Alternative Reading Frame Protein Research Articles

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

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.

Hepatitis C virus F protein: A double-edged sword in the potential contribution of chronic inflammation to carcinogenesis

Persistent hepatitis C virus (HCV) infection can lead to chronic inflammation and even carcinogenesis. This may in part be due to various viral proteins, which affect apoptosis and promote liver cell growth. Recently, researchers reported a novel F protein, an alternative reading frame protein expressed from the HCV core coding sequence. The functional properties of this protein are presently unclear. Thus, we investigated whether F protein participates in the process of chronic HCV inflammation leading to malignant transformation. Serum or peripheral blood from 42 chronic HCV patients or 38 HCV-associated hepatocellular carcinoma (HCC) patients was analyzed for the presence of T lymphocytes and antibodies specific to F protein. Then, the correlation between indicators of F-specific immune response and viral load or serum IL-6 levels was examined. We identified the presence of both T- and B-cell-mediated immune responses specific to such an antigen in subjects with chronic HCV and in HCC patients. Of note, the presence of anti-F-specific antibodies appeared to be negatively correlated with HCV RNA viral load level. Furthermore, circulating IL-6 levels were significantly higher in patients with HCC than in those with chronic HCV. These increased serum IL-6 levels were, in some HCC patients, positively correlated with the presence of anti-F-specific antibodies. Taken together, these results suggest that F protein may participate in viral clearance and have anti-tumor effects, but may also contribute to the persistence of HCV infection in patients with a low viral load. This latter effect may cause hepatic damage leading to carcinogenesis. Elucidation of these F protein-associated contradictory functional activities is required.

Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala 14 and Thr 31) were found to destabilize the protein while two others (Val 24 and Ala 41) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val 24 was required for p53-independent growth suppression whereas multiple residues (Val 24, Thr 31, Ala 41 and His 60) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.

Seroconversion to hepatitis C virus alternate reading frame protein during acute infection #

The existence of hepatitis C virus (HCV) proteins encoded by alternate reading frames overlapping the core-encoding region has been suggested. Several mechanisms of production have been postulated, and the functions of these proteins in the HCV life cycle remain unknown. We analyzed cases of seroconversion to an alternate reading frame protein in a group of 17 patients infected by one of the two HCV genotype 1b strains during an outbreak in a hemodialysis unit. Three patients seroconverted, and antibodies were transiently detected in another patient. Three of these patients were infected by one of the two HCV strains, whereas the strain infecting the remaining patient could not be identified. Quasispecies sequence analysis of the core-coding region showed no differences in the core or +1 reading frame sequences that could explain alternate reading frame protein seroconversion in some but not all of the patients infected by one of the HCV strains, and no such difference was found between the two strains. Because differences in the structure of RNA elements could play a role in frameshift events, we conducted a predictive analysis of RNA folding. No difference was found between the patients who did and did not seroconvert to alternate reading frame protein. Our findings prove that alternate reading frame proteins can be produced during acute HCV infection. However, seroconversion does not occur in all patients for unknown reasons. Alternate reading frame protein could be generated by minority quasispecies variants or variants that occur transiently.

Subcellular localizations of the hepatitis C virus alternate reading frame proteins

Alternate reading frame proteins (ARFPs) resulting either from frameshifting, from transcriptional slippage or from internal initiation in the +1 open reading frame (ORF) of hepatitis C virus (HCV) core protein coding sequence have been described in vitro. As an approach to study the roles of these proteins, we investigate the subcellular localization of ARFPs fused with the green fluorescent protein (GFP) either at their N- or C-terminus. Most GFP fusion products have a diffuse localization, as revealed by confocal microscopy. One GFP chimeric protein, arising from internal initiation at codon 26 in the +1 ORF (ARFP 26–161), is specifically targeted to mitochondria. Mitochondrial localization was confirmed by immunoblot with an anti-ARFP antibody of a mitochondria-enriched cellular fraction. Mitochondrial targeting of ARFP 26–161 mostly involved the N-terminal portion of the protein as revealed by the cellular localization of truncated mutants. Interestingly, ARFP 26–161 from both genotypes 1a and 1b, but not the protein from the genotype 2a JFH1 infectious sequence, exhibit mitochondrial localization. These results are the first concerning the cellular localization and the role of this HCV ARFP; they may serve as a platform for further studies on its mitochondrial effects and their role in the virus life cycle and pathogenesis.

