Innate host response in primary human hepatocytes with hepatitis C virus infection

Background and Aim

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.

Methods

An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.

Results

Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.

Conclusion

Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection.     

HCV infective virions can be carried by human platelets

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.     

Interleukin-27 Is Differentially Associated with HIV Viral Load and CD4+ T Cell Counts in Therapy-Na?ve HIV-Mono-Infected and HIV/HCV-Co-Infected Chinese

Human Immunodeficiency Virus (HIV) infection and the resultant Acquired Immunodeficiency Syndrome (AIDS) epidemic are major global health challenges; hepatitis C virus (HCV) co-infection has made the HIV/AIDS epidemic even worse. Interleukin-27 (IL-27), a cytokine which inhibits HIV and HCV replication in vitro, associates with HIV infection and HIV/HCV co-infection in clinical settings. However, the impact of HIV and HCV viral loads on plasma IL-27 expression levels has not been well characterized. In this study, 155 antiretroviral therapy-naïve Chinese were recruited. Among them 80 were HIV- and HCV-negative healthy controls, 45 were HIV-mono-infected and 30 were HIV/HCV-co-infected. Plasma level HIV, HCV, IL-27 and CD4+ number were counted and their correlation, regression relationships were explored. We show that: plasma IL-27 level was significantly upregulated in HIV-mono-infected and HIV/HCV-co-infected Chinese; HIV viral load was negatively correlated with IL-27 titer in HIV-mono-infected subjects whereas the relationship was opposite in HIV/HCV-co-infected subjects; and the relationships between HIV viral loads, IL-27 titers and CD4⁺ T cell counts in the HIV mono-infection and HIV/HCV co-infection groups were dramatically different. Overall, our results suggest that IL-27 differs in treatment-naïve groups with HIV mono-infections and HIV/HCV co-infections, thereby providing critical information to be considered when caring and treating those with HIV mono-infection and HIV/HCV co-infection.     

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.     

Hepatic HMOX1 Expression Positively Correlates with Bach-1 and miR-122 in Patients with HCV Mono and HIV/HCV Coinfection

Aim

To analyze the expression of HMOX1 and miR-122 in liver biopsy samples obtained from HCV mono-and HIV/HCV co-infected patients in relation to selected clinical parameters, histological examination and IL-28B polymorphism as well as to determine whether HMOX1 expression is dependent on Bach-1.

Materials and Methods

The study group consisted of 90 patients with CHC: 69 with HCV mono and 21 with HIV/HCV co-infection. RT-PCR was used in the analysis of HMOX1, Bach-1 and miR-122 expression in liver biopsy samples and in the assessment of IL-28B single-nucleotide polymorphism C/T (rs12979860) in the blood. Moreover in liver biopsy samples an analysis of HO-1 and Bach-1 protein level by Western Blot was performed.

Results

HCV mono-infected patients, with lower grading score (G<2) and higher HCV viral load (>600000 IU/mL) demonstrated higher expression of HMOX1. In patients with HIV/HCV co-infection, the expression of HMOX1 was lower in patients with lower lymphocyte CD4 count and higher HIV viral load. IL28B polymorphism did not affect the expression of either HMOX1 or miR-122. Higher HMOX1 expression correlated with higher expression of Bach-1 (Spearman’s ρ = 0.586, p = 0.000001) and miR-122 (Spearman’s ρ = 0.270, p = 0.014059).

Conclusions

HMOX1 and miR-122 play an important role in the pathogenesis of CHC in HCV mono-and HIV/HCV co-infected patients. Reduced expression of HMOX1 in patients with HIV/HCV co-infection may indicate a worse prognosis in this group. Our results do not support the importance of Bach-1 in repression of HMOX1 in patients with chronic hepatitis C.     

Hepatitis B and C Co-Infections in Some HIV-Positive Populations in Cameroon,West Central Africa: Analysis of Samples Collected Over More Than a Decade

