ABSTRACT
As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.
Subject(s)
RNA Viruses/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , RNA-Binding Proteins/genetics , Viral Proteins/genetics , Virus Diseases/genetics , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/genetics , Protein Binding , Protein Biosynthesis , RNA Viruses/growth & development , RNA Viruses/pathogenicity , RNA, Messenger/immunology , RNA, Viral/immunology , RNA-Binding Proteins/immunology , Ribosomes/genetics , Ribosomes/metabolism , Signal Transduction , Viral Proteins/metabolism , Virus Assembly/genetics , Virus Diseases/immunology , Virus Diseases/pathology , Virus Diseases/virologyABSTRACT
Vaccination is the most effective tool against infectious diseases. Subunit vaccines are safer compared to live-attenuated vaccines but are less immunogenic and need to be delivered with an adjuvant. Adjuvants are essential for enhancing vaccine potency by improving humoral and cell-mediated immune responses. Only a limited number of adjuvants are licensed for human vaccines, and their mode of action is still not clear. Leishmania eukaryotic initiation factor (LeIF) has been described having a dual role, as a natural adjuvant and as an antigen that possesses advantageous immunomodulatory properties. In this study, we assessed the adjuvant properties of recombinant Leishmania infantum eukaryotic initiation factor (LieIF) through in vitro and in vivo assays. LieIF was intraperitoneally administered in combination with the protein antigen ovalbumin (OVA), and the widely used alum was used as a reference adjuvant. Our in vitro studies using J774A.1 macrophages showed that LieIF induced stimulatory effects as demonstrated by the enhanced surface expression of CD80 and CD86 co-stimulatory molecules and the induced production of the immune mediators NO and MIP-1α. Additionally, LieIF co-administration with OVA in an in vivo murine model induced a proinflammatory environment as demonstrated by the elevated expression of TNF-α, IL-1ß, and NF-κB2 genes in peritoneal exudate cells (PEC). Furthermore, PEC derived from OVA-LieIF-immunized mice exhibited elevated expression of CD80 molecule and production of NO and MIP-1α in culture supernatants. Moreover, LieIF administration in the peritoneum of mice resulted in the recruitment of neutrophils and monocytes at 24 h post-injection. Also, we showed that this immunopotentiating effect of LieIF did not depend on the induction of uric acid danger signal. These findings suggest the potential use of LieIF as adjuvant in new vaccine formulations against different infectious diseases.
Subject(s)
Adjuvants, Immunologic , Eukaryotic Initiation Factors/immunology , Inflammation/immunology , Leishmania infantum/physiology , Leishmaniasis, Visceral/immunology , Macrophages/immunology , Protozoan Proteins/immunology , Animals , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Cell Line , Disease Models, Animal , Eukaryotic Initiation Factors/genetics , Female , Humans , Interleukin-1beta/metabolism , Mice , Mice, Inbred BALB C , Neutrophil Infiltration , Ovalbumin/immunology , Protozoan Proteins/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The microRNA (miRNA) biogenesis pathway is regulated by specific proteins and enzymes, including Dicer, Drosha, DGCR8, Exportin 5 and the Argonaute (AGO) family. In this study, we investigated the AGO family, which is the primary component of RISC (RNA-induced silencing complex) and directly binds to microRNA. We examined the association of polymorphisms in AGO family genes with AGO expression and with the development and prognosis of autoimmune thyroid diseases. We genotyped AGO1 rs636832A/G, AGO2 rs7005286C/T, AGO2 rs11166985A/G and AGO2 rs2292779C/G polymorphisms in 184 Graves' disease (GD) patients, 195 Hashimoto's disease (HD) patients and 122 healthy volunteers using the polymerase chain reaction-restriction fragment length polymorphism method. We also examined the expression of AGO1 and AGO2 mRNAs in peripheral blood mononuclear cells (PBMC) obtained from 52 GD patients, 41 HD patients, and 25 healthy volunteers using quantitative RT-PCR methods. The G allele of AGO1 rs636832 and the A allele of AGO2 rs11166985 polymorphisms were significantly more frequent in GD patients than in healthy controls. The A allele of AGO2 rs11166985 was also significantly more frequent in intractable GD patients than in controls. The C carrier (CC + CG genotypes) and C allele of AGO2 rs2292779 polymorphism were significantly more frequent in intractable GD patients than in patients with GD in remission. Expression of AGO1 mRNA in PBMC was significantly higher in AITD patient than in controls, and that of AGO2 mRNA in PBMC was significantly higher in intractable GD patients than in patients with GD in remission. Furthermore, the expression levels of both the AGO1 and AGO2 genes were significantly correlated with the proportions of Th17 cells in PBMC. In conclusion, the polymorphisms of the AGO1 and AGO2 genes, the expression levels of which correlated with the proportion of Th17 cells, were associated with the development and prognosis of GD. The AGO2 rs2292779 C carrier and C allele were associated with the intractability of GD.
