna

ABSTRACTTemplate-dependent RNA replication mechanisms render picornaviruses susceptible to error catastrophe, an overwhelming accumulation of mutations incompatible with viability. Viral RNA recombination, in theory, provides a mechanism for viruses to counteract error catastrophe. We tested this theory by exploiting well-defined mutations in the poliovirus RNA-dependent RNA polymerase (RDRP), namely, a G64S mutation and an L420A mutation. Our data reveal two distinct mechanisms by which picornaviral RDRPs influence error catastrophe: fidelity of RNA synthesis and RNA recombination. A G64S mutation increased the fidelity of the viral polymerase and rendered the virus resistant to ribavirin-induced error catastrophe, but only when RNA recombination was at wild-type levels. An L420A mutation in the viral polymerase inhibited RNA recombination and exacerbated ribavirin-induced error catastrophe. Furthermore, when RNA recombination was substantially reduced by an L420A mutation, a high-fidelity G64S polymerase failed to make the virus resistant to ribavirin. These data indicate that viral RNA recombination is required for poliovirus to evade ribavirin-induced error catastrophe. The conserved nature of L420 within RDRPs suggests that RNA recombination is a common mechanism for picornaviruses to counteract and avoid error catastrophe.IMPORTANCEPositive-strand RNA viruses produce vast amounts of progeny in very short periods of time via template-dependent RNA replication mechanisms. Template-dependent RNA replication, while efficient, can be disadvantageous due to error-prone viral polymerases. The accumulation of mutations in viral RNA genomes leads to error catastrophe. In this study, we substantiate long-held theories regarding the advantages and disadvantages of asexual and sexual replication strategies among RNA viruses. In particular, we show that picornavirus RNA recombination counteracts the negative consequences of asexual template-dependent RNA replication mechanisms, namely, error catastrophe.

scholarly journals Coronavirus RNA synthesis takes place within membrane-bound sites

2021 ◽  
Author(s):  
Nicole Doyle ◽  
Jennifer Simpson ◽  
Philippa C Hawes ◽  
Helena J Maier
Keyword(s):  
Life Cycle ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Viral Rna ◽  
Host Cells ◽  
Infectious Bronchitis ◽  
Welfare Issue ◽  
Membrane Bound ◽  
Positive Strand Rna

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Under-standing more about the replication of these viruses is imperative in order to effectively find ways to control them.

scholarly journals Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

2010 ◽  
Vol 84 (6) ◽  
pp. 2843-2858 ◽  
Author(s):  
Benjamin P. Steil ◽  
Brian J. Kempf ◽  
David J. Barton
Keyword(s):  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
Data Support ◽  
Reiterative Transcription ◽  
Positive Strand Rna

ABSTRACT A 3′ poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive- and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3′ end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5′ end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5′ ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3′ ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3′ poly(A) tails on progeny RNA genomes.

scholarly journals Noise-induced bistability in the quasineutral coexistence of viral RNA under different replication modes

2018 ◽  
Author(s):  
Josep Sardanyés ◽  
Andreu Arderiu ◽  
Santiago F. Elena ◽  
Tomás Alarcón
Keyword(s):  
Viral Replication ◽  
Evolutionary Dynamics ◽  
Rna Viruses ◽  
Initial Conditions ◽  
Rna Replication ◽  
Viral Rna ◽  
Stochastic Simulations ◽  
Strong Impact ◽  
Deterministic Models ◽  
The Impact

Evolutionary and dynamical investigations on real viral populations indicate that RNA replication can range between two extremes given by so-called stamping machine replication (SMR) and geometric replication (GR). The impact of asymmetries in replication for single-stranded, (+) sense RNA viruses has been up to now studied with deterministic models. However, viral replication should be better described by including stochasticity, since the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasineutral coexistence scenario, with a line of fixed points involving different strands’ equilibrium ratios depending on the initial conditions. Recent research on the quasineutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alters the mean-field scenario, and one of the two species outcompetes the other one. In this manuscript we study this phenomenon for RNA viral replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNA, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication.

scholarly journals Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

2020 ◽  
Vol 117 (31) ◽  
pp. 18680-18691 ◽  
Author(s):  
Nuruddin Unchwaniwala ◽  
Hong Zhan ◽  
Janice Pennington ◽  
Mark Horswill ◽  
Johan A. den Boon ◽  
...  
Keyword(s):  
Protein A ◽  
Rna Replication ◽  
Replication Protein A ◽  
Replication Protein ◽  
Viral Rna ◽  
Interaction Sites ◽  
Basal Lobe ◽  
Apical Lobe ◽  
Positive Strand Rna ◽  
Em Tomography

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter “crown” complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (−) and (+)RNA synthesis.

scholarly journals Yeast Lsm1p-7p/Pat1p Deadenylation-Dependent mRNA-Decapping Factors Are Required for Brome Mosaic Virus Genomic RNA Translation

2003 ◽  
Vol 23 (12) ◽  
pp. 4094-4106 ◽  
Author(s):  
Amine O. Noueiry ◽  
Juana Diez ◽  
Shaun P. Falk ◽  
Jianbo Chen ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Genomic Rna ◽  
Reading Frame ◽  
Mrna Decapping ◽  
Rna Translation ◽  
Positive Strand Rna

ABSTRACT Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5′ and 3′ noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.

scholarly journals Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Maija K. Pietilä ◽  
Martijn J. van Hemert ◽  
Tero Ahola
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rna Viruses ◽  
Viral Rna ◽  
Cellular Membranes ◽  
Positive Strand ◽  
Membrane Markers ◽  
Highly Active ◽  
Replicase Proteins ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCESimilar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA andin vitroreplication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

scholarly journals Noise-induced bistability in the quasi-neutral coexistence of viral RNAs under different replication modes

2018 ◽  
Vol 15 (142) ◽  
pp. 20180129 ◽  
Author(s):  
Josep Sardanyés ◽  
Andreu Arderiu ◽  
Santiago F. Elena ◽  
Tomás Alarcón
Keyword(s):  
Evolutionary Dynamics ◽  
Rna Viruses ◽  
Initial Conditions ◽  
Rna Replication ◽  
Viral Rna ◽  
Stochastic Simulations ◽  
Strong Impact ◽  
Deterministic Models ◽  
Viral Rnas ◽  
The Impact

Evolutionary and dynamical investigations into real viral populations indicate that RNA replication can range between the two extremes represented by so-called ‘stamping machine replication’ (SMR) and ‘geometric replication’ (GR). The impact of asymmetries in replication for single-stranded (+) sense RNA viruses has been mainly studied with deterministic models. However, viral replication should be better described by including stochasticity, as the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasi-neutral coexistence scenario, with a line of fixed points involving different strands' equilibrium ratios depending on the initial conditions. Recent research into the quasi-neutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alter the mean-field scenario, and one of the two species outcompetes the other. In this article, we study this phenomenon for viral RNA replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNAs, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication.

scholarly journals 5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

2009 ◽  
Vol 83 (7) ◽  
pp. 2976-2988 ◽  
Author(s):  
Priscilla M. Van Wynsberghe ◽  
Paul Ahlquist
Keyword(s):  
Complex Formation ◽  
Mutational Analysis ◽  
Protein A ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

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