As people infected with the human immunodeficiency virus (HIV) in Sub-Saharan Africa live longer due to availability of antiretroviral treatment (ART), so is the rise of associated infections with their burdens on patients. But reliable data on the prevalence of co-infection with hepatitis B (HBV) or C (HCV) still remains sparse and many individuals with HIV do not know their co-infection status. This study attempted to estimate the seroprevalence and identify risk factors associated with hepatitis B and/or C co-infections in HIV-infected individuals from five Regions of Cameroon by screening 531 HIV infected subjects for the presence of HBV surface antigen (HBsAg) and antibodies to HCV (HCV-Ab). A Screening and a confirmatory Enzyme linked immunosorbent assay were used to detect presence of markers of infection. CD4 count levels were also examined. The results indicate that of the 531 participants, 68% were females and 32% males. Mean CD4 count was ~400 cells/μl. Seroprevalence rates for HBsAg and HCV-Ab were 23.7%, and 7.2%, respectively. Associations assessed using logistic regression revealed that HBsAg but not HCV-Ab positivity was linked to age, lower CD4 count and residing in an urban rather than in a rural setting. This high prevalence of co-infection with HBV raises the urgent need to systematically screen all newly diagnosed HIV cases for co-infection in Cameroon and other regions of sub-Saharan Africa where HIV accounts for the majority of the global infection, so as to improve management strategies for HBV infection and ART implementation.     

Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core''s YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.     

Umbilical Cord Blood Mononuclear Cell HIV-1 LTR Binding Activities

Vertically transmitted HIV disease constitutes a significant problem in pediatrics. In order to characterize some of the possible host factors involved in HIV replication in fetuses and newborns, we surveyed the HIV-1 LTR binding factors present in nuclear extracts from cord blood mononuclear cells. A series of electrophoretic mobility shift assays (EMSAs) showed that protein extracts from cord blood interacted with several regions of the HIV LTR. The most prominent binding activities involved the NF-kB sites, but other regions of the LTR also showed factor binding with the cord blood extracts. Some of these cord blood extract binding activities displayed qualitative differences when compared to adult peripheral blood mononuclear cell extracts in EMSA and UV cross-linking studies. Transient transfection experiments indicated that the NF-kB and Sp1 sequences were important for wild type levels of expression in cord blood cells, but that additional sequences 5 to the NF-kB sites also contributed activity. Thus, factors that interact with many of the well-known HIV LTR regulatory sites are present in cord blood cells. However, certain qualitative differences distinguished cord blood and adult peripheral blood binding activities and these may contribute to pathogenesis of HIV infection in neonates.     

Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices

Lipid droplets (LD) are dynamic storage organelles that are involved in lipid homeostasis. Hepatitis C virus (HCV) is closely associated with LDs. HCV Core and nonstructural (NS) proteins colocalize with LDs and presumably are involved in virion formation at that site. We demonstrated that HCV NS4B, an integral membrane protein in endoplasmic reticulum (ER), strongly targeted LDs. Confocal imaging studies showed that NS4B localized at the margins of LDs. Biochemical fractionation of HCV-replicating cells suggested that NS4B existed in membranes associated with LDs rather than on the LD surface membrane itself. The N- and C-terminal cytosolic domains of NS4B showed targeting of LDs, with the former being much stronger. In both domains, activity was present in the region containing an amphipathic α-helix, in which 10 hydrophobic residues were identified as putative determinants for targeting LDs. JFH1 mutants with alanine substitutions for the hydrophobic residues were defective for virus replication. W43A mutant with a single alanine substitution showed loss of association of NS4B with LDs and severely reduced release of infectious virions compared with wild-type JFH1. NS4B plays a crucial role in virus replication at the site of virion formation, namely, the microenvironment associated with LDs.     

Possible mechanisms of the reciprocal influence of infections caused by HIV and hepatitis C virus

Literature data on the pathogenesis of mixed infection caused by human immunodeficiency viruses (HIV), type I, and hepatitis C virus (HCV), as well as on possible mechanisms of the reciprocal influence of these viruses necessary to understand their co-existence, are reviewed. The extrahepatic replication of HCV, its role in the infectious process and its possible contribution to the course of mono- and co-infection are considered in detail. Much attention is paid to the specific features of immune response in HIV-HCV infection and the immunomediated mechanisms of the reciprocal influence of the viruses. The problem of the hepatotoxicity of antiretrovirus preparations, actively discussed in literature, is considered from biological positions. The role of the phenomenon of immunity reconstitution in cases of the exacerbation of chronic HCV infection in the course of the treatment of HIV infection as well as of other factors, in particular, infections caused by other parenterally transmitted viruses, in the course of HIV-HCV co-infection is pointed out.     

RNA binding protein 24 regulates the translation and replication of hepatitis C virus

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5′-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5′-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5′- and 3′-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.     

