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1.
Hum Genomics ; 16(1): 59, 2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2119172

ABSTRACT

BACKGROUND: AU-rich elements (AREs) are located in the 3'UTRs of 22% of human mRNAs, including most transiently expressed inflammatory mediators. By default, AREs mark mRNAs for decay and translational inhibition, but this activity can be temporarily inhibited in case of infection to allow the onset of inflammation. Morbidity and mortality in COVID-19 patients have been associated with dysregulated inflammation, a process that may include aberrant ARE activity. RESULTS: RNA-seq data from available transcriptomic studies were analyzed to investigate a possible differential expression of mRNAs that contain AREs in the context of SARS-CoV-2 infections. ARE-mRNAs turned out to be significantly overrepresented among the upregulated mRNAs after SARS-CoV-2 infection (up to 42%). In contrast, ARE-mRNAs were underrepresented (16%) in the downregulated group. Consequently, at a global scale, ARE-mRNAs are significantly more upregulated after SARS-CoV-2 infection compared to non-ARE mRNAs. This observation was apparent in lung cell line models such as A549 and Calu-3 and with infections with other respiratory viruses and cell lines. Most importantly, at the clinical level, the elevated ARE-mRNA response appeared strongest in blood cells of COVID-19 patients with mild disease. It diminished with disease severity and was least apparent in patients in need of intubation and respiratory-related death. Gene function and clustering analysis suggest that the ARE-response is rather global and the upregulated ARE-mRNAs in patients with mild disease do not particularly cluster in specific functional groups. CONCLUSIONS: Compared to the rest of the transcriptome, ARE-containing mRNAs are preferentially upregulated in response to viral infections at a global level. In the context of COVID-19, they are most upregulated in mild disease. Due to their large number, their levels measured by RNA-seq may provide a reliable indication of COVID-19 severity.


Subject(s)
COVID-19 , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , COVID-19/genetics , Up-Regulation/genetics , SARS-CoV-2 , Inflammation
2.
Emerg Microbes Infect ; 11(1): 2529-2543, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2107214

ABSTRACT

Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.


Subject(s)
Middle East Respiratory Syndrome Coronavirus , Middle East Respiratory Syndrome Coronavirus/physiology , AMP-Activated Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , RNA, Messenger/metabolism , Lysosomes/metabolism , Autophagy , Endonucleases/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Adenosine Triphosphate/metabolism
3.
Redox Biol ; 56: 102465, 2022 10.
Article in English | MEDLINE | ID: covidwho-2105815

ABSTRACT

BACKGROUND: The pathophysiologic significance of redox imbalance is unquestionable as numerous reports and topic reviews indicate alterations in redox parameters during corona virus disease 2019 (COVID-19). However, a more comprehensive understanding of redox-related parameters in the context of COVID-19-mediated inflammation and pathophysiology is required. METHODS: COVID-19 subjects (n = 64) and control subjects (n = 19) were enrolled, and blood was drawn within 72 h of diagnosis. Serum multiplex assays and peripheral blood mRNA sequencing was performed. Oxidant/free radical (electron paramagnetic resonance (EPR) spectroscopy, nitrite-nitrate assay) and antioxidant (ferrous reducing ability of serum assay and high-performance liquid chromatography) were performed. Multivariate analyses were performed to evaluate potential of indicated parameters to predict clinical outcome. RESULTS: Significantly greater levels of multiple inflammatory and vascular markers were quantified in the subjects admitted to the ICU compared to non-ICU subjects. Gene set enrichment analyses indicated significant enhancement of oxidant related pathways and biochemical assays confirmed a significant increase in free radical production and uric acid reduction in COVID-19 subjects. Multivariate analyses confirmed a positive association between serum levels of VCAM-1, ICAM-1 and a negative association between the abundance of one electron oxidants (detected by ascorbate radical formation) and mortality in COVID subjects while IL-17c and TSLP levels predicted need for intensive care in COVID-19 subjects. CONCLUSION: Herein we demonstrate a significant redox imbalance during COVID-19 infection affirming the potential for manipulation of oxidative stress pathways as a new therapeutic strategy COVID-19. However, further work is requisite for detailed identification of oxidants (O2•-, H2O2 and/or circulating transition metals such as Fe or Cu) contributing to this imbalance to avoid the repetition of failures using non-specific antioxidant supplementation.