Subject(s)
Alleles , Argonaute Proteins , Eukaryotic Initiation Factors , Gene Expression Regulation/immunology , Graves Disease , Polymorphism, Genetic , Th17 Cells , Adult , Argonaute Proteins/genetics , Argonaute Proteins/immunology , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Female , Graves Disease/genetics , Graves Disease/immunology , Graves Disease/pathology , Hashimoto Disease/genetics , Hashimoto Disease/immunology , Hashimoto Disease/pathology , Humans , Male , Middle Aged , Th17 Cells/immunology , Th17 Cells/pathologyABSTRACT
It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.
Subject(s)
Algorithms , Eukaryotic Initiation Factors/chemistry , Immunogenicity, Vaccine/immunology , Immunomodulation/immunology , Leishmania infantum/chemistry , Peptides/immunology , Eukaryotic Initiation Factors/immunology , Leishmania infantum/immunology , Models, Molecular , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistryABSTRACT
TBK1 is a component of the type I interferon (IFN) signaling pathway, yet the mechanisms controlling its activity and degradation remain poorly understood. Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo. USP38 specifically cleaves K33-linked poly-ubiquitin chains from TBK1 at Lys670, and it allows for subsequent K48-linked ubiquitination at the same position mediated by DTX4 and TRIP. Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signaling. Our findings identify an essential role for USP38 in negatively regulating type I IFN signaling, and they provide insights into the mechanisms by which USP38 regulates TBK1 ubiquitination through the NLRP4 signalosome.
Subject(s)
Immunity, Innate , Interferon Type I/metabolism , Macrophages/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Signal Transduction/immunology , Ubiquitin-Specific Proteases/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/virology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Eukaryotic Initiation Factors/metabolism , Gene Expression Regulation , Herpesvirus 1, Human/growth & development , Herpesvirus 1, Human/immunology , Host-Pathogen Interactions , Interferon Type I/genetics , Interferon Type I/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/immunology , Macrophages/virology , Mice , Mice, Knockout , Phosphorylation , Polyubiquitin/genetics , Polyubiquitin/immunology , Polyubiquitin/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Proteins/genetics , Proteins/immunology , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/immunology , Ubiquitination , Vesiculovirus/growth & development , Vesiculovirus/immunologyABSTRACT
To investigate the immunogenicity of Homo sapiens putative translation initiation factor (Sui1) in hepatocellular carcinoma (HCC), enzyme-linked immunosorbent assay (ELISA) and Western blot were utilized to assess autoantibody responses to Sui1 in sera from HCC patients and healthy individuals. Indirect immunofluorescence (IIF) assay with cancer cells and immunohistochemistry (IHC) study with tissue array slides were performed to examine Sui1 expression profile in cancer cells and tissues. The data confirmed that the frequency of autoantibody to Sui1 in sera of HCC patients was 15.5 % (16/103), which was remarkably higher than that in sera of liver cirrhosis (LC) patients (3.3 %, 1/30), chronic hepatitis (CH) patients (0 %, 0/29), and normal human serum (NHS) (0 %, 0/82) (p < 0.01). IHC study showed that the Sui1 expression in HCC tissues was 26.7 % (16/60). The expression of Sui1 had the trend of increasing along with the cancer grades but no statistical significance (p > 0.05). In immunodiagnosis of HCC, the sensitivity and specificity of the anti-Sui1 antibody were 15.5 and 99.3 %, respectively. If both anti-Sui1 and alpha fetal protein (AFP) were simultaneously utilized as detective markers, 66.7 % (30/45) of HCC patients could be correctly distinguished. The results suggested that anti-Sui1 could be utilized as a supplementary serological marker for the detection of HCC and Sui1 might be associated to HCC carcinogenesis.
