Hepatitis C Virus Infection In Hepatocytes Research Articles

The role of inflammasome in chronic viral hepatitis.

Infections of hepatotropic viruses cause a wide array of liver diseases including acute hepatitis, chronic hepatitis and the consequently developed cirrhosis and hepatocellular carcinoma (HCC). Among the five classical hepatotropic viruses, hepatitis B virus (HBV) and hepatitis C virus (HCV) usually infect human persistently and cause chronic hepatitis, leading to major troubles to humanity. Previous studies have revealed that several types of inflammasomes are involved in the infections of HBV and HCV. Here, we summarize the current knowledge about their roles in hepatitis B and C. NLRP3 inflammasome can be activated and regulated by HBV and HCV. It is found to exert antiviral function or mediates inflammatory response in viral infections depending on different experimental models. Besides NLRP3 inflammasome, IFI16 and AIM2 inflammasomes participate in the pathological process of hepatitis B, and NALP3 inflammasome may sense HCV infection in hepatocytes. The inflammasomes affect the pathological process of viral hepatitis through its downstream secretion of inflammatory cytokines interleukin-1β (IL-1β) and IL-18 or induction of pyroptosis resulting from cleaved gasdermin D (GSDMD). However, the roles of inflammasomes in different stages of viral infection remains mainly unclear. More proper experimental models of viral hepatitis should be developed for specific studies in future, so that we can understand more about the complexity of inflammasome regulation and multifunction of inflammasomes and their downstream effectors during HBV and HCV infections.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

Hepatocytes infected with hepatitis C virus change immunological features in the liver microenvironment.

Hepatitis C virus (HCV) infection is remarkably efficient in establishing viral persistence, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Direct-acting antiviral agents (DAAs) are promising HCV therapies to clear the virus. However, recent reports indicate potential increased risk of HCC development among HCV patients with cirrhosis following DAA therapy. CD8+ T-cells participate in controlling HCV infection. However, in chronic hepatitis C patients, severe CD4+ and CD8+ T-cell dysfunctions have been observed. This suggests that HCV may employ mechanisms to counteract or suppress the host T-cell responses. The primary site of viral replication is within hepatocytes where infection can trigger the expression of costimulatory molecules and the secretion of immunoregulatory cytokines. Numerous studies indicate that HCV infection in hepatocytes impairs antiviral host immunity by modulating the expression of immunoregulatory molecules. Hepatocytes expressing whole HCV proteins upregulate the ligands of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and transforming growth factor β (TGF-β) synthesis compared to those in hepatocytes in the absence of the HCV genome. Importantly, HCV-infected hepatocytes are capable of inducing regulatory CD4+ T-cells, releasing exosomes displaying TGF-β on exosome surfaces, and generating follicular regulatory T-cells. Recent studies report that the expression profile of exosome microRNAs provides biomarkers of HCV infection and HCV-related chronic liver diseases. A better understanding of the immunoregulatory mechanisms and identification of biomarkers associated with HCV infection will provide insight into designing vaccine against HCV to bypass HCV-induced immune dysregulation and prevent development of HCV-associated chronic liver diseases.

Retraction Note: 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.

Repression of MicroRNA-30e by Hepatitis C Virus Enhances Fatty Acid Synthesis.

Chronic hepatitis C virus (HCV) infection often leads to end‐stage liver disease, including hepatocellular carcinoma (HCC). We have previously observed reduced expression of microRNA‐30e (miR‐30e) in the liver tissues and sera of patients with HCV‐associated HCC, although biological functions remain unknown. In this study, we demonstrated that HCV infection of hepatocytes transcriptionally reduces miR‐30e expression by modulating CCAAT/enhancer binding protein β. In silico prediction suggests that autophagy‐related gene 5 (ATG5) is a direct target of miR‐30e. ATG5 is involved in autophagy biogenesis, and HCV infection in hepatocytes induces autophagy. We showed the presence of ATG5 in the miR‐30e–Argonaute 2 RNA‐induced silencing complex. Overexpression of miR‐30e in HCV‐infected hepatocytes inhibits autophagy activation. Subsequent studies suggested that ATG5 knockdown in Huh7.5 cells results in the remarkable inhibition of sterol regulatory element binding protein (SREBP)‐1c and fatty acid synthase (FASN) level. We also showed that overexpression of miR‐30e decreased lipid synthesis‐related protein SREBP‐1c and FASN in hepatocytes. Conclusion: We show new mechanistic insights into the interactions between autophagy and lipid synthesis through inhibition of miR‐30e in HCV‐infected hepatocytes.