Assessment of Specific Antibodies to F Protein in Serum Samples from Chinese Hepatitis C Patients Treated with Interferon plus Ribavarin

The hepatitis C virus (HCV) alternate reading frame protein or F protein of the HCV 1b genotype is a double-frameshift product of the HCV core protein. In order to assess the presence of antibodies specific for F protein and their clinical relevance in sera from HCV patients, we produced recombinant F protein and core protein of the HCV 1b genotype in Escherichia coli. An enzyme-linked immunosorbent assay was developed using purified recombinant HCV core, F protein, and a 99-residue synthetic F peptide (F99). The seroprevalences of anticore, anti-F protein, and anti-F99 synthetic peptide were 95%, 68%, and 36%, respectively, in 168 HCV patients. The prevalence of anti-F antibodies did not correlate with viral load, genotype, or alanine aminotransferase level. Interferon combination therapy induced a decline in the level of anti-F antibodies in 55 responders (P < 0.01). Thirteen responders (24%) lost their anti-F recombinant protein antibodies, and 17 (31%) lost their anti-F synthetic peptide antibodies, whereas no decrease was observed for the 17 nonresponders. These changes were significant between responders and nonresponders (P < 0.05). Meanwhile, no change was found in the anticore antibody titer of the 72 treated patients. The percentage of anti-F-protein-negative patients (15/15 [100%]) who achieved a sustained virological response (SVR) was higher than that of the anti-F-positive patients (70%) (P < 0.05). Based on these findings, HCV F protein elicits a specific antibody response other than the anticore protein response. Our data also suggest that the presence and level of anti-F antibody responses might be influenced by the treatment (interferon plus ribavirin) and associated with an SVR in Chinese hepatitis C patients.

Cooperation between the ribosomal proteins L5 and L11 in the p53 pathway

MDM2 is a ubiquitin ligase that plays a key role in regulating the stability of the p53 tumor suppressor protein. Several proteins have been shown to activate the p53 pathway by interacting with and inhibiting the E3 function of MDM2, thereby leading to an accumulation of p53. These include the alternate reading frame (ARF) proteins and the ribosomal proteins L5 and L11. We found that when overexpressed alone, L11 is much less potent in inhibiting MDM2 than p14(ARF). However, L11 cooperates with L5, resulting in a robust inhibition of the E3 activity of MDM2, and a stabilization and activation of p53 approaching that achieved by p14(ARF). We further showed that the ability of L11 to bind the 5S rRNA is important for the cooperation with L5, and a mutant L11, which cannot bind the 5S rRNA, cannot cooperate with L5 in inhibiting MDM2.

Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies.

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression

Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an ‘alternate reading frame’ (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named 86/88ARFP. Interestingly, 86/88ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle.

Expression studies of the HCV-1a core+1 open reading frame in mammalian cells

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.

Expression of alternate reading frame protein (F1) of hepatitis C virus in Escherichia coli and detection of antibodies for F1 in Indian patients

Apart from the core (21 kD), a novel hepatitis C virus (HCV) frame shift protein (F1) is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frame shift into the −2/+1 reading frame. To date, no information is available on F1 protein of Indian isolates, and hence detection of antibodies for F1 protein in Indian patients assumes great relevance. Specific primers have been designed to amplify sequence coding for 120aa of truncated F1 (tF1). The amplified tF1 has been cloned in bacterial expression vector, pET21b for expression in Escherichia coli. Partially purified expressed protein has been subjected to western blot analysis using patients’ sera. Three HCV positive sera employed in western analysis showed positive signals to tF1, while sera from uninfected individuals failed to give any signals. Further, results of western blots, carried out with patients sera titrated with purified core protein, confirmed the presence of antibodies specific to F1. The positive signal observed for F1 in western analysis with HCV infected sera suggests that F1 protein is synthesized in the natural course of HCV infection in Indian patients as well. Presence of antibodies against F1 protein of subtype 1c has been demonstrated, for the first time, in Indian patients.

CD43 promotes cell growth and helps to evade FAS-mediated apoptosis in non-hematopoietic cancer cells lacking the tumor suppressors p53 or ARF

CD43 is a highly glycosylated transmembrane protein expressed on the surface of most hematopoietic cells. Expression of CD43 has also been demonstrated in many human tumor tissues, including colon adenomas and carcinomas, but not in normal colon epithelium. The potential contribution of CD43 to tumor development is still not understood. Here, we show that overexpression of CD43 increases cell growth and colony formation in mouse and human cells lacking expression of either p53 or ARF (alternative reading frame) tumor-suppressor proteins. In addition, CD43 overexpression also lowers the detection of the FAS death receptor on the cell surface of human cancer cells, and thereby helps to evade FAS-mediated apoptosis. However, when both p53 and ARF proteins are present, CD43 overexpression activates p53 and suppresses colony formation due to induction of apoptosis. These observations suggest CD43 as a potential contributor to tumor development and the functional ARF-p53 pathway is required for the elimination of cells with aberrant CD43 expression.

