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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.

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., (Copyright © 2018 American Society for Microbiology.)

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 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

Hepatitis C virus (HCV) infection is a major cause for chronic liver disease and hepatocellular carcinoma. The HCV-ARF/core+1 protein is an alternative product of HCV core-encoding sequence of unknown biological function. Highly purified HCV core and ARF/core+1 recombinant proteins from HCV genotype 1a and HCV-ARF/core+1 recombinant protein from HCV genotype 3a were expressed in Escherichia coli. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/core+1 antibodies in 90 chronic hepatitis C patients infected with HCV genotypes 1a/1b or 3a, treated with pegylated interferon (Peg-IFN-a-2a) plus ribavirin. Samples derived from 92 healthy blood donors were used as negative controls. All HCV-RNA-positive serum samples reacted with core 1a antigen, while 15 (37.5%) of 40 and 14 (28%) of 50 patients infected with HCV-1a/1b and HCV-3a, respectively, were found to have anti-ARF/core+1 antibodies into their serum before treatment initiation. These antibodies were persistently present during treatment follow-up and linked to elevated levels of HCV-RNA at baseline., (© 2011 Blackwell Publishing Ltd.)

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

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.

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., (Copyright © 2010. Published by Elsevier B.V.)

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., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) is a major disease agent affecting approximately 3% of the world's population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa precore protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome binding sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on Western blots providing a first step towards developing a chloroplast-based HCV diagnostic method., (Copyright 2009 Elsevier B.V. All rights reserved.)

The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome. Core+1/S is expressed as a result of internal initiation at AUG codons (85-87) located downstream of the polyprotein initiator codon, and corresponds to the C-terminal part of most ARFPs. Core+1/S is a highly basic polypeptide, and its function still remains unclear. In this work, untagged recombinant Core+1/S was expressed and purified from Escherichia coli in native conditions, and was shown to react with sera of HCV-positive patients. We subsequently undertook the biochemical and biophysical characterization of Core+1/S. The conformation and oligomeric state of Core+1/S were investigated using size exclusion chromatography, dynamic light scattering, fluorescence, CD, and NMR. Consistent with sequence-based disorder predictions, Core+1/S lacks significant secondary structure in vitro, which might be relevant for the recognition of diverse molecular partners and/or for the assembly of Core+1/S. This study is the first reported structural characterization of an HCV ARFP/Core+1 protein, and provides evidence that ARFP/Core+1/S is highly disordered under native conditions, with a tendency for self-association.

Circulating 'free' non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core(173)-eGFP, eGFP-core(191) and eGFP-core(173) can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells - the main target of HCV - but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.

Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus of the Flaviviridae family. It has a genome of about 9,600 nucleotides encoding a large polyprotein (about 3,000 amino acids) that is processed by cellular and viral proteases into at least 10 structural and nonstructural viral proteins. A novel HCV protein has also been identified by our laboratory and others. This protein--known as ARFP (alternative reading frame protein), F (for frameshift) or core+1 (to indicate the position) protein--is synthesized by an open reading frame overlapping the core gene at nucleotide +1 (core+1 ORF). However, almost 10 years after its discovery, we still know little of the biological role of the ARFP/F/core+1 protein. Abolishing core+1 protein production has no affect on HCV replication in cell culture or uPA-SCID mice, suggesting that core+1 protein is probably not important for the HCV reproductive cycle. However, the detection of specific anti-core+1 antibodies and T-cell responses in HCV-infected patients, as reported by many independent laboratories, provides strong evidence that this protein is produced in vivo. Furthermore, analyses of the HCV sequences isolated from patients with hepatocellular carcinoma and in vitro studies have provided strong preliminary evidence to suggest that core+1 protein plays a role in advanced liver disease and liver cancer. The available in vitro data also suggest that certain core function proteins may depend on production of the core+1 protein. We describe here the discovery of the various forms of the core+1 protein and what is currently known about the mechanisms of their production and their biochemical and functional properties. We also provide a detailed summary of the results of patient-based research.

The hepatitis C virus (HCV) genome possesses an open reading frame overlapping the core gene in the +1 frame (core+1 ORF). Initial studies, mainly in rabbit reticulocyte lysates, indicated that the HCV-1 core+1 ORF is expressed by a -2/+1 frameshift at codons 8-11 during translation elongation of the viral polyprotein, resulting in a protein known as alternative reading frame protein (ARFP), frameshift (F), or core+1. However, subsequent investigation, based on reporter constructs carrying portions of the core+1 ORF, suggested the function of alternative mechanisms for core+1 expression in mammalian cells, including translation initiation from internal codons 85/87 or 26. Because results from these studies have been variable, we sought to re-evaluate expression of the core+1 ORF using constructs carrying the complete core+1 coding sequence fused to GFP or LUC. We showed here that codons 85/87 serve as the predominant initiation sites for internal translation initiation of core+1 ORF in Huh-7 and Huh-7/T7 mammalian cells, which support nuclear or cytoplasmic transcription, respectively. We also showed that internal translation initiation can occur concomitantly with the expression of the core+1/F protein that is produced artificially in Huh-7 or naturally in Huh-7/T7 cells. Furthermore, translation of core+1 ORF is not significantly affected by the presence of the HCV IRES element. The core+1/S-GFP protein is cytoplasmic and exhibits an ER distribution similar to that of the core+1/F-GFP protein.

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal -2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the -2/+1 and -1/+2 frameshift events operating at the HCV-1 core codons 8-11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8-11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.

HCV-1 produces a novel protein, known as ARFP, F, or core+1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the +1 frame (core+1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core+1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core+1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core+1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8-11), the proposed frameshift site for the production of the ARFP/F/core+1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core+1 ORF. Furthermore, we showed that efficient translation initiation of the core+1 ORF is mediated by internal initiation codon(s) within the core/core+1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core+1 protein in transfected cells.