Hepatocyte‐derived exosomes promote T follicular regulatory cell expansion during hepatitis C virus infection

These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. (Hepatology 2018;67:71-85).

RETRACTED ARTICLE: 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.

Hepatitis C virus infection stimulates transforming growth factor-β1 expression through up-regulating miR-192.

The objective of this study was to determine the molecular mechanisms underlying chronic liver injury and fibrosis caused by hepatitis C virus (HCV). This study revealed that miR-192 expreßsion was induced by HCV infection without affecting viral replication. However, viral-induced miR-192 up-regulated transforming growth factor-ß1 (TGF-ß1) expreßsion in liver cells at transcriptional level. TGF-ß1 stimulation by HCV-induced miR-192 was caused through ZEB1 down-regulation and TGF-ß1 increased miR-192 level via positive feedback pathway. Increase in miR-192 expreßsion by HCV infection was due to HCV core protein released and/or expressed by viral infection. TGF-ß1 promoter activity was also increased by HCV core protein in liver cells. Taken together, HCV infection resulted in increased TGF-ß1 transcription in hepatocytes through ZEB1 down-regulation by HCV core-mediated miR-192 stimulation. Importantly, miR-192 inhibition with anti-miR-192 rescued ZEB1 expression down-regulated by HCV infection, thus reducing the level of TGF-ß1 expression increased by HCV infection in hepatocytes. These results suggest a novel mechanism of HCV-mediated liver fibrogenesis with miR-192 being a potential molecular target to ameliorate viral pathogenesis.

Hepatitis C Virus Infection Induces Autophagy as a Prosurvival Mechanism to Alleviate Hepatic ER-Stress Response.

Hepatitis C virus (HCV) infection frequently leads to chronic liver disease, liver cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms by which HCV infection leads to chronic liver disease and HCC are not well understood. The infection cycle of HCV is initiated by the attachment and entry of virus particles into a hepatocyte. Replication of the HCV genome inside hepatocytes leads to accumulation of large amounts of viral proteins and RNA replication intermediates in the endoplasmic reticulum (ER), resulting in production of thousands of new virus particles. HCV-infected hepatocytes mount a substantial stress response. How the infected hepatocyte integrates the viral-induced stress response with chronic infection is unknown. The unfolded protein response (UPR), an ER-associated cellular transcriptional response, is activated in HCV infected hepatocytes. Over the past several years, research performed by a number of laboratories, including ours, has shown that HCV induced UPR robustly activates autophagy to sustain viral replication in the infected hepatocyte. Induction of the cellular autophagy response is required to improve survival of infected cells by inhibition of cellular apoptosis. The autophagy response also inhibits the cellular innate antiviral program that usually inhibits HCV replication. In this review, we discuss the physiological implications of the HCV-induced chronic ER-stress response in the liver disease progression.

Methods to Evaluate Novel Hepatitis C Virus Vaccines.

