12 research outputs found
Post-COVID-19 Parkinsonism and Parkinson's Disease Pathogenesis: The Exosomal Cargo Hypothesis
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease, globally. Dopaminergic neuron degeneration in substantia nigra pars compacta and aggregation of misfolded alpha-synuclein are the PD hallmarks, accompanied by motor and non-motor symptoms. Several viruses have been linked to the appearance of a post-infection parkinsonian phenotype. Coronavirus disease 2019 (COVID-19), caused by emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, has evolved from a novel pneumonia to a multifaceted syndrome with multiple clinical manifestations, among which neurological sequalae appear insidious and potentially long-lasting. Exosomes are extracellular nanovesicles bearing a complex cargo of active biomolecules and playing crucial roles in intercellular communication under pathophysiological conditions. Exosomes constitute a reliable route for misfolded protein transmission, contributing to PD pathogenesis and diagnosis. Herein, we summarize recent evidence suggesting that SARS-CoV-2 infection shares numerous clinical manifestations and inflammatory and molecular pathways with PD. We carry on hypothesizing that these similarities may be reflected in exosomal cargo modulated by the virus in correlation with disease severity. Travelling from the periphery to the brain, SARS-CoV-2-related exosomal cargo contains SARS-CoV-2 RNA, viral proteins, inflammatory mediators, and modified host proteins that could operate as promoters of neurodegenerative and neuroinflammatory cascades, potentially leading to a future parkinsonism and PD development
HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle
Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection
Regulation of the internal ribosome entry site (IRES) of Ηepatitis C Virus
HCV infection is a major public health problem and a leading cause of chronic liver disease and hepatocellular carcinoma, with approximately 180 million infected individuals worldwide. HCV is a positive sense RNA virus that belongs to the genus hepacivirus of the Flaviviridae family. Six major HCV genotypes (1–6) are known, each of which can be further subdivided into several subtypes (1a, 1b, 2a, etc). The HCV genome consists of a large open reading frame (ORF), flanked by highly structured 5’ and 3’ untranslated regions (UTRs). Both UTRs are conserved and control viral translation and replication. The HCV 5'-UTR contains an IRES that initiates cap-independent translation of the viral RNA. IRES-mediated translation of the HCV ORF yields a single polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases, giving rise to mature structural and non structural proteins. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. The aim of this study was to understand the regulation of the HCV IRES dependent translation. Firstly, we tried to delineate the role of the viral proteins on HCV IRES dependent translation that still remains controversial. Clearly our studies demonstrated that HCV NS5A down-regulates IRES activity in a cell-type dependent manner. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. In addition, we concluded that the NS5A-mediated inhibitory effect on IRES-dependent translation was linked with the PKR inactivation. Moreover, as it is already reported that localised hypoxic areas are continuously present in HCC due to its high proliferation rate leading to an altered translation pattern, we investigated modulation of HCV IRES activity under low oxygen settings. Our results provided preliminary evidence that HCV-IRES-dependent translation is negatively regulated by low oxygen levels or under hypoxia-mimicking conditions in cell-specific manner.H λοίμωξη που προκαλείται απο τον ιό της ηπατίτιδας C HCV είναι μείζον πρόβλημα για την δημόσια υγεία όπως επίσης και η κύρια αιτία της χρόνιας ηπατικής νόσου και ηπατοκυτταρικού καρκινώματος, με περίπου 180 εκατομμύρια μολυσμένα άτομα σε όλο τον κόσμο. Ο HCV είναι ένας RNA ιός θετικής πολικότητας που ανήκει στο γένος Hepacivirus της οικογένειας των Flaviviridae. Έξι γονότυποι ( 1-6 ) είναι γνωστοί, καθένας από τους οποίους μπορεί να υποδιαιρεθεί περαιτέρω σε αρκετές υποτύπους. Το γονιδίωμα του HCV αποτελείται από ένα μεγάλο ανοικτό πλαίσιο ανάγνωσης (ORF), πλαισιωμένο από ιδιαίτερα δομημένες 5 'και 3' αμετάφραστες περιοχές (UTRs). Και οι δύο περιοχές UTRs είναι συντηρημένες και έχουν τον έλεγχο της ιογενούς μετάφρασης και αναπαραγωγής. Το 5' UTR του HCV περιέχει μια περιοχή IRES απο την οποία γινεται η έναρξη της cap ανεξάρτητης μετάφρασης του ιικού RNA. Η HCV IRES εξαρτώμενη μετάφραση του ORF παράγει μια ενιαία πρόδρομη πολυπρωτεΐνη που μεσω μετα-μεταφραστικής τροποποίησης από κυτταρικές και ιικές πρωτεάσες, οδηγεί στην ωρίμανση δομικών και μη δομικών πρωτεϊνών. Αρκετές μελέτες έχουν δείξει ότι διαφορετικές κυτταρικές ή ιικές πρωτεΐνες μπορούν να ρυθμίσουν την ενεργότητα του HCV IRES . Στόχος της παρούσας μελέτης ήταν η κατανόηση της ρύθμισης της HCV IRES εξαρτώμενης μετάφρασης. Πρώτον, προσπαθήσαμε να ορίσουμε το