HCV Viral Replication Research Articles

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients.A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

The incidence of cytomegalovirus infection after deceased-donor kidney transplantation from hepatitis-C antibody positive donors to hepatitis-C antibody negative recipients

Background Deceased-donor kidney transplantation (KT) from hepatitis C (HCV)-infected donors into HCV-uninfected recipients (HCV D+/R−) could become standard care in the near future. However, HCV viral replication by viral transmission might lead to a higher incidence of cytomegalovirus (CMV) infection in these recipients. Methods A national-registry-based retrospective cohort study was conducted using the Scientific Registry of Transplant Recipients (SRTR) data set. We assessed the incidence of CMV infection in HCV antibody (Ab) negative recipients receiving kidneys from HCV Ab positive (HCVAb D+/R−) and negative (HCVAb D−/R−) donors. The risk of CMV infection was analyzed by Cox regression analysis in a propensity score (PS) matched-cohort of HCVAb D+/R− (n = 950) versus HCVAb D−/R− (n = 950). Sensitivity analysis was also conducted in the entire cohort (n = 181 082). Results The mean age at baseline was 54 years, 75% were male, and 55% of the patients were African American in PS-matched cohort. Compared to the HCVAb D−/R − patients, recipients with HCVAb D+/R − showed identical probability for the incidence of CMV infection (Hazard Ratio (HR) = 1.00, 95% Confidence Interval (CI): 0.82–1.22). In the sensitivity analysis, compared to the HCVAb D−/R − patients, the HCVAb D+/R − group had a significantly lower risk of CMV infection in the unadjusted analysis (HR = 0.75, 95%CI: 0.65–0.85), while this risk difference disappeared after the adjusted analysis (HR = 0.99, 95%CI: 0.87–1.14). Conclusion The incidence of CMV infection was similar in recipients who received HCVAb D + and HCVAb D − KT. Further studies are needed to assess this association in KT from HCV nucleic acid positive donors.

A Novel Chimeric Anti-HCV Peptide Derived from Camel Lactoferrin and Molecular Level Insight on Its Interaction with E2

In the present study, a novel chimeric peptide was derived from camel lactoferrin designed with a considerable anti-HCV activity and its neutralization mechanism was predicted by molecular modelling tools. A novel anti-HCV peptide derived from camel lactoferrin (cLF36) was designed and expressed it recombinantly in HEK-293-T cells. Anti-viral activity of this peptide was evaluated against hepatitis C virus by Real-time PCR assay in vitro. Finally, to have a better insight into the mode of action of peptide on HCV entry inhibition, we examined the interaction of cLF36 with envelope glycoprotein E2 by molecular dynamic simulation. This chimeric peptide had significant inhibitory effects on both HCV entry (44 µg/mL) and viral replication (88 µg/mL) under in vitro (p > 0.01). Moreover, cLF36 peptide was not toxic to HEK cells as a normal cell at twofold of its anti-viral concentrations for HCV entry and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis (2.5%) against human RBCs (red blood cells). The results of in silico analysis showed that cLF36 interacted with β-sandwich and front layer of E2 protein as two potential CD81 binding sites. We generated and characterized a new camel lactoferrin derived HCV inhibitors. This peptide blocked HCV entry and also intracellular HCV replication in cell culture experiment.

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.

Prevalence of hepatitis C among HIV-1, HIV-2 and dually reactive patients: A multi-country cross-sectional survey in West Africa.