The hepatitis C virus (HCV) is a major cause of severe liver disease worldwide. It is estimated that around 130-170 million individuals are chronic carriers of the infection and they are over time at an increased risk of developing severe liver disease. HCV is often referred to as a silent epidemic because the majority of infected individuals do not develop any symptoms. Hence, many individuals are diagnosed at a late stage and thus in need of immediate treatment. Today we have very effective direct-acting antivirals (DAAs), which cure more than 90-95 % of all treated patients. However, this treatment is associated with high-costs and the use is limited to the patients with most advanced liver disease in high-income countries. Notably, a majority of the chronic HCV carriers live in resource-poor countries and do not have access to the new effective DAAs. We therefore need to develop alternative treatments for chronic HCV infection such as therapeutic vaccines. The idea with therapeutic vaccines is to reactivate the infected patient's own immune system. It is well known that patients with chronic HCV infection have dysfunctional immune responses to the virus. Hence, the vaccine should activate HCV-specific T cells that will home to the liver and eradicate the HCV infected hepatocytes. Importantly, one should also consider the combination of a therapeutic vaccine and DAAs as a treatment strategy to equip the resolving patients with post-cure HCV-specific immune responses. This would provide patients with a better protection against reinfection. Numerous genetic vaccine candidates for HCV have been developed and tested in clinical trials with limited effects on viral load and in general inefficient activation of HCV-specific immune responses. In this chapter we describe the rational of developing highly immunogenic vaccines for HCV. Different strategies to improve vaccine immunogenicity and methods to evaluate vaccine efficacy are described. Detailed description of vaccine delivery by intramuscular immunization in combination with in vivo electroporation/electrotransfer (EP/ET) is covered, as well as immunological analysis of primed immune responses by determination of interferon-γ (IFN-γ) production by ELISpot assay and direct ex vivo quantification of HCV NS3/4A-specific CD8+ T cells by pentamer staining. To analyze the in vivo functionality of primed NS3/4A-specific T cells we utilized the in vivo bioluminescence imaging technology. In conclusion, this chapter describes a method to design HCV vaccines and also a protocol to assess their efficacy.

Inhibition of microRNA-199a-5p reduces the replication of HCV via regulating the pro-survival pathway

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that post-transcriptionally regulate the pathological processes of various liver diseases including hepatitis C virus (HCV) infection. In the present study, we demonstrated that HCV infection enhanced the expression of miR-199a-5p in HCV infected human hepatocytes and Huh7.5.1cells, as well as liver biopsy specimens. Inhibition of miR-199a-5p decreased HCV replication not only in terms of HCV RNA, but also the protein levels of NS3 and NS5A. Furthermore, we discovered that miR-199a-5p knockdown in Huh7.5.1 cells infected with genotype 2a (JFH1) or genotype 1b (SN1a) resulted in the remarkable inhibition of pro-survival pathways, as observed by the down-regulation of p-Akt, p-ERK and β-catenin protein levels. Moreover, pre-treatment with the pro-survival pathway specific activator prominently ablated the inhibition of HCV replication induced by miR-199a-5p knockdown. Collectively, our results highlight the up-regulation of miR-199a-5p expression with HCV infection and the promotion of HCV replication by miR-199a-5p. Moreover, miR-199a-5p may facilitate HCV replication by regulating pro-survival pathways through PI3K/Akt, Ras/ERK and Wnt/β-catenin. miR-199a-5p might be a potential drug target for developing a novel strategy to combat HCV infection.

Identification of Cholesterol 25-Hydroxylase as a Novel Host Restriction Factor and a Part of the Primary Innate Immune Responses against Hepatitis C Virus Infection.

Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, causes chronic liver diseases, including hepatitis, cirrhosis, and cancer. HCV infection is critically dependent on host lipid metabolism, which contributes to all stages of the viral life cycle, including virus entry, replication, assembly, and release. 25-Hydroxycholesterol (25HC) plays a critical role in regulating lipid metabolism, modulating immune responses, and suppressing viral pathogens. In this study, we showed that 25HC and its synthesizing enzyme cholesterol 25-hydroxylase (CH25H) efficiently inhibit HCV infection at a postentry stage. CH25H inhibits HCV infection by suppressing the maturation of SREBPs, critical transcription factors for host lipid biosynthesis. Interestingly, CH25H is upregulated upon poly(I · C) treatment or HCV infection in hepatocytes, which triggers type I and III interferon responses, suggesting that the CH25H induction constitutes a part of host innate immune response. To our surprise, in contrast to studies in mice, CH25H is not induced by interferons in human cells and knockdown of STAT-1 has no effect on the induction of CH25H, suggesting CH25H is not an interferon-stimulated gene in humans but rather represents a primary and direct host response to viral infection. Finally, knockdown of CH25H in human hepatocytes significantly increases HCV infection. In summary, our results demonstrate that CH25H constitutes a primary innate response against HCV infection through regulating host lipid metabolism. Manipulation of CH25H expression and function should provide a new strategy for anti-HCV therapeutics. Recent studies have expanded the critical roles of oxysterols in regulating immune response and antagonizing viral pathogens. Here, we showed that one of the oxysterols, 25HC and its synthesizing enzyme CH25H efficiently inhibit HCV infection at a postentry stage via suppressing the maturation of transcription factor SREBPs that regulate lipid biosynthesis. Furthermore, we found that CH25H expression is upregulated upon poly(I·C) stimulation or HCV infection, suggesting CH25H induction constitutes a part of host innate immune response. Interestingly, in contrast to studies in mice showing that ch25h is an interferon-stimulated gene, CH25H cannot be induced by interferons in human cells but rather represents a primary and direct host response to viral infection. Our studies demonstrate that the induction of CH25H represents an important host innate response against virus infection and highlight the role of lipid effectors in host antiviral strategy.