Hepatitis B and C Co-Infection in HIV Patients from the TREAT Asia HIV Observational Database: Analysis of Risk Factors and Survival

Background

We assessed the effects of hepatitis B (HBV) or hepatitis C (HCV) co-infection on outcomes of antiretroviral therapy (ART) in HIV-infected patients enrolled in the TREAT Asia HIV Observational Database (TAHOD), a multi-center cohort of HIV-infected patients in the Asia-Pacific region.

Methods

Patients testing HBs antigen (Ag) or HCV antibody (Ab) positive within enrollment into TAHOD were considered HBV or HCV co-infected. Factors associated with HBV and/or HCV co-infection were assessed by logistic regression models. Factors associated with post-ART HIV immunological response (CD4 change after six months) and virological response (HIV RNA <400 copies/ml after 12 months) were also determined. Survival was assessed by the Kaplan-Meier method and log rank test.

Results

A total of 7,455 subjects were recruited by December 2012. Of patients tested, 591/5656 (10.4%) were HBsAg positive, 794/5215 (15.2%) were HCVAb positive, and 88/4966 (1.8%) were positive for both markers. In multivariate analysis, HCV co-infection, age, route of HIV infection, baseline CD4 count, baseline HIV RNA, and HIV-1 subtype were associated with immunological recovery. Age, route of HIV infection, baseline CD4 count, baseline HIV RNA, ART regimen, prior ART and HIV-1 subtype, but not HBV or HCV co-infection, affected HIV RNA suppression. Risk factors affecting mortality included HCV co-infection, age, CDC stage, baseline CD4 count, baseline HIV RNA and prior mono/dual ART. Shortest survival was seen in subjects who were both HBV- and HCV-positive.

Conclusion

In this Asian cohort of HIV-infected patients, HCV co-infection, but not HBV co-infection, was associated with lower CD4 cell recovery after ART and increased mortality.     

Viral Co-infection and Leprosy Outcomes: A Cohort Study

Background

The role of the host immunity in determining leprosy clinical forms and complications is well recognized, implying that changes in the immune status may interfere with several aspects of the disease. Therefore, we hypothesized that the presence of viral co-infections and associated immunological changes will have a clinical impact on leprosy outcomes. The aim of our study was to determine the clinical impact of human immunodeficiency virus (HIV), human T cell lymphotrophic virus type 1 (HTLV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection on the development of reactions, neuritis, neuropathy and relapses.

Methodology/Principal Findings

Cohort study in 245 leprosy subjects from Bahia, Brazil. Patients were followed from the time of diagnosis until at least the end of multidrug therapy. Viral co-infection was detected in 36 out of the 245 patients (14.7%). Specific co-infection rates were 10.6% for HBV, 2.9% for HIV, 2.5% for HTLV-1 and 0.8% for HCV. All four groups of co-infected patients had higher rates of neuritis and nerve function impairment compared to non co-infected leprosy subjects. The relapse rate was also higher in the co-infected group (8.3%) versus patients without co-infection (1.9%); relative risk 4.37, 95% confidence interval 1.02–18.74.

Conclusions/Significance

Leprosy patients should be screened for HBV, HCV, HIV and HTLV-1 co-infections. Besides contributing to better health care, this measure will facilitate the early detection of severe complications through targeting of higher risk patients.     

Hepatitis C Virus Infection Induces Inflammatory Cytokines and Chemokines Mediated by the Cross Talk between Hepatocytes and Stellate Cells

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro, we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) β mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPβ target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPβ in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.     

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.     

Ribavirin Contributes to Hepatitis C Virus Suppression by Augmenting pDC Activation and Type 1 IFN Production

Ribavirin is used as a component of combination therapies for the treatment of chronic hepatitis C virus (HCV) infection together with pegylated interferon and/or direct-acting antiviral drugs. Its mechanism of action, however, is not clear. Direct antiviral activity and immunomodulatory functions have been implicated. Plasmacytoid dendritic cells (pDCs) are the principal source of type 1 interferon during viral infection. The interaction of pDCs with HCV-infected hepatocytes is the subject of intense recent investigation, but the effect of ribavirin on pDC activation has not been evaluated. In this study we showed that ribavirin augments toll-like receptors 7 and 9-mediated IFNα/β expression from pDCs and up-regulated numerous interferon-stimulated genes. Using the H77S.3 HCV infection and replication system, we showed that ribavirin enhanced the ability of activated pDCs to inhibit HCV replication, correlated with elevated induction of IFNα. Our findings provide novel evidence that ribavirin contributes to HCV inhibition by augmenting pDCs-derived type 1 IFN production.