ρόλο των ιικών πρωτεϊνών στην HCV IRES εξαρτώμενη μετάφραση που παραμένει αμφιλεγόμενος. Σαφώς, οι μελέτες μας έδειξαν ότι η HCV NS5A ρυθμίζει αρνητικά την ενεργότητα του IRES με κύτταρο-εξαρτώμενο τρόπο. Επιπρόσθετα, υπάρχουν ισχυρές ενδείξεις ότι η ενεργοποιημένη PKR ρυθμίζει θετικά την ενεργότητα του IRES ενώ η καταστολή της έκφρασης της ενδογενούς PKR έχει το αντίθετο αποτέλεσμα. Επιπλέον, καταλήξαμε στο συμπέρασμα ότι η NS5A μεσολαβεί ανασταλτικά στην IRES-εξαρτώμενη μετάφραση και συνδέεται με την αδρανοποίηση της PKR. Τέλος στην παρούσα εργασία ερευνήσαμε τη ρύθμιση της ενεργότητας του HCV IRES σε συνθήκες χαμηλού οξυγόνου, δεδομένου ότι υποξικές περιοχές εντοπίζονται στον ηπατοκυτταρικό καρκίνο (HCC) και συνδέονται με την ύπαρξη ενός εναλλακτικού προφίλ κυτταρικής μετάφρασης Τα αποτελέσματά μας δείχνουν ότι η HCV IRES-εξαρτώμενη μετάφραση ρυθμίζεται αρνητικά σε υποξικές συνθήκες και μάλιστα με κύτταρο-εξαρτώμενο τρόπο
Hepatitis Viruses Control Host Immune Responses by Modifying the Exosomal Biogenesis Pathway and Cargo
The development of smart immune evasion mechanisms is crucial for the establishment of acute and chronic viral hepatitis. Hepatitis is a major health problem worldwide arising from different causes, such as pathogens, metabolic disorders, and xenotoxins, with the five hepatitis viruses A, B, C, D, and E (HAV, HBV, HCV, HDV, and HEV) representing the majority of the cases. Most of the hepatitis viruses are considered enveloped. Recently, it was reported that the non-enveloped HAV and HEV are, in reality, quasi-enveloped viruses exploiting exosomal-like biogenesis mechanisms for budding. Regardless, all hepatitis viruses use exosomes to egress, regulate, and eventually escape from the host immune system, revealing another key function of exosomes apart from their recognised role in intercellular communication. This review will discuss how the hepatitis viruses exploit exosome biogenesis and transport capacity to establish successful infection and spread. Then, we will outline the contribution of exosomes in viral persistence and liver disease progression
Targeting the YXXΦ Motifs of the SARS Coronaviruses 1 and 2 ORF3a Peptides by In Silico Analysis to Predict Novel Virus—Host Interactions
The emerging SARS-CoV and SARS-CoV-2 belong to the family of “common cold” RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue—L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific—yet to be discovered—functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence
HCV-Induced Immunometabolic Crosstalk in a Triple-Cell Co-Culture Model Capable of Simulating Systemic Iron Homeostasis
Iron is crucial to the regulation of the host innate immune system and
the outcome of many infections. Hepatitis C virus (HCV), one of the
major viral human pathogens that depends on iron to complete its life
cycle, is highly skilled in evading the immune system. This study
presents the construction and validation of a physiologically relevant
triple-cell co-culture model that was used to investigate the input of
iron in HCV infection and the interplay between HCV, iron, and
determinants of host innate immunity. We recorded the expression
patterns of key proteins of iron homeostasis involved in iron import,
export and storage and examined their relation to the iron regulatory
hormone hepcidin in hepatocytes, enterocytes and macrophages in the
presence and absence of HCV. We then assessed the transcriptional
profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and
interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10)
under normal or iron-depleted conditions and determined how these were
affected by infection. Our data suggest the presence of a link between
iron homeostasis and innate immunity unfolding among liver, intestine,
and macrophages, which could participate in the deregulation of innate
immune responses observed in early HCV infection. Coupled with
iron-assisted enhanced viral propagation, such a mechanism may be
important for the establishment of viral persistence and the ensuing
chronic liver disease
Alterations in the iron homeostasis network: A driving force for macrophage-mediated hepatitis C virus persistency
Mechanisms that favor Hepatitis C virus (HCV) persistence over clearance
are unclear, but involve defective innate immunity. Chronic infection is
characterized by hepatic iron overload, hyperferraemia and
hyperferittinaemia. Hepcidin modulates iron egress via ferroportin and
its storage in ferritin. Chronic HCV patients have decreased hepcidin,
while HCV replication is modified by HAMP silencing. We aimed to
investigate interactions between HCV and hepcidin, during acute and
chronic disease, and putative alterations in cellular iron homeostasis
that enhance HCV propagation and promote viral persistence. Thus, we
used HCV JFH-1-infected co-cultures of Huh7.5 hepatoma and THP-1
macrophage cells, HCV patients' sera and Huh7 hepcidin-expressing cells
transfected with HCV replicons. Hepcidin levels were elevated in acutely
infected patients, but correlated with viral load in chronic patients.