Little is known on the impact of HIV-2 infection on HCV viral replication. The aim of the study was to compare HCV prevalence and viral replication based on HIV types in West Africa.A cross-sectional survey was conducted within the IeDEA HIV-2 West Africa cohort from March to December 2012. All HIVinfected adult patients who attended participating HIV clinics during the study period were included. Blood samples were collected and re-tested for HIV type discrimination, HCV serology and viral load.A total of 767 patients were enrolled: 186 HIV-1, 431 HIV-2 and 150 HIV-1&2 dually reactive. At time of sampling, 531 (69.2%) were on ART and median CD4+ cell count was 472/mm3. Thirty (3.9%, 95% CI 2.7-5.5) patients were anti-HCV positive (4.3% in HIV-1, 4.0% in HIV-1&2 dually reactive and 3.7% in HIV-2; p=0.91). Detectable HCV RNA was identified in 21 (70.0%) patients (100% in HIV-1 and HIV- 1&2 dually reactive vs. 43.8% in HIV-2; p=0.003).Systematic screening should be promoted and performed in this population, since HCV is now potentially curable in sub- Saharan Africa.

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or “G4” that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3′end of the HCV (−) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy’ of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Up-regulation of circulating miRNA146a correlates with viral load via IRAK1 and TRAF6 in hepatitis C virus-infected patients

BackgroundHepatitis C virus (HCV) is a life threatening human pathogen. It has been found that miRNA146a regulates innate immunity, inflammatory response and antiviral pathway.We evaluated miRNA146a expression by real-time PCR and IL-1 receptor associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) levels by ELISA in serum of 36 HCV viremia patients and 42 age and gender matched healthy controls. ResultsmiRNA146a expression was significantly higher in HCV patients with a best cut off value 1.63 to discriminate between HCV patients and healthy controls. Meanwhile, it was negatively correlated to IRAK1 and TRAF6 levels and positively correlated to viral load in HCV patients. ConclusionsmiRNA146a has a potential role in HCV infection and viral replication through IRAK1 and TRAF6. It can also serve as a new screening method for HCV.

The dynamics of HCV-specific antibody responses in HIV/HCV patients on long-term antiretroviral therapy

Antibody responses have not been fully characterised in chronically HIV/HCV patients receiving antiretroviral therapy (ART). Seventeen HIV/HCV patients receiving ART were followed for a median (range) interval of 597 (186–766) weeks. Prior to ART, HIV/HCV patients had lower levels of antibodies reactive with HCV core and JFH-1, and lower genotype cross-reactive neutralising antibodies (nAb) titres, than HCV patients. Levels of JFH-1 reactive antibody increased on ART, irrespective of CD4+ T-cell counts or changes in serum ALT levels. The appearance of nAb coincided with control of HCV viral replication in five HIV/HCV patients. In other patients, HCV viral loads remained elevated despite nAb responses. Sustained virological responses following HCV therapy were associated with reduced antibody responses to JFH-1 and core but elevated responses to non-structural proteins. We conclude that nAb responses alone may fail to clear HCV, but contribute to control of viral replication in some HIV/HCV patients responding to ART.

HCV non-1b genotypes in injecting drug users from Romania

Chronic hepatitis C cases diagnosed in Romania were mostly related to unsafe parenteral treatments and blood transfusions; HCV genotype 1b was prevalent. During the last decade, an increasing number of HCV infections was reported among people who inject drugs (PWID). The aim of the current study was to test if this epidemiological shift triggered a diversification of the circulating viral strains. HCV genotypes were determined by reverse hybridization in 130 HCV-infected PWID (87.7% males; mean age 27.9 ± 6.7 years, injecting drugs for 8.1 ± 4.8 years). HIV-HCV co-infection was diagnosed in 80.8% of the subjects and 26.9% were HIV-HCV-HBV triple infected. Active HCV viral replication was present in 104 PWID (80%), more frequently in those HIV-co-infected (91.4% vs. 52% in HCV mono-infected, and 77.148.5% in HIV-HCV-HBV triple-infected, p = 0.0001). Non-1b genotypes were prevalent (54.8%), with subtype 1a the most commonly detected (24%), followed by genotypes 3a (14.4%) and 4 (7.7%). Mixed infections with genotypes 1a and 1b were found in nine subjects (8.7%). There was no difference in the genotypes frequencies based on HIV or HBV co-infection status, length of drug usage, or associated risk factors (tattoos, piercing, detention). The continuous surveillance of HCV genotypes in PWID from Romania will add valuable information to the overall European epidemiological picture, with important therapeutic implications.

