RNase L limits host and viral protein synthesis via inhibition of mRNA export

RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Here, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L–mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. The heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L–mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

eIF3a Destabilization and TDP-43 Alter Dynamics of Heat-Induced Stress Granules

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.

PKR-Mediated Stress Response Enhances Dengue and Zika Virus Replication

ABSTRACTThe mechanisms by which Flaviviruses use non-canonical translation to support their replication in host cells are largely unknown. Here we investigated how the integrated stress response (ISR), which promotes translational arrest by eIF2α phosphorylation (p–eIF2α), regulates Flavivirus replication. During Dengue virus (DENV) and Zika virus (ZIKV) infection, eIF2α activation peaked at 24 hours post infection and was dependent on PKR but not type I interferon. The ISR is activated downstream of p-eIF2α during infection with either virus, but translation arrest only occurred following DENV infection. Despite this difference, both DENV and ZIKV replication was impaired in cells lacking PKR, independent of IFN-I/NF-kB signaling or cell viability. By using a ZIKV 5’UTR reporter system as a model, we found that this region of the genome is sufficient to promote an enhancement of viral mRNA translation in the presence of an active ISR. Together we provide evidence that Flaviviruses escape ISR translational arrest and co-opt this response to increase viral replication.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by the subtilase cytotoxin (SubAB). PERK inactivation, increases globally protein synthesis levels and regulates IFN-β expression, while impairing LPS-stimulated DC migration. Although the loss of PERK activity does not impact DC development, the cross talk existing between actin cytoskeleton dynamics; PERK and eIF2α phosphorylation is likely important to adapt DC homeostasis to the variations imposed by the immune contexts.

First person – Shan Ying

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shan Ying is first author on ‘UV damage induces G3BP1-dependent stress granule formation that is not driven by mTOR inhibition-mediated translation arrest’, published in JCS. Shan is a research assistant in the lab of Denys Khaperskyy at Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada where her research interests focus on understanding properties and functions of stress granules and the molecular mechanism of viral host shutoff.

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

UV damage induces G3BP1-dependent stress granule formation that is not driven by mTOR inhibition-mediated translation arrest

ABSTRACTTranslation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work, we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not involve inhibition of the mechanistic target of rapamycin (mTOR) signaling or dissociation of the 48S preinitiation complexes. The general control non-derepressible 2 (GCN2; also known as EIF2AK4) kinase contributes to UV-induced SG formation, which is independent of the phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules requires the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1). Our work reveals that, in UV-treated cells, the mechanisms of translation arrest and SG formation may be unlinked, resulting in SGs that do not contain the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.This article has an associated First Person interview with the first author of the paper.

Influence of nascent polypeptide positive charges on translation dynamics

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.