PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity.

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.

The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life.

The ribosome has recently transitioned from being viewed as a passive, indiscriminate machine to a more dynamic macromolecular complex with specialized roles in the cell. Here, we discuss the historical milestones from the discovery of the ribosome itself to how this ancient machinery has gained newfound appreciation as a more regulatory participant in the central dogma of gene expression. The first emerging examples of direct changes in ribosome composition at the RNA and protein level, coupled with an increased awareness of the role individual ribosomal components play in the translation of specific mRNAs, is opening a new field of study centered on ribosome-mediated control of gene regulation. In this Perspective, we discuss our current understanding of the known functions for ribosome heterogeneity, including specialized translation of individual transcripts, and its implications for the regulation and expression of key gene regulatory networks. In addition, we suggest what the crucial next steps are to ascertain the extent of ribosome heterogeneity and specialization and its importance for regulation of the proteome within subcellular space, across different cell types, and during multi-cellular organismal development.

All eggs in one basket: How potyvirus infection is controlled at a single cap-independent translation event.

Regulation of protein synthesis is a strong determinant of potyviral pathogenicity. The Potyviridae family is the largest family of plant-infecting positive sense RNA viruses. Similar to the animal-infecting Picornaviridae family, the potyviral RNA genome lacks a 5' cap, and instead has a viral protein (VPg) linked to its 5' end. Potyviral genomes are mainly translated into one large polyprotein relying on a single translation event to express all their protein repertoire. In the absence of the 5' cap, the Potyviridae family depends on cis-acting elements in their 5' untranslated regions (UTR) to recruit the translation machinery. In this review, we summarize the diverse 5'UTR-driven, cap-independent translation mechanisms employed by the Potyviridae family including scanning-dependent mechanism, internal initiation, and the stimulatory role of the VPg. These mechanisms have direct implications on potyviral pathogenicity, including host range specificity and resistance. Finally, we discuss how these viral strategies could not only inform new avenues for engineering and/or breeding for crop resistance but would also provide opportunities for the development of biotechnological tools for large-scale protein production in plant systems.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes.

Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5' end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ∼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNAi Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ∼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem-loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem--loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAi Met Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

Specific mechanisms of translation initiation in higher eukaryotes: the eIF4G2 story.

The eukaryotic initiation factor 4G2 (eIF4G2, DAP5, Nat1, p97) was discovered in 1997. Over the past two decades, dozens of papers have presented contradictory data on eIF4G2 function. Since its identification, eIF4G2 has been assumed to participate in noncanonical translation initiation mechanisms, but recent results indicate that it can be involved in scanning as well. In particular, eIF4G2 provides leaky scanning through some upstream open reading frames (uORFs), which are typical for long 5' UTRs of mRNAs from higher eukaryotes. It is likely the protein can also help the ribosome overcome other impediments during scanning of the 5' UTRs of animal mRNAs. This may explain the need for eIF4G2 in higher eukaryotes, as many mRNAs that encode regulatory proteins have rather long and highly structured 5' UTRs. Additionally, they often bind to various proteins, which also hamper the movement of scanning ribosomes. This review discusses the suggested mechanisms of eIF4G2 action, denotes obscure or inconsistent results, and proposes ways to uncover other fundamental mechanisms in which this important protein factor may be involved in higher eukaryotes.

A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE: The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

MicroRNA-22-3p displaces critical host factors from the 5' UTR and inhibits the translation of Coxsackievirus B3 RNA.

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Hydrogen peroxide sensitivity connects the activity of COX5A and NPR3 to the regulation of YAP1 expression.

Reactive oxygen species (ROS) are among the most severe types of cellular stressors with the ability to damage essential cellular biomolecules. Excess levels of ROS are correlated with multiple pathophysiological conditions including neurodegeneration, diabetes, atherosclerosis, and cancer. Failure to regulate the severely imbalanced levels of ROS can ultimately lead to cell death, highlighting the importance of investigating the molecular mechanisms involved in the detoxification procedures that counteract the effects of these compounds in living organisms. One of the most abundant forms of ROS is H2 O2 , mainly produced by the electron transport chain in the mitochondria. Numerous genes have been identified as essential to the process of cellular detoxification. Yeast YAP1, which is homologous to mammalian AP-1 type transcriptional factors, has a key role in oxidative detoxification by upregulating the expression of antioxidant genes in yeast. The current study reveals novel functions for COX5A and NPR3 in H2 O2 -induced stress by demonstrating that their deletions result in a sensitive phenotype. Our follow-up investigations indicate that COX5A and NPR3 regulate the expression of YAP1 through an alternative mode of translation initiation. These novel gene functions expand our understanding of the regulation of gene expression and defense mechanism of yeast against oxidative stress.

An Unchartered Journey for Ribosomes: Circumnavigating Circular RNAs to Produce Proteins.