EFFECTS OF SHORT RNA SEQUENCES ANALOGS AGAINST HCV GENOTYPE 4(A) IN REPLICON CELLS

Aim To determine to what extent short RNA sequences structural analogs could inhibit HCV genotype 4a (ED43) replication in cultured cell with sub-genomic HCV replicons. Method HCV short RNA sequences designed to be structurally similar to X region in the 3’-UTR of HCV 1b genome, were inserted into BamH I and Hind III restriction sites in pSilencer 4.1 CMV puro plasmid, transfected into human hepatoma cells Huh 7.5 with subgenomic replicons HCV genotype 4a (ED43/SG.Feo). 48 hrs after decoy transfection HCV RNA were isolated and quantified by real-time Polymerase chain reaction. Results the RNA structure analogs 5B46, X12, X12a and X12b were able to inhibit RNA levels by 65.5%, 38.5%, 64.5% and 68% respectively, in HCV genotype 4a. Conclusion Despite the sequence dissimilarity of the HCV RNA analogs and HCV genome in the replicon cells, there were enough 3 dimensional structural similarities to inhibit HCV viral replication.

Mutual antagonism between circadian protein period 2 and hepatitis C virus replication in hepatocytes.

BackgroundHepatitis C virus (HCV) infects approximately 3% of the world population and is the leading cause of liver disease, impacting hepatocyte metabolism, depending on virus genotype. Hepatic metabolic functions show rhythmic fluctuations with 24-h periodicity (circadian), driven by molecular clockworks ticking through translational-transcriptional feedback loops, operated by a set of genes, called clock genes, encoding circadian proteins. Disruption of biologic clocks is implicated in a variety of disorders including fatty liver disease, obesity and diabetes. The relation between HCV replication and the circadian clock is unknown.MethodsWe investigated the relationship between HCV core infection and viral replication and the expression of clock genes (Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in two cellular models, the Huh-7 cells transiently expressing the HCV core protein genotypes 1b or 3a, and the OR6 cells stably harboring the full-length hepatitis C genotype 1b replicon, and in human liver biopsies, using qRT-PCR, immunoblotting, luciferase assays and immunohistochemistry.ResultsIn Huh-7 cells expressing the HCV core protein genotype 1b, but not 3a, and in OR6 cells, transcript and protein levels of PER2 and CRY2 were downregulated. Overexpression of PER2 led to a consistent decrease in HCV RNA replicating levels and restoration of altered expression pattern of a subset of interferon stimulated genes (ISGs) in OR6 cells. Furthermore, in liver biopsies from HCV genotype 1b infected patients, PER2 was markedly localized to the nucleus, consistent with an auto-inhibitory transcriptional feedback loop.ConclusionsHCV can modulate hepatic clock gene machinery, and the circadian protein PER2 counteracts viral replication. Further understanding of circadian regulation of HCV replication and rhythmic patterns of host-hosted relationship may improve the effectiveness of HCV antiviral therapy. This would extend to hepatic viral infections the current spectrum of chronotherapies, implemented to treat metabolic, immune related and neoplastic disease.

