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.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(-) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV-1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(-) samples were tested further for HBV serological markers and HBV DNA was quantified by real-time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti-HBc only: 7 donors, anti-HBc/anti-HBs: 7 donors, anti-HBc/anti-HBe: 5 donors, anti-HBc/anti-HBs/anti-HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively.

The 5P P untranslated region of hepatitis C virus RNA forms an extensive secondary structure including several hairpin motifs and mediates translation initiation by an internal ribosome entry site-dependent pathway. We report, here, an extensive mutagenesis analysis of a highly conserved tetraloop in the 5P P untranslated region of hepatitis C virus, namely hairpin IIIe (295P P-GAUA-298P P). Our results demonstrate that hairpin IIIe is essential for the internal ribosome entry site function. Moreover, they indicate the importance of the primary structure of this motif because mutations in all four nucleotides of the loop caused a severe loss of internal ribosome entry site activity. These data represent the first experimental evidence for the functional significance of tetraloops in internal ribosome entry site-driven translation of hepatitis C virus.z 1999 Federation of European Biochemical Societies.

Background and Objectives. Health Related Quality of Life (HRQoL) is an important health outcome measure in haemophilia. The aim of this study was to assess the psychometric properties of the Greek version of Haem-A-QoL, a disease-specific questionnaire for haemophiliacs. Methods. Haem-A-QoL and SF-36 were administered to 118 adult haemophilia patients. Hypothesized scale structure, internal consistency (Cronbach's α), and test-retest reliability, as well as various types of construct validity were evaluated. Results. Scale structure of Haem-A-QoL was confirmed, with good item convergence (87%) and discrimination (80.6%) rates. Cronbach's α was >0.70 for all but one dimension (dealing) and test-retest reliability was significantly high. The strength of Spearman's correlations between Haem-A-QoL and SF-36 scales ranged from 0.25 to 0.75 (P < 0.01). Multiple stepwise linear regression analysis revealed that all but one Haem-A-QoL dimensions were important predictors of SF-36 scales. Known-groups comparisons yielded consistent support of the instruments' construct validity and significant relationships were identified for age, educational level, haemophilia type, disease severity, and viral infections. Conclusion. Overall, the psychometric properties of the Greek version of Haem-A-QoL, resulting from this first time administration of the instrument to Greek adult haemophiliacs, confirmed it as a reliable and valid questionnaire for assessing haemophilia-specific HRQoL in Greece.