Transcriptional regulation of IFN-λ genes in Hepatitis C virus-infected hepatocytes via IRF-3·IRF-7·NF-κB complex

Hepatitis C virus (HCV) infection in hepatocytes stimulates innate antiviral responses including the production of type III interferons (IFN-λ), including IL-28A, IL-28B, and IL-29 (Figure ​(Figure1).1). However, the molecular mechanism(s) regulating the expression of IFN-λ genes in HCV-infected hepatocytes remains undefined. In this study, we examined regulatory elements involved in the induction of IFN-λ genes following HCV infection in hepatocytes and further determined the binding of specific transcription factor(s) to promoter regions of IFN-λ genes. Our studies reveal that the regulatory portion for IL-28A, IL-28B, andIL-29 genes is localized to a 1-kb region in their respective promoters (Figure ​(Figure2,2, ​,4).4). Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28Bgenes (Figure ​(Figure5,5, ​,6),6), whereas NF-κB is an additional requirement for the induction of the IL-29 gene (Figure ​(Figure3).3). Ligation of Toll-like receptors (TLR) 3, 7, 8, and 9, which also activate IRFs and NF-κB, resulted in more robust production of IFN-λ than that observed with HCV infection, verifying the importance of TLR pathways in IFN-λ production (Figure ​(Figure8).8). Furthermore, the addition of IFN-λ to HCV-infected hepatocytes decreased viral replication and produced a concurrent reduction in microRNA-122 (miR-122). The decrease in viral replication was enhanced by the co-administration of IFN-λ and miR-122 inhibitor (miRIDIAN) (Figure ​(Figure7),7), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection. Figure 1 Hepatic induction of IFN-λ genes during HCV infection. A, B. Total RNA was extracted from liver tissues of healthy and chronic HCV patients for IL-28A/B (A) and IL-29 (B) mRNA quantification by real-time PCR. C-F. Human primary hepatocytes were ... Figure 2 Transcriptional activity at the IL-28A, IL-28B, and IL-29 promoter by HCV infection. PH5CH8 cells were transfected with a mixture of the luciferase constructs containing various 5' deletions of the human IL-28A (A), IL-28B (B), or IL-29 (C) promoters ... Figure 3 IRF-7, and NF-κB differentially determine expression of IL-28A/B and IL-29. Nucleotide sequence from 1.0 kb upstream of the transcription start site of human IL-28A, IL-28B, and IL-29. The positions of the IRF, NF-κB, AP-1, and GATA-1 ... Figure 4 Overexpression of IRFs and NF-κB confirms their role in expression of IFN-λ genes. PH5CH8 cells were transiently transfected with plasmids encoding different promoter regions of human IL-28A (pGL3-IL-28A 1kb) (A-C), IL-28B (pGL3-IL-28B ... Figure 5 IRFs are core transcription factors of IFN-λ genes. IL-28B (B), or IL-29 (C) in combination with siRNA specific for IRF-3, IRF-7, RELA(NF-κB p65), or control. At 36 h post-transfection, cells were stimulated with poly(I:C) for 6 hours ... Figure 6 IRFs and NF-κB directly bind the promoters of IFN-λ genes. PH5CH8 cells were stimulated with poly(I:C) (A and C) or infected with JFH-1 (B and D) for the indicated lengths of time. Chromatin was immunoprecipitated with anti-IRF-3, anti-IRF-7, ... Figure 7 miR-122-dependent replication of HCV is suppressed by IFN-λ stimulation. Huh 7.5.1 cells infected with JFH-1 for the indicated number of days and HCV RNA or miR-122 levels were quantified by real time PCR. The mean value of day 1 post-infection ... Figure 8 TLR3, 7, 8, and 9 induce human IFN-λ gene expression in human primary hepatocytes. Human primary hepatocytes were incubated with JFH-1, Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), Poly (I-C) (5 μg/ml), LPS (10 μg/ml), Flagellin ...