Identification of Novel HCV Deletion Mutants in Chronic Hepatitis C Patients

The HCV genome consists of a positive 9.6 kb single-strand of RNA. Nucleotide substitutions in the HCV genome are common and a 2 kb deletion has been reported. A total of 117 chronic hepatitis C (CHC) patients who were treated with pegylated interferon plus ribavirin combination therapy were enrolled in this study. Total RNA was extracted from the patients' sera and reverse transcription and PCR were performed. Statistical analysis was performed to evaluate the effects of HCV deletion mutants on treatment with combination therapy. By amplifying entire HCV genomes using long-distance PCR, novel large deletion mutants were identified. Sequence analysis revealed that these deletions extended approximately 6 kb from the core/E2 region to the NS5A region and that there are three kinds of deletions that are identical at their 3' and 5' extremities. The subgenome virus particles appeared to coexist with full-genome virus particles in the sera of CHC patients despite lacking essential components for HCV viral replication. These short fragments were detected in 26 of 117 patients and were associated with significantly higher HCV RNA levels (P=0.018) and poor response to combination therapy (P=0.043). Moreover, the existence of HCV deletion mutants was significantly associated with virological relapse following combination therapy (P=0.046, OR=3.4). HCV deletion mutants may affect the HCV life cycle and reduce the antiviral effects of interferon therapy for CHC.

PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients

BackgroundTight correlation between host circulating CD8+ T cell-mediated immune response and control of viral replication is classical characteristic of long-term HCV infection. CD8+ T cell maturation/activation markers are expected to be associated with viral replication and disease progression in chronic HCV infection. The aim of the present study was to explore novel markers on CD8+ T cells with ability to evaluate HCV viral replication and disease progression.MethodsPBMCs were isolated from 37 chronic HCV-infected patients and 17 healthy controls. Distributed pattern of CD8+ T cells subsets and expression of PD-1, CD38, HLA-DR and CD127 were analyzed by flow cytometry. The correlation between expression of surface markers and HCV viral load or ALT was studied.ResultsDeclined naïve and increased TEMRA CD8+ T subsets were found in HCV-infected individuals compared with healthy controls. Percentage and MFI of PD-1, CD38 and HLA-DR on all CD8+ T cell subsets were higher in HCV-infected patients than healthy controls. In contrast, CD127 expression on CD8+ TCM showed an opposite trend as PD-1, CD38 and HLA-DR did. In chronic HCV infection, MFI of PD-1 on CD8+ TEM (p < 0.0001) and TEMRA (p = 0.0015) was positively correlated with HCV viral load while HLA-DR expression on non-naive CD8+ T cell subsets (p < 0.05) was negatively correlated with HCV viral load.ConclusionPD-1 level on peripheral CD8+ TEM/TEMRA was highly correlated with HCV viral load in chronic HCV-infected patients, which made PD-1 a novel indicator to evaluate HCV replication and disease progression in chronic hepatitis C patients.

Co-infection avec les hépatites virales

k]HBV and HCV infections are frequent in HIV patients and often contribute significantly to mortality and morbidity. There is no evidence that either HCV or HBV affects HIV disease progression. By contrast, HIV increases HBV and HCV viral replication and leads to higher chronicity rates. It also increases mother-to-child and sexual HCV transmission. HIV speeds the course of HCV and HBV liver disease progression, resulting in accelerated liver fibrosis progression and increased rates of liver decompensation, cirrhosis and hepatocellular carcinoma. Coinfected patients are more likely to have other fibrosis cofactors, such as high alcohol consumption and steatosis. All HIV-infected patients should undergo regular screening for HCV and HBV, using a real-time PCR detection system for HCV RNA and HBV DNA, when possible. Liver damage should be evaluated using liver biopsy or noninvasive methods, such as serum fibrosis marker assays and tissue elastography. HBV and HCV increase HIV drug toxicity. Antiretroviral therapy (ART) has a positive effect on HCV and HBV disease. Didanosine, stavudine and zidovudine should be avoided in HIV treatment in the case of HCV combination therapy with pegylated interferon and ribavirin. Sustained virological response (SVR) is lower in coinfected patients compared with monoinfected patients. Every effort, however, should be made to maintain high-dose therapy, which results in improved SVR. HBV treatment is mainly based on nucleoside/nucleotide analogues. Liver transplantation can be performed in coinfected patients with decompensated cirrhosis. Hepatitis prevention by vaccination against HBV and HAV should be widely considered in the care of HIV-infected patients.