Subject(s)
Autoantibodies/immunology , Carcinoma, Hepatocellular/metabolism , Eukaryotic Initiation Factors/immunology , Eukaryotic Initiation Factors/metabolism , Liver Neoplasms/metabolism , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Adult , Aged , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Carcinogenesis/metabolism , Carcinogenesis/pathology , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/pathology , Female , Fluorescent Antibody Technique, Indirect/methods , Humans , Immunohistochemistry/methods , Immunologic Tests/methods , Liver Cirrhosis/diagnosis , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/diagnosis , Liver Neoplasms/pathology , Male , Middle Aged , Sensitivity and Specificity , Young AdultABSTRACT
Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable.
Subject(s)
Eukaryotic Initiation Factors/immunology , Plant Diseases/virology , Plant Proteins/immunology , Plant Viruses/immunology , Plants/immunology , Eukaryotic Initiation Factors/genetics , Plant Diseases/genetics , Plant Diseases/immunology , Plant Proteins/genetics , Plant Viruses/genetics , Plants/genetics , Plants/virologyABSTRACT
A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19-C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.
Subject(s)
Antineoplastic Agents/pharmacology , Epoxy Compounds/metabolism , Eukaryotic Initiation Factors/metabolism , Macrolides/metabolism , Thiazoles/metabolism , Biological Products , Cell Proliferation , Epoxy Compounds/immunology , Eukaryotic Initiation Factors/immunology , Humans , Macrolides/immunology , Peptide Chain Initiation, Translational , Thiazoles/immunologyABSTRACT
Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.
Subject(s)
Autoantibodies/blood , Autoantigens/analysis , Liver Cirrhosis, Biliary , Protein Array Analysis , Proteome/analysis , Adult , Argonaute Proteins/immunology , Autoantibodies/immunology , Autoantigens/immunology , Autoimmune Diseases/diagnosis , Autoimmune Diseases/immunology , Carrier Proteins/analysis , Carrier Proteins/immunology , Eukaryotic Initiation Factors/immunology , Female , Hexokinase/analysis , Hexokinase/immunology , Humans , Liver Cirrhosis, Biliary/blood , Liver Cirrhosis, Biliary/diagnosis , Liver Cirrhosis, Biliary/immunology , Male , Middle Aged , Proteome/immunology , Repressor Proteins/analysis , Repressor Proteins/immunology , Sensitivity and Specificity , Zinc Fingers/immunologyABSTRACT
Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.
Subject(s)
Depressive Disorder, Major/metabolism , Prefrontal Cortex/physiopathology , TOR Serine-Threonine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Autopsy , Depressive Disorder, Major/genetics , Depressive Disorder, Major/immunology , Eukaryotic Initiation Factors/analysis , Eukaryotic Initiation Factors/biosynthesis , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Family , Female , Humans , Male , Middle Aged , Prefrontal Cortex/immunology , Prefrontal Cortex/pathology , Ribosomal Protein S6 Kinases/analysis , Ribosomal Protein S6 Kinases/biosynthesis , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/immunology , Ribosomal Protein S6 Kinases, 70-kDa/analysis , Ribosomal Protein S6 Kinases, 70-kDa/biosynthesis , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/immunology , Signal Transduction/genetics , Signal Transduction/immunology , TOR Serine-Threonine Kinases/analysis , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/immunologyABSTRACT
Small RNA pathways fulfill a plethora of gene-regulatory functions in a variety of organisms. In the nematode worm, Caenorhabditis elegans, a number of endogenous small RNA pathways have been described, including the microRNA pathway, the 21U/piRNA pathway, the 26G-RNA pathways, and the 22G-RNA pathways. Argonaute proteins are key effector molecules of each pathway that, together with their small RNA cofactors regulate various processes including developmental timing, fertility, transposon silencing, and chromosome segregation. Although several of the 26 Argonautes in the worm have been studied to date, a number have yet to be fully characterized or their small RNA binding complement defined. The identification of small RNAs that copurify with an Argonaute family member is central to understanding the targets and assessing the function of that Argonaute. Here we discuss the rationale for generating reagents to immunoprecipitate Argonaute complexes and provide a cohesive protocol for the cloning and Illumina deep-sequencing of Argonaute-associated small RNAs in C. elegans.