Subject(s)
COVID-19 , Antioxidants/metabolism , Electron Spin Resonance Spectroscopy , Free Radicals , Humans , Hydrogen Peroxide , Intercellular Adhesion Molecule-1/metabolism , Interleukin-17/metabolism , Nitrates , Nitrites , Oxidants/metabolism , Oxidation-Reduction , Oxidative Stress , RNA, Messenger/metabolism , Uric Acid , Vascular Cell Adhesion Molecule-1/metabolism
4.
PLoS Pathog ; 18(11): e1010930, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2098781

ABSTRACT

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , COVID-19/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Cytoplasm/metabolism , Mammals
5.
Int J Mol Sci ; 23(20)2022 Oct 19.
Article in English | MEDLINE | ID: covidwho-2082081

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV, now appears likely to mediate 2019-nCoV entry into human cells. However, inhibitors such as PAP-1 and bergamottin have been discovered; both of them can preferentially bind to ACE2, prevent RBD Spike S protein from binding to ACE2, and reduce the binding sites for RBD Spike S protein. In addition, we investigated the binding energy of PAP-1 and bergamottin with ACE2 through molecular docking with bio-layer interferometry (BLI) and found relatively high binding affinity (KD = 48.5 nM, 53.1 nM) between the PAP-1 and bergamottin groups. In addition, the nanomolar fraction had no effect on growth of the AT-II cell, but 150 µM PAP-1 and 75 µM bergamottin inhibited the proliferation of AT-II cells in vitro by 75% and 68%, respectively. Meanwhile, they significantly reduced ACE2 mRNA and proteins by 67%, 58% and 55%, 41%, respectively. These results indicate that psoralen compounds PAP-1 and bergamottin binding to ACE2 protein could be further developed in the fight against COVID-19 infection during the current pandemic. However, attention should be paid to the damage to human alveolar type II epithelial cells.


Subject(s)
COVID-19 , Furocoumarins , Humans , Angiotensin-Converting Enzyme 2 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Molecular Docking Simulation , COVID-19/drug therapy , Peptidyl-Dipeptidase A/metabolism , Furocoumarins/pharmacology , RNA, Messenger/metabolism , Protein Binding
6.
Viruses ; 14(10)2022 09 30.
Article in English | MEDLINE | ID: covidwho-2066551

ABSTRACT

Molecular therapies exploiting mRNA vectors embody enormous potential, as evidenced by the utility of this technology for the context of the COVID-19 pandemic. Nonetheless, broad implementation of these promising strategies has been restricted by the limited repertoires of delivery vehicles capable of mRNA transport. On this basis, we explored a strategy based on exploiting the well characterized entry biology of adenovirus. To this end, we studied an adenovirus-polylysine (AdpL) that embodied "piggyback" transport of the mRNA on the capsid exterior of adenovirus. We hypothesized that the efficient steps of Ad binding, receptor-mediated entry, and capsid-mediated endosome escape could provide an effective pathway for transport of mRNA to the cellular cytosol for transgene expression. Our studies confirmed that AdpL could mediate effective gene transfer of mRNA vectors in vitro and in vivo. Facets of this method may offer key utilities to actualize the promise of mRNA-based therapeutics.


Subject(s)
Adenoviridae Infections , COVID-19 , Humans , Adenoviridae/genetics , Genetic Vectors/genetics , Gene Transfer Techniques , Polylysine , RNA, Messenger/genetics , RNA, Messenger/metabolism , Pandemics , Capsid Proteins/genetics , Capsid Proteins/metabolism , Biology
7.
Front Immunol ; 13: 989298, 2022.
Article in English | MEDLINE | ID: covidwho-2065518

ABSTRACT

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins , Positive-Strand RNA Viruses , Animals , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Life Cycle Stages , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins , Viral Proteins/metabolism
8.
Kidney Blood Press Res ; 47(9): 565-575, 2022.
Article in English | MEDLINE | ID: covidwho-2064352

