Hepatitis C Core Protein Induces a Genotype-Specific Susceptibility of Hepatocytes to TNF-Induced Death In Vitro and In Vivo.

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo.

A Proteomic Approach to Study the Biological Role of Hepatitis C Virus Protein Core+1/ARFP.

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein-known as ARFP (alternative reading frame protein) or Core+1 protein-is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.

Association between vitamin D receptor gene polymorphisms and fibrosis susceptibility in Greek patients with HCV infection.

Introduction: Hepatitis C virus (HCV) infection is a prime cause of chronic hepatitis worldwide, that often silently progresses to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Notably, the majority of individuals infected with HCV develop symptoms at late stages, often associated with liver damage that cannot revert after virus clearance. Thus, current antiviral therapy alone is rather insufficient to eliminate the global burden of HCV in the near future.During the past few years, vitamin D deficiency as well as certain single nucleotide polymorphisms in the vitamin D receptor (VDR) gene have been associated with liver fibrosis. Therefore, the aim of the present study was to investigate the possible correlation between VDR polymorphisms ApaI (rs7975232) and TaqI (rs731236) and the fibrosis stage of patients with HCV infection from Thrace, Greece. Methods: Eighty-one patients with HCV infection underwent transient elastography for the assessment of their fibrosis stage, and PCR-restriction fragment length polymorphism (RFLP) genotyping for VDR ApaI and TaqI polymorphisms. VDR genotypes were then statistically associated with the patients' fibrosis stage using ordinal regression models. Results: Non-cirrhotic stages were positively correlated with TaqI TT genotype (p=0.003) and negatively correlated with TaqI TC genotype (p=0.007). In the presence of Hardy-Weinberg equilibrium and linkage disequilibrium between the two VDR polymorphisms, mild fibrosis stages (F0-2) were correlated with ApaI/TaqI GG/TT (p=0.002) and TG/TT (p=0.008) genotypes, while cirrhotic stage F4 was associated with ApaI/TaqI TG/TC genotype (p=0.038). Conclusions: TaqI TT and ApaI/TaqI GG/TT, TG/TT and TG/TC genotypes could be explored as prognostic genetic markers for fibrosis susceptibility in HCV patients.

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF.

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

Comparison of Dendritic Cell Activation by Virus-Based Vaccine Delivery Vectors Emphasizes the Transcriptional Downregulation of the Oxidative Phosphorylation Pathway.

Antigen delivery platforms based on engineered viruses or virus-like particles are currently developed as vaccines against infectious diseases. As the interaction of vaccines with dendritic cells (DCs) shapes the immunological response, we compared the interaction of a range of virus-based vectors and virus-like particles with DCs in a murine model of systemic administration and transcriptome analyses of splenic DCs. The transcriptome profiles of DCs separated the vaccine vectors into two distinct groups characterized by high- and low-magnitude differential gene expression, which strongly correlated with (1) the surface expression of costimulatory molecules CD40, CD83, and CD86 on DCs, and (2) antigen-specific T-cell responses. Pathway analysis using PANOGA (Pathway and Network-Oriented GWAS Analysis) revealed that the JAK/STAT pathway was significantly activated by both groups of vaccines. In contrast, the oxidative phosphorylation pathway was significantly downregulated only by the high-magnitude DC-stimulating vectors. A gene signature including exclusively chemokine-, cytokine-, and receptor-related genes revealed a vector-specific pattern. Overall, this in vivo DC stimulation model demonstrated a strong relationship between the levels of induced DC maturation and the intensity of T-cell-specific immune responses with a distinct cytokine/chemokine profile, metabolic shifting, and cell surface expression of maturation markers. It could represent an important tool for vaccine design.

Relationship between antibodies to hepatitis C virus core+1 protein and treatment outcome.

BACKGROUND: It has been suggested that hepatitis C virus (HCV) core+1 protein plays a crucial role in the viral life cycle, potentially affecting liver cirrhosis and the development of hepatocellular carcinoma. METHODS: To investigate its relationship with the outcome of HCV standard combination therapy with peginterferon-α plus ribavirin, we screened 139 consecutive HCV patients (119 with chronic HCV infection and 20 who spontaneously cleared HCV) for the presence of anti-core+1 antibodies (Abs). In addition, liver fibrosis was determined by FibroScan in all but one patients. RESULTS: Twenty-nine patients were cirrhotic (stiffness >12.5 kPa, F4 METAVIR), all of them with mild liver cirrhosis (Child-Pugh score A). Eighty-six of 139 patients were treatment-experienced with standard combination therapy. Fifty of them had achieved a sustained virological response, while 36 were non-responders. The prevalence of anti-core+1 Abs in patients with chronic HCV infection was 22.69% (27/119 patients): 18% (9/50 patients) in responders and 36.11% (13/36 patients) in non-responders (P=0.050). Five (17.24%) of the 29 cirrhotic patients and 22 (24.72%) of the 89 non-cirrhotic patients were positive for anti-core+1 Abs (P=0.405). Furthermore, the presence of anti-core+1 Abs correlated with the poor response interleukin (IL) 28B genotype TT (P=0.040). No correlation between spontaneous clearance and anti-core+1 Abs was observed (P=0.088). CONCLUSION: The presence of anti-core+1 Abs might be correlated with the poor response IL28B TT genotype and may negatively affect the outcome of standard combination treatments in HCV patients, suggesting that core+1 may play a biological role in the course of HCV infection.