CHOLESTATIC HEPATITIS (CH) IN LIVER ALLOGRAFTS OF HEPATITIS C (HCV) PATIENTS: HISTOPATHOLOGIC PROGRESSION AND CORRELATION WITH HCV-RNA VIRAL LEVELS

38 PURPOSE: A subset of HCV liver transplant patients develop a severe form of CH. In this study we characterize the histopathologic features on serial biopsies and investigated the role of HCV viral replication in disease progression. MATERIALS and METHODS: Seven cases of CH were identified among patients transplanted for HCV endstage liver disease. CH was defined as hyperbilirubinemia > 5mgs/dl for a duration of > 1 month. No other causes of cholestasis were identified. All biopsies [median #9 (6-12)] were reviewed as well as native explant and allograft explant livers. The tissues were evaluated for multiple histologic features and overall tissue diagnosis. HCV viral load was measured in 28 native explant, 6 allograft explant liver tissues and 2 allograft biopsies with CH using AMPLICOR HCV MONITOR™ test. RESULTS: 5/7 cases of CH required retransplantation. Two died < 1 month following retransplantation of infections. All three surviving retransplant patients had recurrent CH, of these, two cases developed bridging fibrosis 3 and 20 months following retransplantation. The histopathologic progression of CH in the original allografts was similar in all 7 cases. Mild lobular hepatitis is present initially and precedes the development of ballooning degeneration and cholestasis. Ballooning degeneration dominated the histologic picture during the cholestatic phase and culminated in centrizonal collapse. There was no evidence of chronic rejection in any of the biopsies or allografts. The native explant liver tissue of CH cases had significantly higher HCV levels than an unselected group of 21 HCV native explant livers (P<.013). Four cases had levels >25,000 copies/ug DNA, while all non-cholestatic cases had lower levels. Two cases of CH with <10,000 copies/ug DNA in the native explant did not require retransplantation. HCV levels in 3 allograft explants were >20,000 copies/ug DNA. The 2 cases with the highest levels showed the most ballooning. The levels in the 2 biopsies with CH were >50,000 copies/ug DNA, and were higher than in native explant or allograft explant livers. CONCLUSIONS: 1) CH is an aggressive disease with poor prognosis and recurs after retransplantation. 2) The histopathological progression is predictable. 3) HCV RNA levels >25,000/ug DNA in native explant liver tissue is associated with CH in allografts requiring retransplantation.

Incidence of hepatitis C virus infection in Italian patients with idiopathic pulmonary fibrosis.

A viral cause of idiopathic pulmonary fibrosis (IPF) was recently suggested by a Japanese study in which a high prevalence of anti-hepatitis C virus (HCV) antibodies was detected. A subsequent British study failed to confirm these results. Antibodies to HCV were evaluated in 60 patients with IPF, 130 patients with non-interstitial lung disease, and in 4614 blood donors. HCV-RNA and HCV genotypes were evaluated in the anti-HCV positive patients with IPF. Anti-HCV antibodies were evaluated by ELISA and confirmed by recombinant immunoblotting assay (RIBA). HCV-RNA and genotypes were detected by reverse transcriptase polymerase chain reaction (PCR). Eight patients with IPF had anti-HCV antibodies detected by ELISA (13.3%). In the blood donor control group the prevalence of HCV antibodies was lower (0.3%). In patients with non-interstitial lung disease HCV antibody prevalence was 6.1%. In all eight patients with IPF found to be anti-HCV positive by ELISA, HCV antibodies were also detected by RIBA. Furthermore, all were HCV-RNA positive by PCR assay. HCV genotypes were identified in four of these eight patients. In all four genotype II was present and in two it was associated with genotype III and/or genotype IV. In the remaining four cases the genotype was not identified. Italian patients with IPF show an increased prevalence (approximately 13%) of HCV infection and viral replication, but the prevalence of anti-HCV antibodies does not differ from other lung diseases.