Subject(s)
Caenorhabditis elegans/genetics , Cloning, Molecular , Eukaryotic Initiation Factors/metabolism , MicroRNAs/genetics , Animals , Antibodies/immunology , Antibodies/metabolism , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Epitopes/immunology , Epitopes/metabolism , Eukaryotic Initiation Factors/immunology , Gene Library , Immunoprecipitation , MicroRNAs/isolation & purification , MicroRNAs/metabolism , Quality Control , Reproducibility of ResultsABSTRACT
The final step of B-cell maturation is to differentiate into plasma cells, a process that is accompanied by gross changes in subcellular organization to enable antibody secretion. To better understand this critical step in mounting a humoral immune response, we analyzed proteome dynamics during plasma cell differentiation with combined 2-DE/MS. Thirty-two identified protein spots changed in relative abundance when lipopolysaccharide (LPS)-stimulated primary B cells differentiated into antibody-secreting plasma cells. A correlative analysis of protein and transcript abundance suggested that one third of these proteins are post-transcriptionally regulated. Apart from ER-resident chaperones, lipid metabolic enzymes, and translation initiation factors, we identified several proteins that had not been previously studied in plasma cells. Among them is the transiently upregulated proteasome activator (PA) 28γ, a component of the putative nuclear proteasome. Additionally, we discovered that the non-canonical inflammatory cytokine high-mobility group box 1 (HMG1) was released from plasma cells into the extracellular milieu. This suggests a novel role for plasma cells as pro-inflammatory mediators, which has important implications for various autoimmune diseases and chronic inflammation.
Subject(s)
Autoantigens/immunology , HMGB1 Protein/immunology , Plasma Cells/immunology , Proteasome Endopeptidase Complex/immunology , Proteome/genetics , Proteome/immunology , RNA, Messenger/biosynthesis , Animals , Antibody Formation/genetics , Autoantigens/genetics , Autoantigens/metabolism , Blotting, Western , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Eukaryotic Initiation Factors/metabolism , Gene Expression Profiling , HMGB1 Protein/genetics , HMGB1 Protein/metabolism , Immunity, Humoral/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Lipid Metabolism/genetics , Lipid Metabolism/immunology , Lipopolysaccharides/pharmacology , Mass Spectrometry , Mice , Mice, Inbred C57BL , Molecular Chaperones/genetics , Molecular Chaperones/immunology , Molecular Chaperones/metabolism , Plasma Cells/cytology , Plasma Cells/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Proteome/metabolism , RNA, Messenger/analysisABSTRACT
Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length. The findings show that the length of small RNA duplexes plays a critical role in the activation of both RNAi-related and innate antiviral responses. They also suggest that these two mechanisms of antiviral response may activate the same pathway, requiring Lv-Sid 1 and Lv-Ago 2 induction.
Subject(s)
DNA Virus Infections/immunology , Eukaryotic Initiation Factors/metabolism , Penaeidae , Protein Kinases/metabolism , White spot syndrome virus 1/immunology , Amino Acid Sequence , Animals , Cloning, Molecular , DNA Virus Infections/genetics , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Gene Expression Regulation , Immunity, Innate/genetics , Molecular Sequence Data , Phylogeny , Protein Biosynthesis , Protein Kinases/genetics , Protein Kinases/immunology , RNA Interference , RNA, Double-Stranded/immunology , White spot syndrome virus 1/pathogenicityABSTRACT
The worldwide shrimp culture is beset with diseases mainly caused by white spot syndrome virus (WSSV) and suffered huge economic losses, which bring out an urgent need to develop the novel strategies to better protect shrimps against WSSV. In the present study, CpG-rich plasmid pUC57-CpG, plasmid pUC57 and PBS were employed to pretreat shrimps comparatively to evaluate the protective effects of CpG ODNs on shrimps against WSSV. The survival rates, WSSV copy numbers, and antiviral associated factors (Dicer, Argonaute, STAT and ROS) were detected in Litopenaeus vannamei. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in pUC57-CpG-pretreatment shrimps than those in pUC57- and PBS-pretreatment shrimps after WSSV infection. The Argonaute mRNA expression in pUC57-CpG-, pUC57- and PBS-pretreatment shrimps after WSSV infection was significantly higher than that of shrimps post PBS stimulation on the first day. The ROS levels in pUC57-CpG-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection on the first day. These results together demonstrated that pUC57-CpG induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei.