HAMP expression was up-regulated early in HCV infection in vitro, with
corresponding changes in ferritin and FPN. Hepcidin overexpression
enhanced both viral translation and replication. In HCV-infected
co-cultures, we observed increased hepcidin, reduced hepatoma ferritin
and a concurrent rise in macrophaghic ferritin over time. Altered iron
levels complemented amplified replication in hepatoma cells and one
replication round in macrophages. Iron-loading of macrophages led to
enhancement of hepatic HCV replication through reversed ferritin flow.
Viral transmissibility from infected macrophages to naive hepatoma cells
was induced by iron. We propose that HCV control over iron occurs both
by intracellular iron sequestration, through hepcidin, and intercellular
iron mobilisation via ferritin, as means toward enhanced replication.
Persistence could be achieved through HCV-induced changes in macrophagic
iron that enhances viral replication in these cells
Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1 alpha activity
Background & Aims: HCV relies on host lipid metabolism to complete its
life cycle and HCV core is crucial to this interaction. Liver secreted
ANGPTL-3 is an LXR-and HNF-1 alpha-regulated protein, which plays a key
role in lipid metabolism by increasing plasma lipids via inhibition of
lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3
by HCV core and identify the molecular mechanisms involved.
Methods: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein
levels in HCV patients, the JFH-1 infectious system and liver cell
lines. Transfections, chromatin immunoprecipitation and
immunofluorescence delineated parts of the molecular mechanisms
implicated in the core-mediated regulation of ANGPTL-3 gene expression.
Results: ANGPTL-3 gene expression was decreased in HCV-infected patients
and the JFH-1 infectious system. mRNA and promoter activity levels were
down-regulated by core. The response was lost when an HNF-1 alpha
element in ANGPTL-3 promoter was mutated, while loss of HNF-1 alpha DNA
binding to this site was recorded in the presence of HCV core. HNF-1
alpha mRNA and protein levels were not altered by core. However,
trafficking between nucleus and cytoplasm was observed and then blocked
by an inhibitor of the HNF-1 alpha-specific kinase Mirk/Dyrk1B.
Transactivation of LXR/RXR signalling could not restore coremediated
down-regulation of ANGPTL-3 promoter activity.
Conclusions: ANGPTL-3 is negatively regulated by HCV in vivo and in
vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1
alpha binding activity and blockage of LXR/RXR transactivation. The
putative ensuing increase in serum lipid clearance and uptake by the
liver may sustain HCV virus replication and persistence. (C) 2013
European Association for the Study of the Liver. Published by Elsevier
B.V. All rights reserved
lncRNA NORAD is consistently detected in breastmilk exosomes and its expression is downregulated in mothers of preterm infants
Breast milk is the ideal food for infants and undoubtedly has immediate
and long-term benefits. Breast milk contains extracellular vesicles
(EVs) i.e., exosomes secreted by maternal breast cells. Exosomes carry
genetic material, such as long non-coding RNAs (lncRNAs), which possibly
participate in cell-to-cell communications, as they are known to
regulate critical gene pathways. The aim of the present study was to
screen human breastmilk exosomes for their lncRNA cargo and to examine
exosomal lncRNA levels associated with milk obtained from mothers that
gave birth at term or prematurely (<37 weeks of gestation). Samples were
collected at 3 weeks postpartum from 20 healthy, breastfeeding mothers;
10 mothers had given birth at full-term and 10 mothers preterm. Exosomal
RNA was extracted from all samples and the expression of 88 distinct
lncRNAs was determined using reverse transcription-quantitative PCR. A
total of 13 lncRNAs were detected in >= 85% of the samples, while 31
were detected in >= 50% of the samples. Differential expression
analysis of the lncRNAs between the two groups revealed >= 2-fold
differences, with generally higher lncRNA concentrations found in the
milk of the mothers that gave birth at term compared with those that
gave birth preterm. Among these, the non-coding RNA activated at DNA
damage (NORAD) was prominently detected in both groups, and its
expression was significantly downregulated in the breast milk exosomes
of mothers who delivered preterm. On the whole, the present study
demonstrates that breast milk lncRNAs may be important factors of normal
early human development. Collectively, the presence of lncRNAs in human
breast milk may explain the consistent inability of researchers to fully
‘humanize’ animal milk