ABSTRACT

INTRODUCTION: The angiotensin-converting enzyme 2 (ACE2) as well as the transmembrane protease serine type 2 (TMPRSS2) have been found to play roles in cell entry for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19). SARS-CoV-2 infection risk and severity of COVID-19 might be indicated by the expression of ACE2 and TMPRSS2 in the lung. METHODS: A high-salt diet rat model and renin-angiotensin-aldosterone system (RAAS) blockade were used to test whether these factors affect ACE2 and TMPRSS2 expression in the lung. A normal (0.3% NaCl), a medium (2% NaCl), or a high (8% NaCl) salt diet was fed to rats for 12 weeks, along with enalapril or telmisartan, before examining the lung for histopathological alteration. Using immunofluorescence and qRT-PCR, the localization as well as mRNA expression of ACE2 and TMPRSS2 were investigated. RESULTS: The findings provide evidence that both TMPRSS2 and ACE2 are highly expressed in bronchial epithelial cells as well as ACE2 was also expressed in alveolar type 2 cells. High-salt diet exposure in rats leads to elevated ACE2 expression on protein level. Treatment with RAAS blockers had no effect on lung tissue expression of ACE2 and TMPRSS2. CONCLUSIONS: These findings offer biological support regarding the safety of these drugs that are often prescribed to COVID-19 patients with cardiovascular comorbidity. High salt intake, on the other hand, might adversely affect COVID-19 outcome. Our preclinical data should stimulate clinical studies addressing this point of concern.


Subject(s)
COVID-19 , Renin-Angiotensin System , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Animals , Enalapril/pharmacology , Lung , RNA, Messenger/metabolism , Rats , Renin-Angiotensin System/drug effects , Serine Endopeptidases , Sodium Chloride, Dietary/adverse effects , Telmisartan/pharmacology
9.
Sci Rep ; 12(1): 17145, 2022 Oct 13.
Article in English | MEDLINE | ID: covidwho-2062274

ABSTRACT

mRNA vaccines for SARS-CoV-2 have been widely used and saving millions of people in the world. How efficiently proteins are produced from exogenous mRNAs in the embryonic brain, however, is less known. Here we show that protein expression occurs highly efficiently in neural stem cells, in a very narrow time window after mRNA electroporation in the embryonic mouse brain, where plasmids have been successfully transfected. Protein expression is detected 1 h and 12 h after the electroporation of mRNAs and plasmids, respectively. The delivery of exogenous mRNAs may be useful for not only vaccines but also functional analysis in the brain.


Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Brain/metabolism , COVID-19/genetics , Electroporation , Humans , Mice , Plasmids , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2
10.
Proc Natl Acad Sci U S A ; 119(42): e2202322119, 2022 10 18.
Article in English | MEDLINE | ID: covidwho-2050721

ABSTRACT

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.


Subject(s)
COVID-19 , Neurodegenerative Diseases , RNA-Dependent RNA Polymerase , Ribosomes , Viral Nonstructural Proteins , Alzheimer Disease , Amyotrophic Lateral Sclerosis , Animals , COVID-19/genetics , Drosophila , Humans , Neurodegenerative Diseases/genetics , Pandemics , Parkinson Disease , Proto-Oncogene Proteins c-akt , RNA, Messenger/metabolism , Ribosomes/genetics , Ribosomes/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism
11.
Parasit Vectors ; 15(1): 297, 2022 Aug 24.
Article in English | MEDLINE | ID: covidwho-2038858

