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

Boumlic, Anissa; Vassilaki, Niki; Dalagiorgou, Georgia; Kochlios, Emmanouil; Kakkanas, Athanassios; Georgopoulou, Urania; Markoulatos, Panagiotis; Orfanoudakis, Georges; Mavromara, Penelope

doi:10.1016/j.virusres.2010.10.007

Cited by 15 publications

(17 citation statements)

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“…Among the aforementioned putative initiation codons, those that fitted the expression profiles in the replicon system were codons 25/26 for coreϩ1/L and codons 85/87 for coreϩ1/S (12,13,15). After changing codons 26, 85, and 87 individually to GGG (which has never been reported to serve as an alternative initiation codon), the expression from the coreϩ1 frame was similar to that seen with the control (data not shown).…”

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confidence: 69%

“…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)(14)(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.…”

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confidence: 94%

“…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)(14)(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).…”

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confidence: 94%

“…*, P Ͻ 0.02 (Student's t test). 5=-GGCGTCTTCCCCAGATCTCCATCCTGCCCAGCC-3= coreϩ1 630 JFH1 5=-GGCGTCTTCCCCAGATCTGCCATCCTGCCCAGCC-3= core-1 630 JFH1 5=-GGCGTCTTCCCCAGATCTTCACCTGCCCAGCC-3= coreϩ1 630 H77 5=-GGCGTCTTCCCCAGATCTGCCATCCCGCCCACCC-3= N328 frameshift at codons 8 to 11 (7)(8)(9)33), internal initiation at codon 26 (12,15), a frameshift at codon 42 (16), and a shorter form initiating at codons 85/87 (13,15). As shown in Fig.…”

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confidence: 99%

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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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confidence: 69%

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confidence: 94%

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confidence: 94%

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confidence: 99%

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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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“…The basic amino acids in the cI cluster do not affect virus replication or infectivity when mutated to alanines one at a time or in groups of two or three, suggesting a structural role for this cluster in nucleocapsid formation (Alsaleh et al, 2010). The composition of the (A)-rich tract in the hcv orF determines the production of the alternate reading frame (ArF) of c protein which is being implicated in hcv pathogenesis (Boumlic et al, 2011). In summary, deletion of the 15 n-terminal amino acids from the c124 protein (which are encoded by 45 bases spanning the (A)-rich tract) bring about three important changes in the physical and/ or biochemical characteristics of the protein (Fig.…”

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confidence: 99%

The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Chinnaswamy¹,

Lott²

2012

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Summary. -hepatitis c virus (hcv) core (c) protein is thought to bind to viral rnA before it undergoes oligomerization leading to rnA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal c protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site (IreS). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral rnA by an IreS-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the c protein to bind to viral rnA. Serially truncated c proteins with deletions from 10 up to 45 n-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral rnA by a gel shift assay. The results showed that truncation of the c protein from its n-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the n-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGe. truncation by more than 20 amino acids resulted in a complete loss of ability of tagged c protein to bind to viral rnA. These results provide clues to the early events in the c protein -rnA interactions leading to c protein oligomerization, rnA encapsidation and virion assembly.Keywords: hepatitis c virus; core protein; rnA binding e-mail: sc2@nibmg.ac.in; phone: +91-33-25892151(256). Abbreviations: AM = anomalous migration; ArF = alternate reading frame; c protein = core protein; c124 = n terminal 124 amino acids of c protein; GeMSA = gel electrophoresis mobility shift assay; hcv = hepatitis c virus; IreS = internal ribosome entry site; Iec = ion exchange chromatography; nlP = nucleocapsidlike particles; MAb = monoclonal antibody; orF = open reading frame; Sec = size exclusion chromatography

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Hepatitis C Virus RNA Translation

Niepmann

2013

Current Topics in Microbiology and Immunology

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After infection of a cell, the positive-strand RNA genome of Hepatitis C Virus (HCV) directly serves as the template for translation in the cytosol. By the use of an internal ribosome entry site (IRES) element in the 5'-untranslated region (5'-UTR) of the viral RNA, the HCV RNA bypasses the need for nuclear processing events like capping and directly recruits the translation apparatus to the viral RNA to start translation of the viral proteins. In this review, I discuss the structure and function of the HCV IRES, focusing on (1) the recruitment of the cellular translation machinery to the IRES, including canonical and noncanonical translation initiation factors, (2) noncanonical RNA-binding proteins that modulate IRES activity, and (3) microRNAs that have an influence on the efficiency of HCV RNA translation.

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

Cited by 15 publications

References 36 publications

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

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

The adenine-rich tract in the 5ʹ-end of the hepatitis C virus ORF encodes a peptide regulating the binding of the C protein to RNA

Hepatitis C Virus RNA Translation

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