Delayed ageing through damage protection by the Arf/p53 pathway

The tumour-suppressor pathway formed by the alternative reading frame protein of the Cdkn2a locus (Arf) and by p53 (also called Trp53) plays a central part in the detection and elimination of cellular damage, and this constitutes the basis of its potent cancer protection activity. Similar to cancer, ageing also results from the accumulation of damage and, therefore, we have reasoned that Arf/p53 could have anti-ageing activity by alleviating the load of age-associated damage. Here we show that genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 present strong cancer resistance and have decreased levels of ageing-associated damage. These observations extend the protective role of Arf/p53 to ageing, revealing a previously unknown anti-ageing mechanism and providing a rationale for the co-evolution of cancer resistance and longevity.

Expression of the alternative reading frame protein of Hepatitis C virus induces cytokines involved in hepatic injuries

Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mphi) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mphi and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CDeltaARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1beta, whereas production of these chemokines with AdCDeltaARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1beta in Mphi. Wild-type Core sequence, but not CDeltaARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42-44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.

CARF Binds to Three Members (ARF, p53, and HDM2) of the p53 Tumor‐Suppressor Pathway

The INK4a locus (chromosome 9p21) encodes two structurally distinct tumor-suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a major role in cell cycle control and senescence pathways. We originally identified a novel collaborator of ARF, CARF, from a two-hybrid interactive screen using p19(ARF) as bait and found that CARF interacts with ARF in the perinucleolar region and activates p53 function. In the absence of ARF, it interacts with p53 directly leading to ARF-independent enhancement of p53 function and in turn undergoes a negative feedback regulation. Very recently, we found that CARF interacts with HDM2 and undergoes degradation by an HDM2-dependent proteasome pathway. CARF may exert a vital control on p53-HDM2-p21(WAF1) pathway that is central to the cell cycle control, senescence, and DNA damage response of human cells.

TBP-1 protects the human oncosuppressor p14ARF from proteasomal degradation

The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARF's interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes

InOctober 2004, an expert meeting was convened in parallel with the 11th Symposium onHepatitis C and Related Viruses to discuss how HCV sequence databases could introduce and facilitate a standardized numbering system for HCV nucleotides, proteins and epitopes. Inconsistent and inaccurate numbering of locations in DNA and protein sequences is a problem in the HCV scientific literature. Consistency in numbering is increasingly required for functional and clinical studies of HCV. For example, an unambiguous method for referring to amino acid substitutions at specific positions in NS3 and NS5B coding sequences associated with resistance to specific HCV inhibitors is essential in the investigation of antiviral treatment. This article provides a practical guide to help circumvent these problems in the future, and to bring a common language into discussions in the field. The scope of the current system is limited to the HCV polyprotein and the untranslated regions (UTRs); because of the controversial nature and extreme length variation of the alternate reading frame proteins, numbering for these proteins, if needed, will be decided at a later date. We propose a numbering system adapted from the Los Alamos HIV database,1 with elements from the hepatitis B virus numbering system.2 The system comprises both nucleotides and amino acid sequences and epitopes. It uses the full length genome sequence of isolate H77 (accession number AF009606) as a reference, and includes a method for numbering insertions and deletions relative to this reference sequence. H77 was chosen because it is a commonly used reference strain for many different kinds of functional studies. Furthermore, RNA transcripts from this sequence are of demonstrated infectivity,3,4 providing evidence that the 5 and 3 ends of the sequence are complete. Table 1 lists the boundaries of HCV genomic regions and Fig. 1 provides detailed nucleic acid and amino acid numbering over the complete AF009606 HCV genome sequence.

P.167 Relative contribution of core and alternate reading frame protein of HCV to chemokine release by monocytic cells: Implications for HCV pathogenesis

P.167 Relative contribution of core and alternate reading frame protein of HCV to chemokine release by monocytic cells: Implications for HCV pathogenesis

A Merging Duo in Melanoma Formation

A Merging Duo in Melanoma Formation

An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer

The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor gamma-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.