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.

CD72 is an inhibitory pattern recognition receptor that recognizes ribosomes and suppresses production of anti-ribosome autoantibody.

B cell responses to nucleic acid-containing self-antigens that involve intracellular nucleic acid sensors play a crucial role in autoantibody production in SLE. CD72 is an inhibitory B cell co-receptor that down-regulates BCR signaling, and prevents the development of SLE. We previously showed that CD72 recognizes the RNA-containing self-antigen Sm/RNP, a target of SLE-specific autoantibodies, and induces B cell tolerance to Sm/RNP by specifically inhibiting B cell response to this self-antigen. Here, we address whether CD72 inhibits B cell response to ribosomes because the ribosome is an RNA-containing self-antigen and is a target of SLE-specific autoantibodies as well as Sm/RNP. We demonstrate that CD72 recognizes ribosomes as a ligand, and specifically inhibits BCR signaling induced by ribosomes. Although conventional protein antigens by themselves do not induce proliferation of specific B cells, ribosomes induce proliferation of B cells reactive to ribosomes in a manner dependent on RNA. This proliferative response is down-regulated by CD72. These results suggest that ribosomes activate B cells by inducing dual signaling through BCR and intracellular RNA sensors and that CD72 inhibits B cell response to ribosomes. Moreover, CD72-/- but not CD72+/+ mice spontaneously produce anti-ribosome autoantibodies. Taken together, CD72 induces B cell self-tolerance to ribosomes by recognizing ribosomes and inhibiting RNA-dependent B cell response to this self-antigen. CD72 appears to prevent development of SLE by inhibiting autoimmune B cell responses to multiple RNA-containing self-antigens. Because these self-antigens but not protein self-antigens induce RNA-dependent B cell activation, self-tolerance to RNA-containing self-antigens may require a distinct tolerance mechanism mediated by CD72.

An emerging ribosomopathy affecting the skeleton due to biallelic variations in NEPRO.

Cartilage hair hypoplasia (CHH), anauxetic dysplasia 1, and anauxetic dysplasia 2 are rare metaphyseal dysplasias caused by biallelic pathogenic variants in RMRP and POP1, which encode the components of RNAse-MRP endoribonuclease complex (RMRP) in ribosomal biogenesis pathway. Nucleolus and neural progenitor protein (NEPRO), encoded by NEPRO (C3orf17), is known to interact with multiple protein subunits of RMRP. We ascertained a 6-year-old girl with skeletal dysplasia and some features of CHH. RMRP and POP1 did not harbor any causative variant in the proband. Parents-child trio exomes revealed a candidate biallelic variant, c.435G>C, p.(Leu145Phe) in NEPRO. Two families with four affected individuals with skeletal dysplasia and a homozygous missense variant, c.280C>T, p.(Arg94Cys) in NEPRO, were identified from literature and their published phenotype was compared in detail to the phenotype of the child we described. All the five affected individuals have severe short stature, brachydactyly, skin laxity, joint hypermobility, and joint dislocations. They also have short metacarpals, broad middle phalanges, and metaphyseal irregularities. Protein modeling and stability prediction showed that the mutant protein has decreased stability. Both the reported variants are in the same domain of the protein. Our report delineates the clinical and radiological characteristics of an emerging ribosomopathy caused by biallelic variants in NEPRO.

Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo.

Distinguishing the surface markers of cancer stem cells (CSCs) is a useful method for early diagnosis and treatment of tumors, as CSCs may participate in tumorigenesis and metastasis by migrating into the circulatory system. However, the potential targets of CSCs are expressed at low levels in the natural state and are always changing. Thus, dynamic screening has been reported to be an effective measure for exploring CSC markers. In recent years, diverse single-chain variable fragments (scFvs) have been widely used in immunotherapy. In this study, we determined that the scFvs, screened using RD, had a high affinity to microspheres and could inhibit their progression. We also observed that the selected scFvs underwent evolution in vitro, and antitumor-associated proteins were successfully expressed. Combined with chemotherapy, the scFvs had a synergistic effect on the inhibition of the microspheres' progression in vitro and in vivo, which could be ascribed to their high affinity for stem-like cells and the inhibition of the microspheres' collective behaviors. In addition, proteins inhibiting CD44+ /CD24+ and MAPK were involved. Our data indicated that dynamic screening of the scFvs in a natural state was of great significance in the inhibition of the microspheres in vitro and in vivo.

Hepatitis C virus: Enslavement of host factors.