ABSTRACT

BACKGROUND: The effective transmission mode of Neospora caninum, with infection leading to reproductive failure in ruminants, is vertical transmission. The uterus is an important reproductive organ that forms the maternal-fetal interface. Neospora caninum can successfully invade and proliferate in the uterus, but the molecular mechanisms underlying epithelial-pathogen interactions remain unclear. Accumulating evidence suggests that host long noncoding RNAs (lncRNAs) play important roles in cellular molecular regulatory networks, with reports that these RNA molecules are closely related to the pathogenesis of apicomplexan parasites. However, the expression profiles of host lncRNAs during N. caninum infection has not been reported. METHODS: RNA sequencing (RNA-seq) analysis was used to investigate the expression profiles of messenger RNAs (mRNAs) and lncRNAs in caprine endometrial epithelial cells (EECs) infected with N. caninum for 24 h (TZ_24h) and 48 h (TZ_48 h), and the potential functions of differentially expressed (DE) lncRNAs were predicted by using Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of their mRNA targets. RESULTS: RNA-seq analysis identified 1280.15 M clean reads in 12 RNA samples, including six samples infected with N. caninum for 24 h (TZ1_24h-TZ3_24h) and 48 h (TZ1_48h-TZ3_48h), and six corresponding control samples (C1_24h-C3_24h and C1_48h-C3_48h). Within the categories TZ_24h-vs-C_24h, TZ_48h-vs-C_48h and TZ_48h-vs-TZ_24h, there were 934 (665 upregulated and 269 downregulated), 1238 (785 upregulated and 453 downregulated) and 489 (252 upregulated and 237 downregulated) DEmRNAs, respectively. GO enrichment and KEGG analysis revealed that these DEmRNAs were mainly involved in the regulation of host immune response (e.g. TNF signaling pathway, MAPK signaling pathway, transforming growth factor beta signaling pathway, AMPK signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway), signaling molecules and interaction (e.g. cytokine-cytokine receptor interaction, cell adhesion molecules and ECM-receptor interaction). A total of 88 (59 upregulated and 29 downregulated), 129 (80 upregulated and 49 downregulated) and 32 (20 upregulated and 12 downregulated) DElncRNAs were found within the categories TZ_24h-vs-C_24h, TZ_48h-vs-C_48h and TZ_48h-vs-TZ_24h, respectively. Functional prediction indicated that these DElncRNAs would be involved in signal transduction (e.g. MAPK signaling pathway, PPAR signaling pathway, ErbB signaling pathway, calcium signaling pathway), neural transmission (e.g. GABAergic synapse, serotonergic synapse, cholinergic synapse), metabolism processes (e.g. glycosphingolipid biosynthesis-lacto and neolacto series, glycosaminoglycan biosynthesis-heparan sulfate/heparin) and signaling molecules and interaction (e.g. cytokine-cytokine receptor interaction, cell adhesion molecules and ECM-receptor interaction). CONCLUSIONS: This is the first investigation of global gene expression profiles of lncRNAs during N. caninum infection. The results provide valuable information for further studies of the roles of lncRNAs during N. caninum infection.


Subject(s)
Coccidiosis , Neospora , RNA, Long Noncoding , Animals , Coccidiosis/veterinary , Cytokines/genetics , Epithelial Cells/metabolism , Female , Gene Expression Profiling , Goats , Humans , Neospora/genetics , Neospora/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytokine/genetics , Sequence Analysis, RNA
12.
Int J Mol Sci ; 23(17)2022 Aug 25.
Article in English | MEDLINE | ID: covidwho-2023745

ABSTRACT

Discovery of the microbiota-gut-brain axis has led to proposed microbe-based therapeutic strategies in mental health, including the use of mood-altering bacterial species, termed psychobiotics. However, we still have limited understanding of the key signaling pathways engaged by specific organisms in modulating brain function, and evidence suggests that bacteria with broadly similar neuroactive and immunomodulatory actions can drive different behavioral outcomes. We sought to identify pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut-brain signaling pathways but have distinct effects on behaviour. We used RNAseq to identify mRNAs differentially expressed in the blood and hippocampus of mice following Lacticaseibacillus rhamnosus JB-1, and Limosilactobacillus reuteri 6475 treatment and performed Gene Set Enrichment Analysis (GSEA) to identify enrichment in pathway activity. L. rhamnosus, but not L. reuteri treatment altered several pathways in the blood and hippocampus, and the rhamnosus could be clearly distinguished based on mRNA profile. In particular, L. rhamnosus treatment modulated the activity of interferon signaling, JAK/STAT, and TNF-alpha via NF-KB pathways. Our results highlight that psychobiotics can induce complex changes in host gene expression, andin understanding these changes, we may help fine-tune selection of psychobiotics for treating mood disorders.


