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

Vassilaki, Niki; Mavromara, Penelope

doi:10.1002/iub.201

Cited by 51 publications

(52 citation statements)

References 116 publications

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“…This copies the viral positive-strand RNA into a negative-strand intermediate that serves as the template for the synthesis of progeny genomes. The alternative reading frame (ARFP) or coreϩ1 and minicore proteins, with as-yet-unknown functions, appear to be synthesized from the core region by alternative translation mechanisms (6,7).…”

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confidence: 99%

Low Oxygen Tension Enhances Hepatitis C Virus Replication

Vassilaki

Kalliampakou

Kotta‐Loizou

et al. 2013

J Virol

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; Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, South Korea g Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infectionbased assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-␣) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCVhost cell interaction. H epatitis C virus (HCV) infection causes a wide range of clinical manifestations, from a healthy carrier state to acute and chronic hepatitis that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Nearly 3% of the world's population is chronically infected with HCV (1, 2), and current therapeutic approaches are not broadly effective (3).HCV is a positive-strand RNA virus with a 9.6-kb genome that is flanked at both termini by conserved, nontranslated regions (NTRs), required for RNA translation and replication. The 5= NTR comprises an internal ribosome entry site (IRES) that directs the expression of a polyprotein precursor (4, 5). The polyprotein is cleaved into structural (core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins that, in association with cellular factors, form a membrane-associated replicase complex. This copies the viral positive-strand RNA into a negative-strand intermediate that serves as the template for the synthesis of progeny genomes. The alternative reading frame (ARFP) or coreϩ1 and minicore proteins, with as-yet-unknown functions, appear to be synthesized from the core region by alternative translation mechanisms (6, 7).Studies of the...

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confidence: 99%

Low Oxygen Tension Enhances Hepatitis C Virus Replication

Vassilaki

Kalliampakou

Kotta‐Loizou

et al. 2013

J Virol

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“…Recently, it was shown that HCV downregulates hepcidin expression through reactive oxygen species induction and histone deacetylase-mediated displacement of CCAAT/enhancer binding protein (C/ EBP)a and signal transducer and activator of transcription 3 (STAT3) from the hepcidin promoter, and this effect is mediated, at least partly, through core protein (Miura et al, 2008). As there is substantial evidence that core+1/ARFP acts as a transcriptional modulator (Vassilaki & Mavromara, 2009), we sought to investigate the effect of core+1/ARFP on hepcidin gene expression.…”

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confidence: 99%

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

Kotta‐Loizou

Vassilaki

Pissas

et al. 2013

Journal of General Virology

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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.

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“…For example, a form of the core ?1 protein was reported to be generated by initiation of translation at internal codons 85/87. This protein is shorter than that produced by the ?1 frameshift mechanism (F) and was named as core?1/S (short) [27]. Another form of this protein was also described as the ARFP/DF (double frameshift) protein.…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients

et al. 2015

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HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

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

Cited by 51 publications

References 116 publications

Low Oxygen Tension Enhances Hepatitis C Virus Replication

Low Oxygen Tension Enhances Hepatitis C Virus Replication

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

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients

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