Hepatitis C virus (HCV) has infected over 170 million people world-wide. This infection causes severe liver damage that can progress to hepatocellular carcinoma leading to death of the infected patients. Development of a cell culture model system for the study of HCV infection in the recent past has helped the researchers world-wide to understand the biology of this virus. Studies over the past decade have revealed the tricks played by the virus to sustain itself, for as long as 40 years, in the host setup without being eliminated by the immune system. Today we understand that the host organelles and different cellular proteins are affected during HCV infection. This cytoplasmic virus has all the cellular organelles at its disposal to successfully replicate, from ribosomes and intracellular membranous structures to the nucleus. It modulates these organelles at both the structural and the functional levels. The vast knowledge about the viral genome and viral proteins has also helped in the development of drugs against the virus. Despite the achieved success rate to cure the infected patients, we struggle to eliminate the cases of recurrence and the non-responders. Such cases might emerge owing to the property of the viral genome to accumulate mutations during its succeeding replication cycles which favours its survival. The current situation calls an urgent need for alternate therapeutic strategies to counter this major problem of human health. © 2017 IUBMB Life, 70(1):41-49, 2018.

'Z-DNA like' fragments in RNA: a recurring structural motif with implications for folding, RNA/protein recognition and immune response.

Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.

Re-examining class-I presentation and the DRiP hypothesis.

MHC class I molecules present peptides derived from intracellular proteins, enabling immune surveillance by CD8(+) T cells and the elimination of virus-infected and cancerous cells. It has been argued that the dominant source of MHC class I-presented peptides is through proteasomal degradation of newly synthesized defective proteins, termed defective ribosomal products (DRiPs). Here, we critically examine the DRiP hypothesis and discuss recent studies indicating that antigenic peptides are generated from the entire proteome and not just from failures in protein synthesis or folding.

Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures.

Lupus-specific anti-ribosomal P (anti-Rib-P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)-region signatures of secreted autoantibody proteomes specific for the Rib-P heterocomplex and investigate the molecular basis of the reported cross-reactivity with Sm autoantigen. Anti-Rib-P immunoglobulins (IgGs) were purified from six anti-Rib-P-positive sera by elution from enzyme-linked immunosorbent assay (ELISA) plates coated with either native Rib-P proteins or an 11-amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope. Rib-P- and 11-C peptide-specific IgGs were analysed for heavy (H) and light (L) chain clonality and V-region expression using an electrophoretic and de-novo and database-driven mass spectrometric sequencing workflow. Purified anti-Rib-P and anti-SmD IgGs were tested for cross-reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti-Rib-P autoantibody proteomes were IgG1 kappa-restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH-terminal epitope, while the second shared the same pairing signature as a recently reported anti-SmD clonotype, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-Rib-P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti-Rib-P and anti-Sm responses suggest a common B cell clonal origin for subsets of these lupus-specific autoantibodies.

Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway.

The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.

SOCS3 regulates BAFF in human enterocytes under ribosomal stress.

Although the activation of B cells in the gastrointestinal tract is of great importance in the context of immunity to pathogens and mucosal inflammatory diseases, little is known about the mechanisms responsible for the local activation of B cells in the subepithelial area of the intestine. Epithelium-derived BAFF is the major modulator of B cell development and Ig class switching. The present study was performed to address the molecular mechanism of BAFF expression in gut epithelial cells in the presence of proinflammatory stimuli. Inflammation-induced BAFF expression in mucosal epithelial cells might be responsible for diverse mucosa-associated diseases linked to intestinal inflammation and autoimmunity. Although BAFF was marginally expressed in unstimulated epithelial cells, BAFF mRNA was significantly upregulated by proinflammatory IFN-γ. Furthermore, IFN-γ triggered JAK/STAT1 signals via the cytokine receptor, which contributed to epithelial BAFF upregulation. In terms of signaling intervention, ribosomal insult attenuated IFN-γ-activated JAK/STAT signal transduction and subsequent BAFF induction in gut epithelial cells. Ribosomal insults led to the superinduction of SOCS3 by enhancing its mRNA stability via HuR RNA-binding protein. Upregulated SOCS3 then contributed to the blocking of the JAK/STAT-linked signal, which mediated BAFF suppression by ribosomal stress. All of these findings show that ribosomal stress-induced SOCS3 plays a novel regulatory role in epithelial BAFF production, suggesting that epithelial ribosomal dysfunction in association with SOCS3 may be a promising therapeutic point in BAFF-associated human mucosal diseases.

DRiPs solidify: progress in understanding endogenous MHC class I antigen processing.

Defective ribosomal products (DRiPs) are a subset of rapidly degraded polypeptides that provide peptide ligands for major histocompatibility complex (MHC) class I molecules. Here, recent progress in understanding DRiP biogenesis is reviewed. These findings place DRiPs at the center of the MHC class I antigen processing pathway, linking immunosurveillance of viruses and tumors to mechanisms of specialized translation and cellular compartmentalization. DRiPs enable the immune system to rapidly detect alterations in cellular gene expression with great sensitivity.

Eukaryotic ribosome display for antibody discovery: A review.

Monoclonal antibody biologics have significantly transformed the therapeutic landscape within the biopharmaceutical industry, partly due to the utilisation of discovery technologies such as the hybridoma method and phage display. While these established platforms have streamlined the development process to date, their reliance on cell transformation for antibody identification faces limitations related to library diversification and the constraints of host cell physiology. Cell-free systems like ribosome display offer a complementary approach, enabling antibody selection in a completely in vitro setting while harnessing enriched cellular molecular machinery. This review aims to provide an overview of the fundamental principles underlying the ribosome display method and its potential for advancing antibody discovery and development.

