scholarly journals RNA processing bodies are disassembled during Old World alphavirus infection

2019 ◽  
Vol 100 (10) ◽  
pp. 1375-1389 ◽  
Author(s):  
Lifeng Liu ◽  
Eva Weiss ◽  
Marc D. Panas ◽  
Benjamin Götte ◽  
Stina Sellberg ◽  
...  
Keyword(s):  
Rna Processing ◽  
Nuclear Import ◽  
Semliki Forest Virus ◽  
Early Stage ◽  
Processing Bodies ◽  
Old World ◽  
P Bodies ◽  
Infected Cells ◽  
As Stress ◽  
Alphavirus Infection

RNA processing bodies (P-bodies) are non-membranous cytoplasmic aggregates of mRNA and proteins involved in mRNA decay and translation repression. P-bodies actively respond to environmental stresses, associated with another type of RNA granules, known as stress granules (SGs). Alphaviruses were previously shown to block SG induction at late stages of infection, which is important for efficient viral growth. In this study, we found that P-bodies were disassembled or reduced in number very early in infection with Semliki Forest virus (SFV) or chikungunya virus (CHIKV) in a panel of cell lines. Similar to SGs, reinduction of P-bodies by a second stress (sodium arsenite) was also blocked in infected cells. The disassembly of P-bodies still occurred in non-phosphorylatable eIF2α mouse embryonal fibroblasts (MEFs) that are impaired in SG assembly. Studies of translation status by ribopuromycylation showed that P-body disassembly is independent of host translation shutoff, which requires the phosphorylation of eIF2α in the SFV- or CHIKV-infected cells. Labelling of newly synthesized RNA with bromo-UTP showed that host transcription shutoff correlated with P-body disassembly at the same early stage (3–4 h) after infection. However, inhibition of global transcription with actinomycin D (ActD) failed to disassemble P-bodies as effectively as the viruses did. Interestingly, blocking nuclear import with importazole led to an efficient P-bodies loss. Our data reveal that P-bodies are disassembled independently from SG formation at early stages of Old World alphavirus infection and that nuclear import is involved in the dynamic of P-bodies.

scholarly journals Effects of Palmitoylation of Replicase Protein nsP1 on Alphavirus Infection

2000 ◽  
Vol 74 (15) ◽  
pp. 6725-6733 ◽  
Author(s):  
Tero Ahola ◽  
Pekka Kujala ◽  
Minna Tuittila ◽  
Titta Blom ◽  
Pirjo Laakkonen ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Rna Replication ◽  
Cell Types ◽  
Alkaline Solutions ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Replication Sites ◽  
Infected Cells ◽  
Alphavirus Infection

ABSTRACT The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

scholarly journals Biogenesis of the Semliki Forest Virus RNA Replication Complex

2001 ◽  
Vol 75 (8) ◽  
pp. 3873-3884 ◽  
Author(s):  
Pekka Kujala ◽  
Anne Ikäheimonen ◽  
Neda Ehsani ◽  
Helena Vihinen ◽  
Petri Auvinen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Polymerase ◽  
Semliki Forest Virus ◽  
Rna Replication ◽  
Double Labeling ◽  
Virus Rna ◽  
Infected Cells ◽  
Em Autoradiography ◽  
The Individual ◽  
Alphavirus Infection

ABSTRACT The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326–338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

scholarly journals Coordinated Changes in mRNA Turnover, Translation, and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation

2008 ◽  
Vol 28 (24) ◽  
pp. 7414-7426 ◽  
Author(s):  
Yuxin Zhai ◽  
Zhenping Zhong ◽  
Chyi-Ying A. Chen ◽  
Zhenfang Xia ◽  
Ling Song ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Inflammation ◽  
Rna Processing ◽  
Physiological Role ◽  
Bronchial Epithelial Cells ◽  
Interleukin 4 ◽  
Translation Rate ◽  
Processing Bodies ◽  
Bronchial Epithelial ◽  
P Bodies

ABSTRACT Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

scholarly journals RNA helicase DDX6 in P-bodies is essential for the assembly of stress granules

2021 ◽  
Author(s):  
Vladimir Majerciak ◽  
Tongqing Zhou ◽  
Zhi-Ming Zheng
Keyword(s):  
Rna Processing ◽  
Stress Granules ◽  
The Other ◽  
Helicase Activity ◽  
Dual Roles ◽  
Processing Bodies ◽  
P Bodies ◽  
Rna Granules ◽  
Cytoplasmic Rna ◽  
Conceptual Advance

