Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.

The canonical antiviral protein oligoadenylate synthetase 1 elicits antibacterial functions by enhancing IRF1 translation.

An important property of the host innate immune response during microbial infection is its ability to control the expression of antimicrobial effector proteins, but how this occurs post-transcriptionally is not well defined. Here, we describe a critical antibacterial role for the classic antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1). Human OAS1 and its mouse ortholog, Oas1b, are induced by interferon-γ and protect against cytosolic bacterial pathogens such as Francisella novicida and Listeria monocytogenes in vitro and in vivo. Proteomic and transcriptomic analysis showed reduced IRF1 protein expression in OAS1-deficient cells. Mechanistically, OAS1 binds and localizes IRF1 mRNA to the rough endoplasmic reticulum (ER)-Golgi endomembranes, licensing effective translation of IRF1 mRNA without affecting its transcription or decay. OAS1-dependent translation of IRF1 leads to the enhanced expression of antibacterial effectors, such as GBPs, which restrict intracellular bacteria. These findings uncover a noncanonical function of OAS1 in antibacterial innate immunity.

CD8+ T cell tolerance: It doesn't translate.

The mechanisms that make and break CD8+ T cell tolerance to self-antigens remain unclear. In this issue of Immunity, Van Der Byl et al. show that tolerant CD8+ T cells rapidly adopt an epigenetically and transcriptionally distinct cell state and exhibit impaired protein translation. Breaking tolerance requires both inflammation and increased antigen exposure to augment MYC expression and restore translation.

The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Pervasive downstream RNA hairpins dynamically dictate start-codon selection.

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1-4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Translation-A tug of war during viral infection.

Viral reproduction is contingent on viral protein synthesis that relies on the host ribosomes. As such, viruses have evolved remarkable strategies to hijack the host translational apparatus in order to favor viral protein production and to interfere with cellular innate defenses. Here, we describe the approaches viruses use to exploit the translation machinery, focusing on commonalities across diverse viral families, and discuss the functional relevance of this process. We illustrate the complementary strategies host cells utilize to block viral protein production and consider how cells ensure an efficient antiviral response that relies on translation during this tug of war over the ribosome. Finally, we highlight potential roles mRNA modifications and ribosome quality control play in translational regulation and innate immunity. We address these topics in the context of the COVID-19 pandemic and focus on the gaps in our current knowledge of these mechanisms, specifically in viruses with pandemic potential.

mRNA translation from an antigen presentation perspective: A tribute to the works of Nilabh Shastri.

The field of mRNA translation has witnessed an impressive expansion in the last decade. The once standard model of translation initiation has undergone, and is still undergoing, a major overhaul, partly due to more recent technical advancements detailing, for example, initiation at non-AUG codons. However, some of the pioneering works in this area have come from immunology and more precisely from the field of antigen presentation to the major histocompatibility class I (MHC-I) pathway. Despite early innovative studies from the lab of Nilabh Shastri demonstrating alternative mRNA translation initiation as a source for MHC-I peptide substrates, the mRNA translation field did not include these into their models. It was not until the introduction of the ribo-sequence technique that the extent of non-canonical translation initiation became widely acknowledged. The detection of peptides on MHC-I molecules by CD8 + T cells is extremely sensitive, making this a superior model system for studying alternative mRNA translation initiation from specific mRNAs. In view of this, we give a brief history on alternative initiation from an immunology perspective and its fundamental role in allowing the immune system to distinguish self from non-self and at the same time pay tribute to the works of Nilabh Shastri.

Translational Control of Immune Evasion in Cancer.

Mechanisms that control translation play important roles in tumor progression and metastasis. Emerging evidence has revealed that dysregulated translation also impacts immune evasion in response to cellular or oncogenic stress. Here, we summarize current knowledge regarding the translational control of immune checkpoints and implications for cancer immunotherapies.

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

A crayfish ALF inhibits the proliferation of microbiota by binding to RPS4 and MscL of E. coli.

Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.

Human Schlafen 11 exploits codon preference discrimination to attenuate viral protein synthesis of prototype foamy virus (PFV).

Recently, the Schlafen (SLFN) proteins have been identified as a novel interferon-stimulated family with antiviral properties. In this study, we reported that SLFN11 inhibited prototype foamy virus (PFV) replication. Over-expression of human SLFN11 reduced viral production, while knockdown of SLFN11 enhanced viral infectivity. In addition, SLFN11 from cattle and African green monkey also suppressed PFV production. Both the ATPase activity and helicase activity of SLFN11 were required for its inhibitory function. Dephosphorylation activated the antiviral activity of SLFN11. More importantly, SLFN11 inhibited the expression of viral protein, which was rescued by viral gene codon optimization. Together, our results demonstrated that SLFN11 impaired PFV viral protein synthesis by exploiting the distinct codon usage between the virus and the host. These findings further broaden our understanding of the antiviral properties of the SLFN family and the molecular mechanism of PFV latent infection.

Immunomodulatory gene expression analysis in LPS-stimulated human polymorphonuclear leukocytes treated with antibiotics commonly used for multidrug-resistant strains.

Conventional antibiotics used for the treatment of severe infections such as sepsis and septic shock confer immunomodulatory benefits. However, the growing problem of multidrug resistant infections has led to an increase in the administration of non-conventional last-resort antibiotics, including quinolones, aminoglycosides, and polypeptides, and the effects of these drugs on immunomodulatory gene expression in activated human polymorphonuclear leukocytes (PMNs) have not been reported. In this study, lipopolysaccharide-stimulated PMNs were incubated with piperacillin, rifampicin, fosfomycin (FOM), levofloxacin (LVFX), minocycline (MINO), colistin, tigecycline, or amikacin, and the mRNA expression levels of pattern recognition receptors (TLR2, TLR4, and CD14), inflammatory cytokines (TNFα and IL6), and chemokine receptors (IL8Rs and ITGAM) in these cells were quantitated using real-time qPCR. Many of the tested antibiotics altered the expression of the investigated cytokines. Notably, FOM, LVFX, and MINO significantly downregulated the expression of IL6, which is associated with pro- and anti-inflammatory defense mechanisms. Treatment of FOM and LVFX reduced IL-6 production as well as observed for IL6 gene expression. These findings indicated transcription and translation cooperation under the used experimental conditions. Therefore, our findings suggest that administration of these antibiotics suppresses the host anti-inflammatory response.

Anti-tumour immunity induces aberrant peptide presentation in melanoma.

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.

ABC-F translation factors: from antibiotic resistance to immune response.

Energy-dependent translational throttle A (EttA) from Escherichia coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here, we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members.

Type I Interferon (IFN)-Regulated Activation of Canonical and Non-Canonical Signaling Pathways.

For several decades there has been accumulating evidence implicating type I interferons (IFNs) as key elements of the immune response. Therapeutic approaches incorporating different recombinant type I IFN proteins have been successfully employed to treat a diverse group of diseases with significant and positive outcomes. The biological activities of type I IFNs are consequences of signaling events occurring in the cytoplasm and nucleus of cells. Biochemical events involving JAK/STAT proteins that control transcriptional activation of IFN-stimulated genes (ISGs) were the first to be identified and are referred to as "canonical" signaling. Subsequent identification of JAK/STAT-independent signaling pathways, critical for ISG transcription and/or mRNA translation, are denoted as "non-canonical" or "non-classical" pathways. In this review, we summarize these signaling cascades and discuss recent developments in the field, specifically as they relate to the biological and clinical implications of engagement of both canonical and non-canonical pathways.

Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts.

Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.