Transcriptional Regulation of IFN-λ Genes in Hepatitis C Virus-infected Hepatocytes via IRF-3·IRF-7·NF-κB Complex

Hepatitis C virus (HCV) infection in hepatocytes stimulates innate antiviral responses including the production of type III interferons (IFN-λ), including IL-28A, IL-28B, and IL-29. However, the molecular mechanism(s) regulating the expression of IFN-λ genes in HCV-infected hepatocytes remains undefined. In this study, we examined regulatory elements involved in the induction of IFN-λ genes following HCV infection in hepatocytes and further determined the binding of specific transcription factor(s) to promoter regions of IFN-λ genes. Our studies reveal that the regulatory portion for IL-28A, IL-28B, and IL-29 genes is localized to a 1-kb region in their respective promoters. Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28B genes, whereas NF-κB is an additional requirement for the induction of the IL-29 gene. Ligation of Toll-like receptors (TLR) 3, 7, 8, and 9, which also activate IRFs and NF-κB, resulted in more robust production of IFN-λ than that observed with HCV infection, verifying the importance of TLR pathways in IFN-λ production. Furthermore, the addition of IFN-λ to HCV-infected hepatocytes decreased viral replication and produced a concurrent reduction in microRNA-122 (miR-122). The decrease in viral replication was enhanced by the co-administration of IFN-λ and miR-122 inhibitor (miRIDIAN), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection.

Genome Wide Identification of FGFR2 Alternative Splicing in Hepatitis C: Potential Roles in Malignant Transformation

Hepatocellular carcinoma (HCC) is one of the few tumors in which the incidence is on the rise worldwide, especially in the US [1]. The increasing incidence of this disease is associated with the rise in hepatitis C virus (HCV) infection [2]. It is estimated that 3.2 million people in the US are infected with HCV; a blood-borne disease linked to 12,000 US deaths a year [3]. More concerning are projections that rate will quadruple in the next 10 years, to over 40,000 cases per year. Therefore, there is a great need to understand the molecular mechanisms for HCV-induced malignant transformation. Many studies have shown that malignant transformation of HCV infected hepatocytes occurs through a pathway of increased liver cell turnover induced by chronic liver injury and regeneration in a context of inflammation and oxidative DNA damage. In this context, the host immune response against the various HCV-encoded proteins (HCV core, E2, NS3, and NS5A) becomes relevant for HCC development and tumor progression. HCV proteins interact with many host cells factors and affect a wide range of cellular and humoral activities, including cell signaling, transcriptional modulation, transformation, apoptosis, membrane rearrangements, vesicular trafficking and cytokine & chemokine production. Inflammatory cells and immunomodulatory mediators present in the microenvironment polarize host immune response toward specific phenotypes impacting initiation and progression of the disease [4].

Hepatitis C Virus NS5A Protein Modulates IRF-7-Mediated Interferon-α Signaling

Hepatitis C virus (HCV) establishes chronic infection in a large number of infected individuals. We have previously shown that HCV infection in hepatocytes blocks poly (I-C) or interferon (IFN)-α-mediated IRF-7 nuclear translocation (Raychoudhuri and others 2010). However, the mechanism of IRF-7 regulation by HCV remained unknown. In this study, we have observed that HCV NS5A physically associates with IRF-7. A subsequent study suggested that the HCV NS5A protein blocks IRF-7-mediated IFN-α14 promoter activation. Further analyses demonstrated that site-specific mutagenesis of the 2 basic arginine residues (amino acids Arg(216) and Arg(217)) in the NS5A is critical for IRF-7-mediated IFN-α14 promoter regulation. Together, our results suggested that the HCV NS5A protein limits the IFN-α-signaling pathway in association with IRF-7, and may, in part, be responsible for the establishment of chronic infection.

