Unmet needs in the post-DAA era: the risk and molecular mechanisms of hepatocellular carcinoma after HCV eradication.

Hepatitis C virus (HCV) is one of the major etiologies of hepatocellular carcinoma with approximately 30% of HCC being due to HCV infection worldwide. HCV eradication by antivirals greatly reduces the risk of HCC; nevertheless, HCC remains to occur in CHC patients who have achieved a sustained virological response (SVR). The proportion of post-SVR HCC among newly diagnosed HCC patients is increasing in the DAA era and might be due to preexisting inflammatory and fibrotic liver background, immune dysregulation between host and virus interaction, as well as host epigenetic scar, genetic predispositions and alternations. By means of applying surrogate markers and adopting risk stratification, HCC surveillance should be consistently performed in high-risk populations. In this review, we discuss the possible molecular mechanism, risk factors and surveillance strategy for HCC development after HCV eradication.

Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity.

Introduction: The success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. Methods: We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls. Results: In contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. Discussion: These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.

High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. METHODS: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. RESULTS: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. CONCLUSIONS: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

HCV Infection Increases the Expression of ACE2 Receptor, Leading to Enhanced Entry of Both HCV and SARS-CoV-2 into Hepatocytes and a Coinfection State.

Recent studies suggest the enhancement of liver injury in COVID-19 patients infected with Hepatitis C virus (HCV). Hepatocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor, raising the possibility of HCV-SARS-CoV-2 coinfection in the liver. This work aimed to explore whether HCV and SARS-CoV-2 coinfect hepatocytes and the interplay between these viruses. We demonstrate that SARS-CoV-2 coinfects HCV-infected Huh7.5 (Huh7.5HCV) cells. Both viruses replicated efficiently in the coinfected cells, with HCV replication enhanced in coinfected compared to HCV-mono-infected cells. Strikingly, Huh7.5HCV cells were eight fold more susceptible to SARS-CoV-2 pseudoviruses than naive Huh7.5 cells, suggesting enhanced SARS-CoV-2 entry into HCV-preinfected hepatocytes. In addition, we observed increased binding of spike receptor-binding domain (RBD) protein to Huh7.5HCV cells, as well as enhanced cell-to-cell fusion of Huh7.5HCV cells with spike-expressing Huh7.5 cells. We explored the mechanism of enhanced SARS-CoV-2 entry and identified an increased ACE2 mRNA and protein levels in Huh7.5HCV cells, primary hepatocytes, and in data from infected liver biopsies obtained from database. Importantly, higher expression of ACE2 increased HCV infection by enhancing its binding to the host cell, underscoring its role in the HCV life cycle as well. Transcriptome analysis revealed that shared host signaling pathways were induced in HCV-SARS-CoV-2 coinfection. This study revealed complex interactions between HCV and SARS-CoV-2 infections in hepatocytes, which may lead to the increased liver damage recently reported in HCV-positive COVID-19 patients. IMPORTANCE Here, we provide the first experimental evidence for the coexistence of SARS-CoV-2 infection with HCV, and the interplay between them. The study revealed a complex relationship of enhancement between the two viruses, where HCV infection increased the expression of the SARS-CoV-2 entry receptor ACE2, thus facilitating SARS-CoV-2 entry, and potentially, also HCV entry. Thereafter, SARS-CoV-2 infection enhanced HCV replication in hepatocytes. This study may explain the aggravation of liver damage that was recently reported in COVID-19 patients with HCV coinfection and suggests preinfection with HCV as a risk factor for severe COVID-19. Moreover, it highlights the possible importance of HCV treatment for coinfected patients. In a broader view, these findings emphasize the importance of identifying coinfecting pathogens that increase the risk of SARS-CoV-2 infection and that may accelerate COVID-19-related co-morbidities.

Fold-change of chromatin condensation in yeast is a conserved property.