Subject(s)
Lactobacillus rhamnosus , Probiotics , Affect , Animals , Brain/metabolism , Hippocampus , Male , Mice , Probiotics/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism
13.
Methods Mol Biol ; 2573: 77-87, 2022.
Article in English | MEDLINE | ID: covidwho-2007118

ABSTRACT

Modified mRNA (modRNA) is a safe and effective vector for gene-based therapies. Notably, the safety of modRNA has been validated through COVID-19 vaccines which incorporate modRNA technology to translate spike proteins. Alternative gene delivery methods using plasmids, lentiviruses, adenoviruses, and adeno-associated viruses have suffered from key challenges such as genome integration, delayed and uncontrolled expression, and immunogenic responses. However, modRNA poses no risk of genome integration, has transient and rapid expression, and lacks an immunogenic response. Our lab utilizes modRNA-based therapies to promote cardiac regeneration following myocardial infarction and heart failure. We have also developed and refined an optimized and economical method for synthesis of modRNA. Here, we provide an updated methodology with improved translational efficiency for in vitro and in vivo application.


Subject(s)
COVID-19 Vaccines , COVID-19 , COVID-19/therapy , Gene Transfer Techniques , Genetic Therapy/methods , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism
14.
FASEB J ; 36(9): e22494, 2022 09.
Article in English | MEDLINE | ID: covidwho-1997082

ABSTRACT

In a rat middle cerebral artery occlusion (MACo) model of ischemic stroke, intracerebroventricular administration of human recombinant hepatocyte growth factor (HGF) mitigated motor impairment and cortical infarction. Recombinant HGF reduced MCAo-induced TNFα and IL1ß expression, and alleviated perilesional reactivation of microglia and astrocyte. All of the aforementioned beneficial effects of HGF were antagonized by an inhibitor to the type II transmembrane serine protease matriptase (MTP). MCAo upregulated MTP mRNA and protein in the lesioned cortex. MTP protein, not the mRNA, was increased further by recombinant HGF but reduced when MTP inhibitor (MTPi) was added to the treatment. Changes of the endogenous active HGF by MCAo, HGF or MTPi paralleled with the changes of MTP protein under the same conditions whilst neither HGF mRNA nor the total endogenous HGF protein were altered. These data showed that the therapeutic effects of HGF in stroke brain is attributed to its proteolytic activation and that MTP is a main protease of the event. MCAo enhanced MTP mRNA and thus protein expression; the initial use of the recombinant active HGF stabilized MCAo-induced MTP protein and subsequent activation of endogenous latent HGF which in turn stabilized further MTP protein. A reciprocal regulation between MTP and HGF appears to be present where MTP promotes HGF activation and the active HGF prevents MTP protein turnover. This study, for the first time, shows that MTP can participate in neural protection in stroke brain through activation of HGF. The cycles of HGF-MTP regulation achieved preservation of the neurological activity.


Subject(s)
Hepatocyte Growth Factor , Stroke , Animals , Brain/metabolism , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Humans , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/metabolism , Neuroprotection , RNA, Messenger/metabolism , Rats , Serine Endopeptidases , Serine Proteases/metabolism , Stroke/metabolism
15.
BMC Genomics ; 23(1): 586, 2022 Aug 13.
Article in English | MEDLINE | ID: covidwho-1993328

ABSTRACT

BACKGROUND: Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. MicroRNAs and long noncoding RNAs are two relevant non-coding RNAs (ncRNAs) class and play crucial roles in a variety of physiological processes. Increased evidence indicates a complex interaction between mRNA and ncRNA. However, our understanding of the function of ncRNA involved in host-PEDV interaction is limited. RESULTS: A total of 1,197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Moreover, we constructed a miRNA-mRNA-pathway network using bioinformatics, including 12 DE mRNAs, 120 DE miRNAs, and 11 pathways. Finally, the target genes of DE miRNAs were screened by bioinformatics, and we constructed immune-related lncRNA-miRNA-mRNA ceRNA networks. Then, the selected DE genes were validated by qRT-PCR, which were consistent with the results from RNA-Seq data. CONCLUSIONS: This study provides the comprehensive analysis of the expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.