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate storage of translationally arrested mRNAs and are intimately related. In this study, we found the dependence of SG formation on PB in the cells under arsenite (ARS) stress, but not the other way around. GW182, 4E-T and DDX6 essential for PB formation differentially affect SG formation in the cells under ARS stress, with DDX6 being the most prominent. The cells with DDX6 deficiency display irregular shape of SG which could be rescued by ectopic wt DDX6, but not its helicase mutant E247A DDX6, which induces SG in the cells without stress, indicating that DDX6 helicase activity is essential for PB, but suppressive for SG. DDX6's dual roles are independent of DDX6 interactors EDC3, CNOT1, and PAT1B. This study provides a conceptual advance of how DDX6 involves in the biogenesis of PB and SG.

Focusing on mRNA Granules and Stalled Polysomes Amidst Diverse Mechanisms Underlying mRNA Transport, mRNA Storage, and Local Translation

2020 ◽  
Author(s):  
Mina N. Anadolu ◽  
Wayne S. Sossin
Keyword(s):  
Protein Synthesis ◽  
Liquid Phase ◽  
Mrna Translation ◽  
Rna Transport ◽  
P Bodies ◽  
Rna Granules ◽  
Mrna Granules ◽  
Transport Particle ◽  
As Stress ◽  
The Individual

In neurons, mRNAs are transported to distal sites to allow for localized protein synthesis. There are many diverse mechanisms underlying this transport. For example, an individual mRNA can be transported in an RNA transport particle that is tailored to the individual mRNA and its associated binding proteins. In contrast, some mRNAs are transported in liquid-liquid phase separated structures called neuronal RNA granules that are made up of multiple stalled polysomes, allowing for rapid initiation-independent production of proteins required for synaptic plasticity. Moreover, neurons have additional types of liquid-liquid phase–separated structures containing mRNA, such as stress granules and P bodies. This chapter discusses the relationships between all of these structures, what proteins distinguish them, and the possible roles they play in the complex control of mRNA translation at distal sites that allow neurons to use protein synthesis to refine their local proteome in many different ways.

scholarly journals Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

2010 ◽  
Vol 21 (15) ◽  
pp. 2624-2638 ◽  
Author(s):  
Cornelia Kilchert ◽  
Julie Weidner ◽  
Cristina Prescianotto-Baschong ◽  
Anne Spang
Keyword(s):  
Osmotic Stress ◽  
Secretory Pathway ◽  
Small Gtpase ◽  
Processing Bodies ◽  
P Bodies ◽  
Intracellular Signals ◽  
Intracellular Ca ◽  
Response To Stress ◽  
Cellular Stresses ◽  
Translational Block

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca2+ mimicked this increase in PB number. Conversely, intracellular Ca2+-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca2+ required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of ∼40–100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.

scholarly journals The arrested state of processing bodies supports mRNA regulation in early development

2021 ◽  
Author(s):  
M. Sankaranarayanan ◽  
Ryan J. Emenecker ◽  
Marcus Jahnel ◽  
Irmela R. E. A. Trussina ◽  
Matt Wayland ◽  
...  
Keyword(s):  
Physical Properties ◽  
Electrostatic Interactions ◽  
Processing Bodies ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Premature Release ◽  
Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

scholarly journals Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

2008 ◽  
Vol 89 (3) ◽  
pp. 676-686 ◽  
Author(s):  
Kristi Tamm ◽  
Andres Merits ◽  
Inga Sarand
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Structural Protein ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Replicase Complex

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

EV71 2A Protease inhibits P-body formation to promote viral RNA synthesis

2021 ◽  
Author(s):  
Shanshan Fan ◽  
Zihang Xu ◽  
Pengfei Liu ◽  
Yali Qin ◽  
Mingzhou Chen
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Enterovirus 71 ◽  
Viral Rna ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Body Components ◽  
2A Protease ◽  
Enterovirus 71 Infection

Several viruses were proved to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proved by the result that infection of EV71-2A C110S , the 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we showed DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and capturing the nascent RNA assay. Altogether, our data firstly demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulating gene expression and mRNA degradation. P-bodies are the structure that viruses to manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection and its activity was essential. When the assembly of P-bodies was blocked by 2A, DDX6 and 4E-T which were required for P-body formation bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

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