Differential regulation of the Wnt/ß-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes.

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/ß-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/ß-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several ß-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of ß-catenin nuclear translocation varied in accordance with ß-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/ß-catenin pathway in relevant infection systems.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.

High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma.

Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.

Murine Bone Marrow-Derived Dendritic Cells Transduced by Light-Helper-Dependent Herpes Simplex Virus-1 Amplicon Vector Acquire a Mature Dendritic Cell Phenotype.

Dendritic cells (DCs) turn into the most potent antigen-presenting cells following a complex transforming process, which leads to their maturation. Herpes simplex virus-1 (HSV-1) amplicon vectors represent highly versatile viral vector platforms with the ability to transduce immature DCs at exceedingly high efficiencies, while the efficiency of infection of mature DCs is significantly low. However, the bacterial artificial chromosome (BAC)-dependent (BD) amplicon vectors tested so far do not result in the maturation of mouse bone marrow-derived DCs (BMDCs) in vitro. In this study we investigated the effects of light-helper-dependent (LHD) amplicon vectors produced with the replication-defective HSV-1 LaLΔJ helper virus system. First, we observed that transgene expression in BMDC cultures was equally potent between the LHD and the BD amplicon vectors. We determined that the percentage of transduced cells and the duration of transgene expression were negatively influenced by the presence of increasing levels of helper virus. Second, infection by the LHD amplicon vector as well as the helper HSV-1 LaLΔJ virus alone resulted in the phenotypic maturation of BMDCs and the expression of both interferon-stimulated genes and proinflammatory cytokines. Further comparisons of the gene expression of infected DCs showed that while interferon-stimulated genes such as Ifit1, Ifit3, Mx2, Isg15, and Cxcl10 were induced by both BD and LHD amplicon vectors, early proinflammatory cytokine gene expression (Tnfa, Il1a, Il1b, Il6, Il10, Il12b, Cxcl1, and Cxcl16) and DC maturation were mediated only by the LHD amplicons.

Expression of the novel hepatitis C virus core+1/ARF protein in the context of JFH1-based replicons.

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

HCV NS5A co-operates with PKR in modulating HCV IRES-dependent translation.

Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site.

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

Synonymous mutations in the core gene are linked to unusual serological profile in hepatitis C virus infection.

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of anti-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.

Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b.

The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.

Novel tumour-specific promoters for transcriptional targeting of hepatocellular carcinoma by herpes simplex virus vectors.

BACKGROUND: Hepatocellular carcinoma (HCC) is a cancer of poor prognosis, with limited success in patient treatment, which it makes an excellent target for gene therapy and viral oncolysis. Accordingly, herpes virus simplex type-1 (HSV-1) is one of the most promising viral platforms for transferring therapeutic genes and the development of oncolytic vectors that can target, multiply in, and eradicate hepatoma cells via their lytic cycle. Enhanced efficacy and specificity of HSV-1-based vectors towards HCC may be achieved by using HCC-specific gene promoters to drive selective viral gene expression and accomplish conditional replication and/or to control the expression of therapeutic genes. However, careful verification of promoter function in the context of the replication-competent HSV-1 vectors is required. The present study aimed to identify novel HCC-specific promoters that could efficiently direct transgene expression to HCC cells and maintain their activity during active viral replication. METHODS: Publicly available microarray data from human HCC biopsies were analysed in order to detect novel candidate genes induced primarily in HCC compared to normal liver. HCC specificity and promoter activity were evaluated by RT-PCR and chromatin immunoprecipitation. Additionally, transcriptional activity of promoters was further evaluated in the context of HSV-1 genome, using luciferase assays in cultured cells and animal models. RESULTS: Eight HCC-specific genes were characterised in this study: Angiopoietin-like-3, Cytochrome P450, family 2, subfamily C, polypeptide 8, Vitronectin, Alcohol dehydrogenase 6-class V, Apolipoprotein B, Fibrinogen beta chain, Inter-alpha-globulin-inhibitor H3 and Inter-alpha-globulin-inhibitor H1. Specific HCC expression and active gene transcription were confirmed in human liver and non-liver cell lines and further evaluated in primary neoplastic cells from hepatitis C and B virus (HCV- and HBV)-associated HCC patients. High promoter activity and specificity in the presence of HSV-1 infection and from within the viral genome, was validated, both in vitro and in vivo. CONCLUSIONS: We identified and experimentally characterized novel hepatoma-specific promoters, which were valuable for cancer-specific gene therapy, using HSV-1 vectors.

Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa.

BACKGROUND: According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10-43 in the core protein and 1689-1740 or 1921-1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. OBJECTIVE OF THIS STUDY: To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects. STUDY DESIGN: Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative). RESULTS: The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3-65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested). CONCLUSIONS: Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually.

Hepatitis C virus controls interferon production through PKR activation.

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2alpha initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2alpha-dependent (IRES(EMCV)) or independent (IRES(HCV)) RNA showed a specific HCV-mediated inhibition of eIF2alpha-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2alpha at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.