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

Analysis of Protein Interaction Networks for the Detection of Candidate Hepatitis B and C Biomarkers

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the major causes of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC). The resolution or chronicity of acute infection is dependent on a complex interplay between virus and innate/adaptive immunity. The mechanisms that lead a significant proportion of patients to more severe liver disease are not clearly defined and involve virus induced host gene/protein alterations. The utilization of protein interaction networks (PINs) is expected to identify novel aspects of the disease concerning the patients’ immune response to virus as well as the main pathways that are involved in the development of fibrosis and HCC. In this study, we designed several PINs for HBV and HCV and employed topological, modular, and functional analysis techniques in order to determine significant network nodes that correspond to prominent candidate biomarkers. The networks were built using data from various interaction databases. When the overall PINs of HBV and HCV were compared, 48 nodes were found in common. The implementation of a statistical ranking procedure indicated that three of them are of higher importance

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

Mechanical stimulation of polycystin-1 induces human osteoblastic gene expression via potentiation of the calcineurin/NFAT signaling axis

Mechanical forces trigger biological responses in bone cells that ultimately control osteoblastogenesis and bone program. Although several mechanosensors have been postulated, the precise mechanotransduction pathway remains obscure as the initial mechanosensing event has not yet been identified. Studies in kidney cells have shown that polycystin-1 (PC1), via its extracellular N-terminal part, may function as an “antenna-like” protein providing a linkage between environmental cues and their conversion into biochemical responses that regulate various cellular processes via the calcineurin/NFAT pathway. Here we explored the involvement of PC1 in mechanical load (stretching)-induced signaling cascades that control osteoblastogenesis/bone formation. FACS and TransAM Transcription Factor ELISA analyses employing extracts from primary human osteoblast-like, PC1 expressing cells subjected to mechanical stretching (0-6 h) revealed that unphosphorylated/DNA-binding competent NFATc1 increased at 0.5-1 h and returned to normal at 6 h. In accordance with the activation mechanism of NFATc1, stretching of cultured cells pre-treated with cyclosporin A (CsA, a specific inhibitor of the calcineurin/NFAT pathway) abrogated the observed decrease in the abundance of the cytoplasmic pNFATc1 (phosphorylated/inactive) species. Furthermore, stretching of osteoblastic cells pre-treated with an antibody against the mechanosensing N-terminal part of PC1 also abrogated the observed decrease in the cytoplasmic levels of the inactive pNFATc1 species. Importantly, under similar conditions (pre-incubation of stretched cells with the inhibitory anti-PC1 antibody), the expression of the key osteoblastic, NFATc1-target gene runx2 decreased compared to untreated cells. Therefore, PC1 acts as chief mechanosensing molecule that modulates osteoblastic gene transcription and hence bone-cell differentiation through the calcineurin/NFAT signaling cascade

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

Phenotypic and functional alterations of primary human PBMCs induced by HCV non-enveloped capsid-like particles uptake

Hepatitis C virus non-enveloped particles circulate in the serum of HCV-infected patients and are believed to be involved in viral persistence. It was previously demonstrated that recombinant HCVne particles can efficiently enter T cells. In this study we investigated the effect of this entry on the phenotype and function of PBMCs, focused on the CD4+ and CD8+ T-cells. We have generated recombinant HCVne in the absence of other viral proteins. PBMCs from healthy donors were sampled after incubation either with HCVne or the control at different time points. Levels of expression of CD107a, CD25, CTLA-4, and T regulatory cells were estimated and cytokine expression and secretion were also monitored. Peripheral T cells expressed elevated CD127. The intracellular expression of the inhibitory marker CTLA-4 (CD152) increased significantly on peripheral T cells at late hours post-treatment, compared to the respective non-treated group. Despite the fact that there was an initial immune response due to HCVne uptake, T cells were driven to a partial exhausted phenotype. A significant induction of CD4+CD25+(hi)CD127-regulatory T cells at late hours was observed. Consistently, Foxp3+CD4+ T cells were also increased. In parallel, a significant transcriptional activation and increased secretion of IL-2, IL-10, and IFN-gamma, was recorded. Moreover, mRNA transcription of TGF-beta was considerably elevated. HCVne particles have the potential to shape the immune response by modifying specific phenotypic and functional markers mainly on CD4+ T cells and driving them to partial exhaustion as well as to Treg expansion

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

Impact of Interferon-α Receptor-1 Promoter Polymorphisms on the Transcriptome of the Hepatitis B Virus-Associated Hepatocellular Carcinoma

Background and aimsGenetic polymorphisms within the promoter of interferon-α receptor type-1 (IFNAR1) have been associated with the susceptibility to and the outcome of chronic hepatitis B virus (HBV) infection. However, the impact of these polymorphisms in the transcriptome of the HBV-associated hepatocellular carcinoma (HCC) remains largely unexplored.MethodsUsing whole-genome and exome sequencing data from The Cancer Genome Atlas project, we characterized three single-nucleotide polymorphisms (SNPs: −568G/C, −408C/T, −3C/T) and one variable number tandem repeat [VNTR: −77(GT)n] within the IFNAR1 promoter sequence in 49 HCC patients. RNAseq data from 10 genotyped HCC samples were grouped according to their −77VNTR or −3SNP genotype to evaluate the impact of these polymorphisms on the differential expression on the HCC transcriptome.ResultsThere is a fourfold higher impact of the −77VNTR on the HCC transcriptome compared to the −3SNP (q < 0.1, p < 0.001). The expression of the primary IFNAR1 transcript is not affected by these polymorphisms but a secondary, HCC-specific transcript is expressed only in homozygous −77VNTR ≤8/≤8(GT)n samples (p < 0.05). At the same time, patients carrying at least one −77VNTR >8(GT) allele, presented a strong upregulation of the fibronectin-1 (FN-1) gene, which has been associated with the development of HCC. Gene Ontology and pathway enrichment analysis of the differentially expressed genes revealed a strong disruption of the PI3K–AKT signaling pathway, which can be partially triggered by the extracellular matrix FN-1.ConclusionThe IFNAR-1 promoter polymorphisms are not involved in the expression levels of the main IFNAR-1 transcript. The −77VNTR has a regulatory role on the expression of a secondary, truncated, HCC-specific transcript, which in turn coincides with disruptions in cancer-associated pathways and in FN-1 expression modifications