Molecular cloning of a defective hepatitis C virus genome from the ascitic fluid of a patient with hepatocellular carcinoma.

Yeh, Chau‐Ting; Lu, Su-Chuan; Chu, Chia–Ming; Liaw, Yun–Fan

doi:10.1099/0022-1317-78-11-2761

Cited by 32 publications

(28 citation statements)

References 32 publications

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“…At the moment, we have no information about the molecular events that might lead to the initiation of ARFP synthesis; however, several possibilities can be considered+ For example, the AUG of the main ORF, or an up-stream AUG, could be the start site of translation, with the reading frame slipping forward to bypass the stop codon at bases 2-4 (in the ϩ1 reading frame)+ Frame shifts are known to occur during gene expression of many viruses (Brierley, 1995)+ Alternatively, translation of ARFP might involve a non-AUG initiator codon; in fact, the HCV IRES has the power to direct translation from codons other than AUG (Reynolds et al+, 1995)+ Moreover, a recent report indicates that a novel translation initiation mechanism can lead to the synthesis of out-of-frame peptides (even some without upstream AUG initiation codons), which are then expressed by MHC class I molecules and stimulate immune responses (Malarkannan et al+, 1999)+ This report describes a series of non-AUG codons that allow translation of out-of-frame peptides at levels that elicit robust T cell responses+ One of these unusual start codons is AUC+ This unusual start codon is the third in-frame codon in .90% of the ϩ1 alternate reading frames of the reported HCV sequences+ If this were the start codon for the ARFP, then the expected product would range from 122 to 158 amino acids in the majority of sequences+ Finally, RNA stuttering could modify the sequence of HCV RNA during RNA replication, generating a new RNA that has ARFP as its translation product+ Stuttering gives rise to the RNA sequences required for the expression of proteins in most members of the Paramyxoviridae family (Liston & Briedis, 1995)+ If stuttering occurs during HCV replication, it could lead to the expression of chimeric proteins with amino terminal portions of the HCV core protein linked to carboxy-terminal portions of ARFP+ HCV sequences that could give rise to such chimeric proteins have been detected in PCR products from hepatocellular carcinoma (Ruster et al+, 1996) and the ascitic fluid of a patient with hepatocellular carcinoma (Yeh et al+, 1997)+ Our studies do not indicate whether ARFP plays a role in the HCV life cycle, or is synthesized "fortuitously+" However, the ORF is present in all reported HCV genotypes, and all the infectious clones described to date+ If ARFP has an impact on cellular or viral function, its highly basic nature and lack of a known nuclear localization signal suggests an interaction with cytoplasmic RNA, perhaps HCV RNA itself+ A possible role of ARFP in cellular transformation must be considered in light of studies showing chimeric proteins containing portions of ARFP might be expressed in HCC tissue and ascitic fluid (Ruster et al+, 1996; Yeh et al+, 1997)+ ,0+025 a HCV (13/100) vs+ total non-HCV (3/104)…”

Section: Arfp Properties and Synthesismentioning

confidence: 99%

Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame

Walewski

Keller

Stump

et al. 2001

RNA

240

227

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Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p , 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

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Section: Arfp Properties and Synthesismentioning

confidence: 99%

Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame

Walewski

Keller

Stump

et al. 2001

RNA

240

227

View full text Add to dashboard Cite

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“…In addition, phylogenetic analysis with the core11 amino-acid sequence has shown that the core11 variants obtained from HCC samples differ from those in nontumor cells, consistent with the selection of core11 variants with modified functional properties in HCV-induced HCC. Previous studies also reported the accumulation of characteristic mutations in specific regions of the HCV core/core11 RNA sequence in patients with HCC, these mutations being associated with an increase in the production of alternative forms of core/core11 proteins in cell-free systems in several cases (46)(47)(48)(49)(50)(51).…”

Section: Evidence For Core11 Protein Production During Natural Hcv Inmentioning

confidence: 99%

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

Vassilaki

Mavromara

2009

IUBMB Life

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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. © 2009 IUBMB IUBMB Life, 61(7): 739–752, 2009

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“…These studies suggest a link between coreϩ1/ARFP and development of liver cancer (22)(23)(24)(25)(26)(27). Conversely, coreϩ1/ARFP has no obvious effect on virus replication following electroporation of Huh7-Lunet cells with fulllength JFH1 wild-type and mutated viral RNAs or in mice xenografted with human liver tissue (28).…”

mentioning

confidence: 99%

Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

Kotta‐Loizou

Karakasiliotis

Vassilaki

et al. 2015

J Virol

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Hepatitis C virus (HCV) infection is a major cause of non-A, non-B hepatitis, which frequently leads to chronic liver disease and hepatocellular carcinoma (1); no vaccine is available, while the recent broadly effective therapy is highly expensive (2, 3). The single-stranded positive-sense RNA genome of HCV (9.6 kb) encodes a polyprotein precursor of about 3,000 amino acids flanked by conserved untranslated regions (UTRs) (4, 5). The UTRs are required for RNA translation and replication, and an internal ribosome entry site (IRES) within the 5= UTR controls translation initiation (6). The polyprotein is processed by cellular and viral proteases to yield at least 10 structural and nonstructural proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (4, 5).Previous studies from our laboratory and others have shown that HCV possesses a second functional open reading frame (ORF) within the core region of the polyprotein encoding an additional protein designated coreϩ1/alternative reading frame protein (ARFP) (7-9). This alternate ORF is present in all six HCV genotypes (9) but has been studied only in subtypes 1a and 1b. At first, a 17-kDa protein (p17), initially named ARFP (alternative reading frame protein), F (frameshift), or coreϩ1 (to indicate its position), was shown to be synthesized in rabbit reticulocyte lysate (RRL) from the initiating codon of the polyprotein sequence of a genotype 1a HCV type 1 (HCV-1) strain by a ϩ1 frameshift occurring in an A-rich core-encoding region (codons 8 to 11) (7-10). However, the expression of this form of coreϩ1/ARFP (known as F) is limited to the HCV-1 isolate, which is unique in carrying a stretch of 10 consecutive adenine nucleotides within codons 8 to 11 (7,11,12), and its expression in transfected cells is dependent on cytoplasmic transcription (11). Interestingly, other coreϩ1/ARFP forms are expressed independently of the A-rich sequence by an alternative translation mechanism(s). Internal translation initiation at methionine codons 85/87 of HCV-1a and HCV-1b, resulting in a shorter form of coreϩ1/ARFP, has been observed in transfected hepatoma cells (13-15). Furthermore, codon 26 of HCV-1a (12) and HCV-1b (15) was also found to function as an internal translation initiation site in mammalian cells. Additionally, a ϩ1 frameshift at codon 42 of HCV-1b, followed by a rephrasing into the core ORF at stop codon 144, was observed in Escherichia coli (16).The detection of specific anti-coreϩ1/ARFP antibodies and T-cell responses in HCV-infected patients (especially with hepatocellular carcinoma), as reported by many independent laboratories, provides strong evidence that coreϩ1/ARFP is produced during natural infection (7-9, 17-21). These studies suggest a link between coreϩ1/ARFP and development of liver cancer (22-27). Conversely, coreϩ1/ARFP has no obvious effect on virus replication following electroporation of Huh7-Lunet cells with fulllength JFH1 wild-type and mutated viral RNAs or in mice xenografted with human liver tissue (28). Those studies were based on a ...

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Molecular cloning of a defective hepatitis C virus genome from the ascitic fluid of a patient with hepatocellular carcinoma.

Cited by 32 publications

References 32 publications

Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame

Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

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