The circulatory orbit of micro-RNAs in hepatitis C

The circulatory orbit of micro-RNAs in hepatitis C

Thromboxane A2 Synthase Inhibitors Prevent Production of Infectious Hepatitis C Virus in Mice With Humanized Livers

A 3-dimensional (3D) culture system for immortalized human hepatocytes (HuS-E/2 cells) recently was shown to support the lifecycle of blood-borne hepatitis C virus (HCV). We used this system to identify proteins that are active during the HCV lifecycle under 3D culture conditions. We compared gene expression profiles of HuS-E/2 cells cultured under 2-dimensional and 3D conditions. We identified signaling pathways that were activated differentially in the cells, and analyzed their functions in the HCV lifecycle using a recombinant HCV-producing cell-culture system, with small interfering RNAs and chemical reagents. We investigated the effects of anti-HCV reagents that altered these signaling pathways in mice with humanized livers (carrying human hepatocytes). Microarray analysis showed that cells cultured under 2-dimensional vs 3D conditions expressed different levels of messenger RNAs encoding prostaglandin synthases. Small interfering RNA-mediated knockdown of thromboxane A2 synthase (TXAS) and incubation of hepatocytes with a TXAS inhibitor showed that this enzyme is required for production of infectious HCV, but does not affect replication of the HCV genome or particle release. The TXAS inhibitor and a prostaglandin I2 receptor agonist, which has effects that are opposite those of thromboxane A2, reduced serum levels of HCV and inhibited the infection of human hepatocytes by blood-borne HCV in mice. An inhibitor of the prostaglandin synthase TXAS inhibits production of infectious HCV particles in cultured hepatocytes and HCV infection of hepatocytes in mice with humanized livers. It therefore might be therapeutic for HCV infection.

HCV+ Hepatocytes Induce Human Regulatory CD4+ T Cells through the Production of TGF-β

BackgroundHepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4+ regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown.Methodology/Principal FindingsHCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4+ T cells. The production of IFN-γ was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4+ T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV+ hepatocytes upregulated the production of TGF-β and blockade of TGF-β abrogated Treg phenotype and function.Conclusions/SignificanceThese results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.

RIG-I mediated hepatic innate immune signaling that controls HCV infection.

Hepatitis C virus (HCV) infection is one of the most serious public health problems in the world. HCV leads patients to develop hepatic cirrhosis and precipitates hepatocellular carcinoma. HCV establishes persistent infection by impairing host innate and adaptive immune responses. HCV infected hepatocytes sense the infection through Pathogen Associated Molecular Patterns (PAMPs). The sensor molecules, Pattern Recognition Receptors (PRRs) contain two distinct categories, toll like receptors (TLR) and cytoplasmic Retinoic Acid inducible Gene I (RIG-I) like helicases (RLHs). In the hepatocyte, the cytoplasmic PRR, Retinoic Acid inducible Gene I (RIG-I) plays the central role of HCV viral genome recognition, resulting in activation of signaling to induce type I interferon and proinflammatory cytokines. Type I IFN induces more than 300 effector molecules known as interferon stimulated genes (ISGs) that establish an antiviral state in infected cells and neighboring cells. The activation of innate immunity is also critical for the mounting of innate and adaptive immunity. However, a variety of viral strategies of HCV disrupt host innate immune signaling and ISG function, resulting in a dysfunctional immune response against HCV and poor responses to the current type I IFN based therapy. Many studies have reported immune dysfunction during HCV infection in cell culture, animal models and patients. This review article focuses on understanding how the hepatic innate immunity sensor, PRR, associates with HCV PAMPs, and how HCV escapes from host immunity.