Subject(s)
MicroRNAs , Porcine epidemic diarrhea virus , RNA, Long Noncoding , Animals , Gene Regulatory Networks , MicroRNAs/genetics , MicroRNAs/metabolism , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Swine
16.
Nat Commun ; 13(1): 4782, 2022 08 15.
Article in English | MEDLINE | ID: covidwho-1991594

ABSTRACT

The emergence of heavily mutated SARS-CoV-2 variants of concern (VOCs) place the international community on high alert. In addition to numerous mutations that map in the spike protein of VOCs, expression of the viral accessory proteins ORF6 and ORF9b also elevate; both are potent interferon antagonists. Here, we present the crystal structures of Rae1-Nup98 in complex with the C-terminal tails (CTT) of SARS-CoV-2 and SARS-CoV ORF6 to 2.85 Å and 2.39 Å resolution, respectively. An invariant methionine (M) 58 residue of ORF6 CTT extends its side chain into a hydrophobic cavity in the Rae1 mRNA binding groove, resembling a bolt-fitting-hole; acidic residues flanking M58 form salt-bridges with Rae1. Our mutagenesis studies identify key residues of ORF6 important for its interaction with Rae1-Nup98 in vitro and in cells, of which M58 is irreplaceable. Furthermore, we show that ORF6-mediated blockade of mRNA and STAT1 nucleocytoplasmic transport correlate with the binding affinity between ORF6 and Rae1-Nup98. Finally, binding of ORF6 to Rae1-Nup98 is linked to ORF6-induced interferon antagonism. Taken together, this study reveals the molecular basis for the antagonistic function of Sarbecovirus ORF6, and implies a strategy of using ORF6 CTT-derived peptides for immunosuppressive drug development.


Subject(s)
Active Transport, Cell Nucleus , SARS Virus , SARS-CoV-2 , Viral Proteins , Interferons/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/metabolism , Viral Proteins/chemistry
17.
Int Immunopharmacol ; 111: 109054, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-1983253

ABSTRACT

The objective was to evaluate effects of niacin on the intestinal epithelial barrier, intestinal immunity, and microbial community in weaned piglets challenged by Porcine Deltacoronavirus (PDCoV). In this study, fifteen weaned piglets were randomly assigned to 1 of 3 groups, (1) control group, normal diet; (2) PDCoV group, infected with 1 × 107 TCID50 of the PDCoV CHN-HN-17 strain by oral administration; (3) NA + PDCoV group, infected with 1 × 107 TCID50 of the PDCoV CHN-HN-17 strain by oral administration following administration of 40 mg of niacin for three days. The results showed that PDCoV infection induced diarrhea and other clinical symptoms with intestinal villi shedding and atrophy in weaned piglets. Niacin alleviated the symptoms of diarrhea and intestinal damage of PDCoV-infected weaned piglets. Additionally, PDCoV increased (P < 0.05) the mRNA expression of tight junction proteins [zonula occludens-1 (ZO-1) and Claudin] and antimicrobial peptides [porcine ß defensin 1 (pBD1), pBD2, proline-arginine rich 39-amino acid peptide (PR39) and protegrin 1-5 (PG1-5) in the jejunum and ileum of weaned piglets, while niacin increased (P < 0.05) the expression of PG1-5 compared with PDCoV. PDCoV increased (P < 0.05) the contents of serum interleukin-1ß (IL-1ß), IL-8 and intestinal IL-8, and up-regulated the mRNA expression of tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, IL-10, IL-12, and IL-18 in ileum of weaned piglets compared with control. However, niacin decreased (P < 0.05) the contents of serum IL-1ß, IL-6 and intestinal IL-10 and IL-8, and also reduced (P < 0.05) the mRNA expression of ileal TNF-α, IL-10 and IL-12 in the PDCoV-infected piglets. Compared with control, PDCoV up-regulated (P < 0.05) the mRNA expression of key genes related to innate immune and antiviral molecules [toll-like receptor 4 (TLR4), NOD1, NOD2, DDX58, CCL2, STAT2, Mx1, IFN-γ, and protein kinase R (PKR) in the ileum of weaned piglets. Niacin decreased (P < 0.05) the mRNA expression of NOD1, NOD2, STAT2, IFN-γ, and PKR in PDCoV-infected weaned piglets. Moreover, the mRNA expression of IL-6 decreased (P < 0.05) and 2'-5'-oligoadenylate synthetase (OAS), IFN-α, and PKR increased (P < 0.05) in PDCoV-infected IPEC-J2 cells treated with niacin in vitro. Furthermore, niacin decreased (P < 0.05) the elevation of protein expression including inducible NOS (iNOS), nuclear factor-κB (NF-κB p65), inhibitor kappa B (IKKß), histone deacetylase [Sirtuin 1 (SIRT1) and histone deacetylase 7 (HDAC7) and phosphorylation of histone H3 at serine s10 (pH3s10) in the ileum of PDCoV-infected piglets, and increased (P < 0.05) the expression of G protein-coupled receptor (GPR109A). PDCoV disrupted the composition and structure of microflora in the colon of weaned piglets, and reduced the relative abundance of the beneficial bacteria Spirobacterium, but niacin could improve the intestinal microbial flora of the PDCoV-infected piglets associated with increasing the relative abundance of Lactobacillus. Overall, niacin could alleviate diarrhea, intestinal barrier damages, intestinal immune response and colonic microflora disfunction in PDCoV-infected weaned piglets.