Subject(s)
Hemocytes/metabolism , Pandalidae/immunology , Virus Diseases/metabolism , White spot syndrome virus 1/physiology , Animals , Cells, Cultured , Eukaryotic Initiation Factors/biosynthesis , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Hemocytes/drug effects , Hemocytes/immunology , Hemocytes/pathology , Immunity, Innate/drug effects , Oligodeoxyribonucleotides/administration & dosage , Reactive Oxygen Species/immunology , Ribonuclease III/biosynthesis , Ribonuclease III/genetics , Ribonuclease III/immunology , STAT Transcription Factors/biosynthesis , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology , Toll-Like Receptor 9/agonists , Virus Diseases/drug therapy , Virus Diseases/genetics , Virus Diseases/immunology , Virus Replication/drug effects , White spot syndrome virus 1/pathogenicityABSTRACT
BACKGROUND: In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. RESULTS: We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain. CONCLUSION: The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.
Subject(s)
Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/immunology , Interspersed Repetitive Sequences/genetics , Interspersed Repetitive Sequences/immunology , Prokaryotic Cells/immunology , Sequence Homology, Amino Acid , Amino Acid Sequence , Bacteriophages/genetics , Deoxyribonucleases/metabolism , Gene Transfer, Horizontal/genetics , Genome/genetics , Immune System , Molecular Sequence Data , Phylogeny , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Eukaryotic initiation factor (eIF) 4B is known to interact with multiple initiation factors, mRNA, rRNA, and poly(A) binding protein (PABP). To gain a better understanding of the function of eIF4B, the two isoforms from Arabidopsis (Arabidopsis thaliana) were expressed and analyzed using biophysical and biochemical methods. Plant eIF4B was found by ultracentrifugation and light scattering analysis to most likely be a monomer with an extended structure. An extended structure would facilitate the multiple interactions of eIF4B with mRNA as well as other initiation factors (eIF4A, eIF4G, PABP, and eIF3). Eight mRNAs, barley (Hordeum vulgare) alpha-amylase mRNA, rabbit beta-hemoglobin mRNA, Arabidopsis heat shock protein 21 (HSP21) mRNA, oat (Avena sativa) globulin, wheat (Triticum aestivum) germin, maize (Zea mays) alcohol dehydrogenase, satellite tobacco necrosis virus RNA, and alfalfa mosaic virus (AMV) 4, were used in wheat germ in vitro translation assays to measure their dependence on eIF4B and eIF4F isoforms. The two Arabidopsis eIF4B isoforms, as well as native and recombinant wheat eIF4B, showed similar responses in the translation assay. AMV RNA 4 and Arabidopsis HSP21 showed only a slight dependence on the presence of eIF4B isoforms, whereas rabbit beta-hemoglobin mRNA and wheat germin mRNA showed modest dependence. Barley alpha-amylase, oat globulin, and satellite tobacco necrosis virus RNA displayed the strongest dependence on eIF4B. These results suggest that eIF4B has some effects on mRNA discrimination during initiation of translation. Barley alpha-amylase, oat globulin, and rabbit beta-hemoglobin mRNA showed the highest activity with eIF4F, whereas Arabidopsis HSP21 and AMV RNA 4 used both eIF4F and eIF(iso)4F equally well. These results suggest that differential or optimal translation of mRNAs may require initiation complexes composed of specific isoforms of initiation factor gene products. Thus, individual mRNAs or classes of mRNAs may respond to the relative abundance of a particular initiation factor(s), which in turn may affect the amount of protein translated. It is likely that optimal multifactor initiation complexes exist that allow for optimal translation of mRNAs under a variety of cellular conditions.