Rustless translation.

ATP binding cassette proteins are a large and diverse family of molecular machines and include transmembrane transporter, chromosome maintenance and DNA repair proteins, and translation factors. However, the function of the ABCE1, the only member of subfamily E of ABC proteins, remained mysterious for over a decade, even though it is perhaps the most conserved ABC protein in eukaryotes and archaea. Recent results have now identified ABCE1 as the ribosome-recycling factor of eukaryotes and archaea. Thus, two iron-sulfur clusters - the hallmark feature of ABCE1 - help catalyze an integral step of the translational cycle at the core of the protein synthesis machinery.

Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display.

A single-chain variable fragment (ScFv) complementary DNA (cDNA) library against fenitrothion was constructed, and ScFvs specific for fenitrothion were selected by ribosome display from the library. After three rounds of ribosome display, the ScFv genes were cloned into Escherichia coli for expression. The expressed ScFvs of 160 clones were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Of these, 40 clones produced antibodies with relatively high activity against fenitrothion, and 3 were selected for Biacore and ELISA analysis. These 3 antibodies-ScFv-AF50, ScFv-AF93, and ScFv-AF132-had IC(50) values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The IC(50) values and cross-reactivity were lower than achieved previously with polyclonal or monoclonal antibodies against fenitrothion. The equilibrium dissociation constant (K(D)) values determined by Biacore analysis were 4.56×10(-10)M for ScFv-AF50, 1.42×10(-9)M for ScFv-AF93, and 2.66×10(-10)M for ScFv-AF132. These results demonstrate that the ribosome display has great potential in selection of ScFvs against pesticides. Recoveries of fenitrothion from fortified rice and cucumber were in the range 80.6 to 108%, indicating that the ELISAs with the isolated ScFvs can accurately determine fenitrothion in food samples after the simple and rapid extraction procedure.

[Ribosome display screening of a novel human anti-IgE scFv fragment].

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Do ribosomopathies explain some cases of common variable immunodeficiency?

The considerable clinical heterogeneity of patients with common variable immunodeficiency disorders (CVID) shares some similarity with bone-marrow failure disorders such as Diamond-Blackfan anaemia (DBA) and Shwachman-Diamond syndrome (SDS), now recognized as defects in ribosome biogenesis or ribosomopathies. The recognition of a patient with DBA who subsequently developed CVID lends support to our previous finding of a heterozygous mutation in the SBDS gene of SBDS in another CVID patient, suggesting that ribosome biogenesis defects are responsible for a subset of CVID. Genetic defects in the ribosomal translational machinery responsible for various bone marrow failure syndromes are recognized readily when they manifest in children, but diagnosing these in adults presenting with complex phenotypes and hypogammaglobulinaemia can be a challenge. In this perspective paper, we discuss our clinical experience in CVID patients with ribosomopathies, and review the immunological abnormalities in other conditions associated with ribosomal dysfunction. With genetic testing available for various bone marrow failure syndromes, our hypothesis that ribosomal abnormalities may be present in patients with CVID could be proved in future studies by testing for mutations in specific ribosomal genes. New knowledge might then be translated into novel therapeutic strategies for patients in this group of immunodeficiency disorders.

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.

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach.

Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

Ribosomal versus non-ribosomal cellular antigens: factors determining efficiency of indirect presentation to CD4+ T cells.

Proteins released from dying cells can be taken up and presented by antigen-presenting cells (APC) to T cells. While the presentation of such self antigens may lead to beneficial anti-tumour responses, in autoimmune disease it leads to pathological immune responses. The sub-set of self proteins targeted in autoimmune disease is circumscribed, and certain cellular components such as ribonucleoprotein (RNP) complexes are often targeted. Although explanations for this antigen selectivity have been proposed, there has been little direct testing of these hypotheses. We and others previously showed that ribosomal proteins, targeted in autoimmune disease, are also targets of anti-tumour T-cell responses. We asked whether particular properties of ribosomal proteins such as incorporation into RNP complexes or sub-cellular localization enhance ribosomal protein presentation by APC to CD4(+) T cells. Ribosomal protein antigens within purified intact ribosomes or free of the ribosomes were equally well taken up and presented by APC, demonstrating that inclusion of ribosomal proteins into an RNP complex does not confer an advantage. However, antigens localized to ribosomes within apoptotic cells were less efficiently taken up and presented by APC than the same antigens localized diffusely throughout the cell. This suggests that presentation of ribosomal proteins is somehow down-regulated, possibly to facilitate presentation of other less-abundant intracellular proteins. Consequently, the explanation for the frequent targeting of ribosomal proteins by both autoimmune and anti-tumour T-cell responses is not at the level of uptake from apoptotic cells and must be sought elsewhere.

Lupus autoantibodies target ribosomal P proteins.

All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.