The majority of hepatitis C virus (HCV) isolates contain an open reading frame (ORF) overlapping with the core coding sequences in the ؉1 frame, which was assumed to be untranslated. We present evidence supporting the expression of this ORF (designated core؉1 ORF) via novel translation mechanisms. First, fusion of the luciferase gene with the HCV-1 core؉1 ORF followed by in vitro translation resulted in the synthesis of a chimeric protein (core؉1-luciferase) that exhibited ϳ54% luciferase activity relative to the positive control (coreluciferase). Second, antisera raised against two different synthetic core؉1 peptides recognized the previously identified p16 (but not p21) core protein band expressed from HCV-1, indicating the presence of epitopes from the core؉1 ORF within the p16 protein. Third, HCVpositive sera specifically recognized lysates of Escherichia coli cells expressing recombinant core؉1 protein, suggesting the presence of anti-core؉1 antibodies in HCV-infected patients. Finally, luciferase tagging experiments designed to assess for ؊1 frameshifting combined with site-directed mutagenesis experiments supported the presence of ؉1/؊1 ribosomal frameshift translation mechanisms within the core coding region. In conclusion, our data provide evidence for novel translation mechanisms within the core coding region and demonstrate the expression of the core؉1 ORF, at least for some HCV isolates.

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core؉1/ARFP/F). To study the importance of the core؉1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core؉1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core؉1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core؉1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.Hepatitis C virus (HCV) infection causes a wide spectrum of clinical manifestations, ranging from a healthy carrier state to acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (17). More than 170 million people worldwide are chronically infected with the virus, which is responsible for more than 100,000 cases of liver cancer per year (39). A vaccine against the virus is not available at present, and therapeutic approaches are still limited (13,18).HCV is classified within the genus Hepacivirus of the family Flaviviridae (51). Seven major HCV genotypes and more than 100 subtypes have been identified (42). The single-stranded positive-sense RNA genome of HCV (ϳ9.6 kb in length) is flanked at both termini by conserved, highly structured nontranslated regions (NTRs) and encodes a polyprotein precursor of about 3,000 amino acids (4, 37). The NTRs are required for RNA translation and replication. An internal ribosome entry site (IRES) residing in the 5Ј NTR controls translation initiation, which is influenced by multiple transacting cellular factors via interaction either with the IRES sequence or core encoding sequences (20,33,40). Furthermore, long-range RNA-RNA interactions between the IRES and distally located sequences residing in the core coding region or 3Ј NTR, as well as interactions with the viral-protein core, nonstructural protein 4A (NS4A), NS4B, and NS5A, appear to modulate IRES activity.In the N-terminal region, the polyprotein, is processed by cellular proteases to yield the structural-protein core (C) and enve...

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.

; 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...

l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process.

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

Anti-SARS-CoV-2 spike RBD (receptor-binding domain) IgG antibody levels were monitored in 1643 volunteer healthcare workers of Eginition, Evangelismos, and Konstantopoulio General Hospitals (Athens, Greece), who underwent vaccination with two doses of COVID-19 BNT162b2 mRNA vaccine (Pfizer) and had no history of SARS-CoV-2 infection. Venous blood was collected 20–30 days after the second vaccine dose and anti-RBD IgG levels were determined using CMIA SARS-CoV-2 IgG II Quant (Abbott) on ARCHITECT i System or ADVIA Centaur SARS-CoV-2 IgG (Siemens) on Centaur XP platform. From the total population of 1643 vaccinees (533 M/1110 F; median age = 49; interquartile range-IQR = 40–56), 1636 (99.6%) had anti-SARS-CoV-2 IgG titers above the positivity threshold of the assay used. One-Way ANOVA Kruskal-Wallis H test showed a statistically significant difference in the median of antibody titers between the different age groups (p < 0.0001). Consistently, Spearman’s correlation coefficient (r) for IgGs and age as continuous variables was −0.2380 (p = 1.98 × 10−17). Moreover, antibody titers were slightly higher by 1.2-mean fold (p = 3 × 10−6) in the total female population of the three hospitals (median = 1594; IQR = 875–2584) as compared to males (median = 1292; IQR = 671.9–2188). The present study supports that BNT162b2 vaccine is particularly effective in producing high anti-SARS-CoV-2 IgG levels in healthy individuals, and this humoral response is age- and gender-dependent.

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.