During mitosis, chromatin is condensed and organized into mitotic chromosomes. Condensation is critical for genome stability and dynamics, yet the degree of condensation is significantly different between multicellular and single-cell eukaryotes. What is less clear is whether there is a minimum degree of chromosome condensation in unicellular eukaryotes. Here, we exploited two-photon microscopy to analyze chromatin condensation in live and fixed cells, enabling studies of some organisms that are not readily amenable to genetic modification. This includes the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, and Candida albicans, as well as a protist Trypanosoma brucei. We found that mitotic chromosomes in this range of species are condensed about 1.5-fold relative to interphase chromatin. In addition, we used two-photon microscopy to reveal that chromatin reorganization in interphase human hepatoma cells infected by the hepatitis C virus is decondensed compared to uninfected cells, which correlates with the previously reported viral-induced changes in chromatin dynamics. This work demonstrates the power of two-photon microscopy to analyze chromatin in a broad range of cell types and conditions, including non-model single-cell eukaryotes. We suggest that similar condensation levels are an evolutionarily conserved property in unicellular eukaryotes and important for proper chromosome segregation. Furthermore, this provides new insights into the process of chromatin condensation during mitosis in unicellular organisms as well as the response of human cells to viral infection.

Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies.

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.

Anti-TNFα Treatment Impairs Long-Term Immune Responses to COVID-19 mRNA Vaccine in Patients with Inflammatory Bowel Diseases.

Patients with inflammatory bowel disease (IBD) treated with anti-tumor-necrosis factor-alpha (TNFα) exhibited lower serologic responses one-month following the second dose of the COVID-19 BNT162b2 vaccine compared to those not treated with anti-TNFα (non-anti-TNFα) or to healthy controls (HCs). We comprehensively analyzed long-term humoral responses, including anti-spike (S) antibodies, serum inhibition, neutralization, cross-reactivity and circulating B cell six months post BNT162b2, in patients with IBD stratified by therapy compared to HCs. Subjects enrolled in a prospective, controlled, multi-center Israeli study received two BNT162b2 doses. Anti-S levels, functional activity, specific B cells, antigen cross-reactivity, anti-nucleocapsid levels, adverse events and IBD disease score were detected longitudinally. In total, 240 subjects, 151 with IBD (94 not treated with anti-TNFα and 57 treated with anti-TNFα) and 89 HCs participated. Six months after vaccination, patients with IBD treated with anti-TNFα had significantly impaired BNT162b2 responses, specifically, more seronegativity, decreased specific circulating B cells and cross-reactivity compared to patients untreated with anti-TNFα. Importantly, all seronegative subjects were patients with IBD; of those, >90% were treated with anti-TNFα. Finally, IBD activity was unaffected by BNT162b2. Altogether these data support the earlier booster dose administration in these patients.

Autoimmune Disease Classification Based on PubMed Text Mining.

Autoimmune diseases (AIDs) are often co-associated, and about 25% of patients with one AID tend to develop other comorbid AIDs. Here, we employ the power of datamining to predict the comorbidity of AIDs based on their normalized co-citation in PubMed. First, we validate our technique in a test dataset using earlier-reported comorbidities of seven knowns AIDs. Notably, the prediction correlates well with comorbidity (R = 0.91) and validates our methodology. Then, we predict the association of 100 AIDs and classify them using principal component analysis. Our results are helpful in classifying AIDs into one of the following systems: (1) gastrointestinal, (2) neuronal, (3) eye, (4) cutaneous, (5) musculoskeletal, (6) kidneys and lungs, (7) cardiovascular, (8) hematopoietic, (9) endocrine, and (10) multiple. Our classification agrees with experimentally based taxonomy and ranks AID according to affected systems and gender. Some AIDs are unclassified and do not associate well with other AIDs. Interestingly, Alzheimer's disease correlates well with other AIDs such as multiple sclerosis. Finally, our results generate a network classification of autoimmune diseases based on PubMed text mining and help map this medical universe. Our results are expected to assist healthcare workers in diagnosing comorbidity in patients with an autoimmune disease, and to help researchers in identifying common genetic, environmental, and autoimmune mechanisms.