In this issue of Molecular Cell, Legnini et al. (2017) and Pamudurti et al. (2017) demonstrate that endogenous circular RNAs may generate proteins, thereby expanding the eukaryotic proteome and revealing novel modes of cap-independent translation.

Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.

Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression.

False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5' UTR annotations.

Hyperconserved genomic sequences have great promise for understanding core biological processes. It has been recently proposed that scores of hyperconserved 5' untranslated regions (UTRs), also known as transcript leaders (hTLs), encode internal ribosome entry sites (IRESes) that drive cap-independent translation, in part, via interactions with ribosome expansion segments. However, the direct functional significance of such interactions has not yet been definitively demonstrated. We provide evidence that the putative IRESes previously reported in Hox gene hTLs are rarely included in transcript leaders. Instead, these regions function independently as transcriptional promoters. In addition, we find the proposed RNA structure of the putative Hoxa9 IRES is not conserved. Instead, sequences previously shown to be essential for putative IRES activity encode a hyperconserved transcription factor binding site (E-box) that contributes to its promoter activity and is bound by several transcription factors, including USF1 and USF2. Similar E-box sequences enhance the promoter activities of other putative Hoxa gene IRESes. Moreover, we provide evidence that the vast majority of hTLs with putative IRES activity overlap transcriptional promoters, enhancers, and 3' splice sites that are most likely responsible for their reported IRES activities. These results argue strongly against recently reported widespread IRES-like activities from hTLs and contradict proposed interactions between ribosomal expansion segment ES9S and putative IRESes. Furthermore, our work underscores the importance of accurate transcript annotations, controls in bicistronic reporter assays, and the power of synthesizing publicly available data from multiple sources.

Heterogeneous nuclear ribonucleoprotein A3 binds to the internal ribosomal entry site of enterovirus A71 and affects virus replication in neural cells.

Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.

Engineering circular RNA for molecular and metabolic reprogramming.

The role of messenger RNA (mRNA) in biological systems is extremely versatile. However, it's extremely short half-life poses a fundamental restriction on its application. Moreover, the translation efficiency of mRNA is also limited. On the contrary, circular RNAs, also known as circRNAs, are a common and stable form of RNA found in eukaryotic cells. These molecules are synthesized via back-splicing. Both synthetic circRNAs and certain endogenous circRNAs have the potential to encode proteins, hence suggesting the potential of circRNA as a gene expression machinery. Herein, we aim to summarize all engineering aspects that allow exogenous circular RNA (circRNA) to prolong the time that proteins are expressed from full-length RNA signals. This review presents a systematic engineering approach that have been devised to efficiently assemble circRNAs and evaluate several aspects that have an impact on protein production derived from. We have also reviewed how optimization of the key components of circRNAs, including the topology of vector, 5' and 3' untranslated sections, entrance site of the internal ribosome, and engineered aptamers could be efficiently impacting the translation machinery for molecular and metabolic reprogramming. Collectively, molecular and metabolic reprogramming present a novel way of regulating distinctive cellular features, for instance growth traits to neoplastic cells, and offer new possibilities for therapeutic inventions.

UBE4B regulates p27 expression in A549 NSCLC cells through regulating the interaction of HuR and the p27 5' UTR.

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.

Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication.

Hepatitis A virus (HAV) infection often causes acute hepatitis, which results in a case fatality rate of 0.2% and fulminant hepatitis in 0.5% of cases. However, no specific potent anti-HAV drug is available on the market to date. In the present study, we focused on inhibition of HAV internal ribosomal entry site (IRES)-mediated translation and investigated novel therapeutic drugs through drug repurposing by screening for inhibitors of HAV IRES-mediated translation and cell viability using a reporter assay and cell viability assay, respectively. The initial screening of 1,158 drugs resulted in 77 candidate drugs. Among them, nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells. This promising drug also inhibited HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. In the present study, we found that nicotinamide inhibited the activation of activator protein 1 (AP-1) and that knockdown of c-Jun, which is one of the components of AP-1, inhibited HAV HM175 genotype IB IRES-mediated translation and HAV HA11-1299 genotype IIIA and HAV HM175 genotype IB replication. Taken together, the results showed that nicotinamide inhibited c-Jun, resulting in the suppression of HAV IRES-mediated translation and HAV replication, and therefore, it could be useful for the treatment of HAV infection. IMPORTANCE Drug screening methods targeting HAV IRES-mediated translation with reporter assays are attractive and useful for drug repurposing. Nicotinamide (vitamin B3, niacin) has been shown to effectively inhibit HAV replication. Transcription complex activator protein 1 (AP-1) plays an important role in the transcriptional regulation of cellular immunity or viral replication. The results of this study provide evidence that AP-1 is involved in HAV replication and plays a role in the HAV life cycle. In addition, nicotinamide was shown to suppress HAV replication partly by inhibiting AP-1 activity and HAV IRES-mediated translation. Nicotinamide may be useful for the control of acute HAV infection by inhibiting cellular AP-1 activity during HAV infection processes.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.