Subject(s)
Microbiota , Niacin , Animals , Diarrhea/metabolism , Histone Deacetylases/metabolism , Interleukin-10/metabolism , Interleukin-12/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Intestinal Mucosa/metabolism , Niacin/pharmacology , RNA, Messenger/metabolism , Swine , Tumor Necrosis Factor-alpha/metabolism
18.
Cell Rep ; 40(9): 111300, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-1982705

ABSTRACT

Synthetic mRNA technology is a promising avenue for treating and preventing disease. Key to the technology is the incorporation of modified nucleotides such as N1-methylpseudouridine (m1Ψ) to decrease immunogenicity of the RNA. However, relatively few studies have addressed the effects of modified nucleotides on the decoding process. Here, we investigate the effect of m1Ψ and the related modification pseudouridine (Ψ) on translation. In a reconstituted system, we find that m1Ψ does not significantly alter decoding accuracy. More importantly, we do not detect an increase in miscoded peptides when mRNA containing m1Ψ is translated in cell culture, compared with unmodified mRNA. We also find that m1Ψ does not stabilize mismatched RNA-duplex formation and only marginally promotes errors during reverse transcription. Overall, our results suggest that m1Ψ does not significantly impact translational fidelity, a welcome sign for future RNA therapeutics.


Subject(s)
COVID-19 Vaccines , COVID-19 , COVID-19/prevention & control , Humans , Nucleotides , Proteins , Pseudouridine/genetics , RNA , RNA, Messenger/genetics , RNA, Messenger/metabolism , Vaccines, Synthetic , mRNA Vaccines
19.
Viruses ; 14(7)2022 Jul 08.
Article in English | MEDLINE | ID: covidwho-1964117

ABSTRACT

The SARS-CoV-2 infection generates up to nine different sub-genomic mRNAs (sgRNAs), in addition to the genomic RNA (gRNA). The 5'UTR of each viral mRNA shares the first 75 nucleotides (nt.) at their 5'end, called the leader, but differentiates by a variable sequence (0 to 190 nt. long) that follows the leader. As a result, each viral mRNA has its own specific 5'UTR in term of length, RNA structure, uORF and Kozak context; each one of these characteristics could affect mRNA expression. In this study, we have measured and compared translational efficiency of each of the ten viral transcripts. Our data show that most of them are very efficiently translated in all translational systems tested. Surprisingly, the gRNA 5'UTR, which is the longest and the most structured, was also the most efficient to initiate translation. This property is conserved in the 5'UTR of SARS-CoV-1 but not in MERS-CoV strain, mainly due to the regulation imposed by the uORF. Interestingly, the translation initiation mechanism on the SARS-CoV-2 gRNA 5'UTR requires the cap structure and the components of the eIF4F complex but showed no dependence in the presence of the poly(A) tail in vitro. Our data strongly suggest that translation initiation on SARS-CoV-2 mRNAs occurs via an unusual cap-dependent mechanism.


Subject(s)
COVID-19 , RNA, Guide , 5' Untranslated Regions , Genomics , Humans , Protein Biosynthesis , RNA, Messenger/metabolism , SARS-CoV-2/genetics
20.
Proc Natl Acad Sci U S A ; 119(31): e2121453119, 2022 08 02.
Article in English | MEDLINE | ID: covidwho-1960614

ABSTRACT

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.


Subject(s)
Epithelial-Mesenchymal Transition , RNA, Messenger , RNA, Viral , RNA-Binding Proteins , Unfolded Protein Response , Epithelial-Mesenchymal Transition/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nuclear/metabolism
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