Rapid electrochemical immunodetection of SARS-CoV-2 using a pseudo-typed vesicular stomatitis virus model.

The COVID-19 pandemic has highlighted the need for reliable and accurate diagnostic tools that provide quantitative results at the point of care. Real-time RT-PCR requires large laboratories, a skilled workforce, complex and costly equipment, and labor-intensive sample processing. Despite tremendous efforts, scaling up RT-PCR tests is seemingly unattainable. To date, hundreds of millions of COVID-19 tests have been performed globally, but the demand for timely, accurate testing continues to outstrip supply. Antigen-based rapid diagnostic testing is emerging as an alternative to RT-PCR. However, the performance of these tests, namely their sensitivity, is still inadequate. To overcome the limitations of currently employed diagnostic tests, new tools that are both sensitive and scalable are urgently needed. We have developed a miniaturized electrochemical biosensor based on the integration of specific monoclonal antibodies with a biochip and a measurement platform, and applied it in the detection of Spike S1 protein, the binding protein of SARS-CoV-2. Using electrochemical impedance spectroscopy, quantitative detection of sub-nanomolar concentrations of Spike S1 was demonstrated, exhibiting a broad detection range. To demonstrate the applicability of the biosensor, we have further developed a SARS-CoV-2 pseudovirus based on Spike protein-pseudo-typed VSV platform. Specific detection of different concentrations of pseudovirus particles was feasible in <30 min. This new tool may largely contribute to the fight against COVID-19 by enabling intensive testing to be performed and alleviating most of the hurdles that plague current diagnostics.

Lower Serologic Response to COVID-19 mRNA Vaccine in Patients With Inflammatory Bowel Diseases Treated With Anti-TNFα.

BACKGROUND & AIM: Patients with inflammatory bowel diseases (IBD), specifically those treated with anti-tumor necrosis factor (TNF)α biologics, are at high risk for vaccine-preventable infections. Their ability to mount adequate vaccine responses is unclear. The aim of the study was to assess serologic responses to messenger RNA-Coronavirus Disease 2019 vaccine, and safety profile, in patients with IBD stratified according to therapy, compared with healthy controls (HCs). METHODS: Prospective, controlled, multicenter Israeli study. Subjects enrolled received 2 BNT162b2 (Pfizer/BioNTech) doses. Anti-spike antibody levels and functional activity, anti-TNFα levels and adverse events (AEs) were detected longitudinally. RESULTS: Overall, 258 subjects: 185 IBD (67 treated with anti-TNFα, 118 non-anti-TNFα), and 73 HCs. After the first vaccine dose, all HCs were seropositive, whereas ∼7% of patients with IBD, regardless of treatment, remained seronegative. After the second dose, all subjects were seropositive, however anti-spike levels were significantly lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (both P < .001). Neutralizing and inhibitory functions were both lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (P < .03; P < .0001, respectively). Anti-TNFα drug levels and vaccine responses did not affect anti-spike levels. Infection rate (∼2%) and AEs were comparable in all groups. IBD activity was unaffected by BNT162b2. CONCLUSIONS: In this prospective study in patients with IBD stratified according to treatment, all patients mounted serologic response to 2 doses of BNT162b2; however, its magnitude was significantly lower in patients treated with anti-TNFα, regardless of administration timing and drug levels. Vaccine was safe. As vaccine serologic response longevity in this group may be limited, vaccine booster dose should be considered.

BNT162b2 mRNA vaccine elicited antibody response in blood and milk of breastfeeding women.

The importance of breastmilk in postnatal life lies in the strong association between breastfeeding and the reduction in the risk of infection and infection-related infant mortality. However, data regarding the induction and dynamics of breastmilk antibodies following administration of the Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine is scarce, as pregnant and lactating women were not included in the initial vaccine clinical trials. Here, we investigate the dynamics of the vaccine-specific antibody response in breastmilk and serum in a prospective cohort of ten lactating women who received two doses of the mRNA vaccine. We show that the antibody response is rapid and highly synchronized between breastmilk and serum, reaching stabilization 14 days after the second dose. The response in breastmilk includes both IgG and IgA with neutralization capacity.