Subject(s)
Arabidopsis/metabolism , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factors/metabolism , Genetic Variation , Protein Biosynthesis , Triticum/metabolism , Antibodies , Biological Assay , Blotting, Western , Chromatography, Gel , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Eukaryotic Initiation Factor-4F/isolation & purification , Eukaryotic Initiation Factors/immunology , Light , Molecular Weight , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Scattering, Radiation , Sequence Alignment , UltracentrifugationABSTRACT
The plasticity that is needed by the cell to respond to rapid changes in its environment cannot only be provided by means of transcriptional regulation, which generally confers on cells a set of stable properties. Alternatively, the control of mRNA translation allows the cell to modulate rapidly and over short period of time its gene expression program, without invoking the slower nuclear pathways for mRNA synthesis and transport. Several recent findings indicate that regulation of translation affects directly antigen presentation, cytokine production, as well as the survival of dendritic cells. I describe here some of the regulatory mechanisms that control translation in response to microbial products or cytokine exposure and their contribution to the overall immune response.
Subject(s)
Antigen Presentation , Dendritic Cells/immunology , Eukaryotic Initiation Factors/immunology , Immunity, Cellular , Transcriptional Activation , Animals , Cell Differentiation/immunology , Cytotoxicity, Immunologic , Dendritic Cells/metabolism , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Proteasome Endopeptidase Complex , Protein Biosynthesis/immunology , Protein Interaction Domains and Motifs/immunology , Protein Processing, Post-Translational , Proteins , Response Elements/immunology , TOR Serine-Threonine Kinases , Transcription Factors/genetics , Transcription Factors/immunologyABSTRACT
Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.
Subject(s)
Alphavirus Infections/immunology , Eukaryotic Initiation Factors/immunology , Prokaryotic Initiation Factor-3/immunology , Semliki forest virus/immunology , Vaccination , Alphavirus Infections/prevention & control , Animals , Brucella abortus/genetics , Eukaryotic Initiation Factors/genetics , Genetic Engineering , Immunity, Active/genetics , Mice , Mice, Inbred BALB C , Prokaryotic Initiation Factor-3/genetics , Recombination, Genetic , Semliki forest virus/pathogenicity , VirulenceABSTRACT
Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990 s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.
Subject(s)
Immunity , RNA Virus Infections/immunology , RNA Viruses/immunology , RNA, Small Interfering/metabolism , RNA, Viral/immunology , Ribonuclease III/metabolism , Animals , Argonaute Proteins , Eukaryotic Cells/enzymology , Eukaryotic Cells/virology , Eukaryotic Initiation Factors/immunology , Host-Pathogen Interactions/immunology , Humans , Immunity/genetics , Prokaryotic Cells/enzymology , Prokaryotic Cells/virology , RNA Interference/immunology , RNA Processing, Post-Transcriptional/immunology , RNA Virus Infections/enzymology , RNA Virus Infections/prevention & control , RNA, Small Interfering/immunology , RNA, Viral/metabolism , Receptors, Pattern Recognition/immunology , Ribonuclease III/chemistry , Ribonuclease III/genetics , Ribonuclease III/immunology , Virus Diseases/immunologyABSTRACT
We investigated the role of two repressors of translation initiation in granulocytic differentiation using mice with a null mutation in the 4E-BP1 gene or with a null mutation in the 4E-BP2 gene. We show that 4E-BP1(-/-) and 4E-BP2(-/-) mice exhibit an increased number of immature granulocytic precursors, associated with a decreased number of mature granulocytic elements compared with wild-type mice, which is suggestive of an impaired granulocytic differentiation. Clonogenetic analyses revealed a reduced number of granulocytic colonies and concomitant increase in granulo-monocytic colonies in 4E-BP(-/-) mice. Finally, a slight expansion of monocytic cells was observed in the 4E-BP2(-/-) mice. In contrast, we did not observe any significant difference in thymocyte maturation in these mice. These results, together with the fact that 4E-BPs are markedly induced during granulo-monocytic differentiation of myeloid cells in vitro, highlight the pivotal role of 4E-BP1 and 4E-BP2 in the early phases of myelopoiesis. These results represent the first in vivo evidence of the involvement of translation in the early phases of granulo-monocytic differentiation and further extend the role of translation in haematopoietic differentiation.