Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production.

Infectious HCV carrying reporter genes have further applications in understanding the HCV life cycle including replication, viral assembly and release. In this study, a full-length 3039bp LacZ gene was inserted into the derivative of JFH1-AM120 to develop an additional reporter virus. The results showed that the recombinant reporter virus JFH1-AM120-LacZ can replicate and produce lower titers of infectious virus. However, insertion of the LacZ gene in the C-terminal region of the NS5A in HCV JFH1-AM120-LacZ decreased viral replication and dramatically impaired the production of infectious viral particles. Moreover, the JFH1-AM120-LacZ reporter virus lost the LacZ gene after serial passage. Nevertheless, the JFH1-AM120-LacZ reporter virus displayed the entire life cycle of HCV, from replication to production of infectious virus, in Huh7.5 cells. This study demonstrates that the NS5A region of HCV JFH1-AM120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site.

Activation of PERK-Nrf2 oncogenic signaling promotes Mdm2-mediated Rb degradation in persistently infected HCV culture.

The mechanism of how chronic hepatitis C virus (HCV) infection leads to such a high rate of hepatocellular carcinoma (HCC) is unknown. We found that the PERK axis of endoplasmic reticulum (ER) stress elicited prominent nuclear translocation of Nrf2 in 100% of HCV infected hepatocytes. The sustained nuclear translocation of Nrf2 in chronically infected culture induces Mdm2-mediated retinoblastoma protein (Rb) degradation. Silencing PERK and Nrf2 restored Mdm2-mediated Rb degradation, suggesting that sustained activation of PERK/Nrf2 axis creates oncogenic stress in chronically infected HCV culture model. The activation of Nrf2 and its nuclear translocation were prevented by ER-stress and PERK inhibitors, suggesting that PERK axis is involved in the sustained activation of Nrf2 signaling during chronic HCV infection. Furthermore, we show that HCV clearance induced by interferon-α based antiviral normalized the ER-stress response and prevented nuclear translocation of Nrf2, whereas HCV clearance by DAAs combination does neither. In conclusion, we report here a novel mechanism for how sustained activation of PERK axis of ER-stress during chronic HCV infection activates oncogenic Nrf2 signaling that promotes hepatocyte survival and oncogenesis by inducing Mdm2-mediated Rb degradation.

Interferon lambda 4 expression is suppressed by the host during viral infection.

Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.

Tannic Acid Inhibits Hepatitis C Virus Entry into Huh7.5 Cells.

Chronic infection with the hepatitis C virus (HCV) is a cause of cirrhosis and hepatocellular carcinoma worldwide. Although antiviral therapy has dramatically improved recently, a number of patients remain untreated and some do not clear infection with treatment. Viral entry is an essential step in initiating and maintaining chronic HCV infections. One dramatic example of this is the nearly 100% infection of newly transplanted livers in patients with chronic hepatitis C. HCV entry inhibitors could play a critical role in preventing HCV infection of newly transplanted livers. Tannic acid, a polymer of gallic acid and glucose molecules, is a plant-derived polyphenol that defends some plants from insects and microbial infections. It has been shown to have a variety of biological effects, including antiviral activity, and is used as a flavoring agent in foods and beverages. In this study, we demonstrate that tannic acid is a potent inhibitor of HCV entry into Huh7.5 cells at low concentrations (IC50 5.8 µM). It also blocks cell-to-cell spread in infectious HCV cell cultures, but does not inhibit HCV replication following infection. Moreover, experimental results indicate that tannic acid inhibits an early step of viral entry, such as the docking of HCV at the cell surface. Gallic acid, tannic acid's structural component, did not show any anti-HCV activity including inhibition of HCV entry or replication at concentrations up to 25 µM. It is possible the tannin structure is related on the effect on HCV inhibition. Tannic acid, which is widely distributed in plants and foods, has HCV antiviral activity in cell culture at low micromolar concentrations, may provide a relative inexpensive adjuvant to direct-acting HCV antivirals and warrants future investigation.

Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection.

Viral entry requires co-operative interactions of several host cell factors. Interferon (IFN) and the IFN-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We examined the effect of interferon-α inducible protein 6 (IFI6) against HCV infection in human hepatoma cells. HCV RNA level or infectious foci were inhibited significantly by ectopic expression of IFI6. IFI6 impaired CD81 co-localization with claudin-1 (CLDN1) upon HCV infection or CD81 cross-linking by specific antibody. Activation of epidermal growth factor receptor (EGFR), a co-factor involved in CD81/CLDN1 interactions, was reduced in IFI6 expressing cells in response to HCV infection or CD81 cross linking by antibody, but not by treatment with EGF. Taken together, the results from our study support a model where IFI6 inhibits HCV entry by impairing EGFR mediated CD81/CLDN1 interactions. This may be relevant to other virus entry processes employing EGFR.

HCV infection selectively impairs type I but not type III IFN signaling.

A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK-STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.

Genome-wide transcriptome analysis identifies novel gene signatures implicated in human chronic liver disease.

The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a hepatic tumor suppressor and a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. The goal of this study was to identify novel SHP-regulated genes that are involved in the development and progression of chronic liver disease. To achieve this, we conducted the first comprehensive RNA sequencing (RNA-seq) analysis of Shp(-/-) mice, compared the results with human hepatitis C cirrhosis RNA-seq and nonalcoholic steatohepatitis (NASH) microarray datasets, and verified novel results in human liver biospecimens. This approach revealed new gene signatures associated with chronic liver disease and regulated by SHP. Several genes were selected for validation of physiological relevance based on their marked upregulation, novelty with regard to liver function, and involvement in gene pathways related to liver disease. These genes include peptidoglycan recognition protein 2, dual specific phosphatase-4, tetraspanin 4, thrombospondin 1, and SPARC-related modular calcium binding protein-2, which were validated by qPCR analysis of 126 human liver specimens, including steatosis, fibrosis, and NASH, alcohol and hepatitis C cirrhosis, and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies new genes that are regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable transcriptome information for characterizing mechanisms of these diseases.

A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses.

The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses.

RNA-sequencing analysis of 5' capped RNAs identifies many new differentially expressed genes in acute hepatitis C virus infection.

We describe the first report of RNA sequencing of 5' capped (Pol II) RNAs isolated from acutely hepatitis C virus (HCV) infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05). Most of the differentially expressed genes (>80%) and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling) were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.

[Research advances on RB1 gene].

The retinoblastoma1 (RB1) gene is the first cloned tumor suppressor gene. As a negative regulator of the cell cycle, RB1 gene could maintain a balance between cell growth and development through binding to transcription factors and regulating the expression of genes involved in cell proliferation and differentiation. Thus, it is involved in cell cycle, cell senescence, growth arrest, apoptosis and differentiation. This review summarizes recent advances on the structure, expression, and function of RB1 gene.