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Tracking Down the Epigenetic Footprint of HCV-Induced Hepatocarcinogenesis.

Hepatitis C virus (HCV) is a major cause of death and morbidity globally and is a leading cause of hepatocellular carcinoma (HCC). Incidence of HCV infections, as well as HCV-related liver diseases, are increasing. Although now, with new direct acting antivirals (DAAs) therapy available, HCV is a curable cancer-associated infectious agent, HCC prevalence is expected to continue to rise because HCC risk still persists after HCV cure. Understanding the factors that lead from HCV infection to HCC pre- and post-cure may open-up opportunities to novel strategies for HCC prevention. Herein, we provide an overview of the reported evidence for the induction of alterations in the transcriptome of host cells via epigenetic dysregulation by HCV infection and describe recent reports linking the residual risk for HCC post-cure with a persistent HCV-induced epigenetic signature. Specifically, we discuss the contribution of the epigenetic changes identified following HCV infection to HCC risk pre- and post-cure, the molecular pathways that are epigenetically altered, the downstream effects on expression of cancer-related genes, the identification of targets to prevent or revert this cancer-inducing epigenetic signature, and the potential contribution of these studies to early prognosis and prevention of HCC as an approach for reducing HCC-related mortality.

Pseudoviruses for the assessment of coronavirus disinfection by ozone.

The COVID-19 pandemic has severely impacted public health worldwide. Evidence of SARS-CoV-2 transmission via aerosols and surfaces has highlighted the need for efficient indoor disinfection methods. For instance, the use of ozone gas as a safe and potent disinfectant against SARS-CoV-2 virus is of particular interest. Here we tested the use of pseudoviruses as a model for evaluating ozone disinfection of the coronavirus at ozone concentrations of 30, 100, and 1000 ppmv. Results show that ozone disinfection rate of pseudoviruses was similar to that of coronavirus 229E (HuCoV-229E) at short contact times, below 30 min. Viral infection decreased by 95% following ozone exposure for 20 min at 1000 ppmv, 30 min at 100 ppmv and about 40 min at 30 ppmv. This findings mean that ozone is a powerful disinfectant toward the enveloped pseudovirus even at low ozone exposure. We also showed that viral disinfection occurs on various contaminated surfaces, with a positive association between disinfection and surface hydrophilicity. Infected surfaces made of aluminum alloy, for example, were better disinfected with ozone as compared to brass, copper, and nickel surfaces. Lastly, we demonstrate the advantage of ozone over liquid disinfectants by showing similar viral disinfection on top, side, bottom, and interior surfaces. Overall, our study demonstrates the potential use of ozone gas disinfection to combat the COVID-19 outbreak.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Hepatitis C Virus Enhances the Invasiveness of Hepatocellular Carcinoma via EGFR-Mediated Invadopodia Formation and Activation.

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer.

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic "scarring" of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

Dysregulation of the cohesin subunit RAD21 by Hepatitis C virus mediates host-virus interactions.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

Studying the Hepatitis C Virus-Induced Epigenetic Signature After Cure with Direct-Acting Antivirals.

Hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC). While direct-acting antiviral (DAA) therapy efficiently eradicates HCV infection, epidemiological studies show that sustained virological response (SVR) following anti-HCV treatment reduces, but does not eliminate, the risk for HCC. We have recently demonstrated that HCV infection induces genome-wide epigenetic changes that reprogram host gene expression and persist as "epigenetic signature" following virus eradication by DAAs. We suggest that this epigenetic signature underlie the residual risk for HCC post-SVR. Here, we provide a methodology to study the HCV-induced epigenetic signature. We describe a ChIP-seq protocol to evaluate changes in epigenome profile following HCV infection, its cure with DAA, and after treatment with epigenetic modifier inhibitor. We also describe evaluation of changes in the gene expression profile using RNA-seq. The integration between detected alterations in epigenetic marks and gene expression allows for identification of biological processes that are involved in HCV-driven oncogenesis before and after cure.