Occult infection of peripheral B cells by hepatitis C variants which have low translational efficiency in cultured hepatocytes.

Background: Plasma hepatitis C virus (HCV) originates from hepatocytes. However, in certain subjects, B cells may harbour both plasma strains and occult HCV strains tha t are not detected in the plasma. The internal ribosome entry site (IRES) of these latter strains is mutated, suggesting that the efficiency of viral translation could drive the cellular tropism of HCV.

A cell-based bicistronic lentiviral reporter system for identification of inhibitors of the hepatitis C virus internal ribosome entry site.

This report describes the development, optimization and implementation of a persistent cell-based system to test inhibitors of hepatitis C (HCV) translation. The assay is based on a heterologous human immunodeficiency virus-1/simian immunodeficiency virus (HIV-1/SIV) lentiviral vector expressing the bicistronic cassette containing the firefly and renilla luciferase genes, respectively, as reporters, and the HCV internal ribosome entry site (IRES) inserted in between, under the control of the cytomegalovirus (CMV) promoter. The drug target in this assay is the HCV IRES, the activity of which leads to modulation of the renilla luciferase gene expression under its control, which is monitored by luminometry.

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors.

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation.

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5' untranslated region: correlation with reduced in vitro replication.

Computer analysis of 158 hepatitis C virus (HCV) 5' untranslated region (5' UTR) sequences from the six genotypes showed that the 5' UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5' UTR of the plus-strand RNA and the 3' end of the minus strand involved in the initiation of plus-strand RNA synthesis.

Genetic heterogeneity of the hypervariable region I of Hepatitis C virus and lymphoproliferative disorders.

B-cell lymphoproliferative disorders (BCLD) have been associated with chronic hepatitis C virus (HCV) infection. The HCV glycoprotein E2 (gpE2) hypervariable region I (HVR-I) may be a potential antigenic candidate to promote B-cell proliferation. The purpose of this study was to analyze the influence of HVR-I sequence variability in the development of BCLD.

Effect of the 5' non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells.

The effect of the 5' non-translated region (5'NTR) on hepatitis C virus (HCV) morphogenesis in insect cells is investigated in this study. Expression in baculovirus-infected cells of a sequence encoding the C and E1 structural proteins under the control of the very late promoter P10 (AcSLP10-C-E1) led to the synthesis of C and C-E1 complexes, essentially found in dense reticular material associated with the ER and sedimenting at a density of 1.24-1.

In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir.

Emergence of lamivudine-resistant hepatitis B virus (HBV) is a major concern in human immunodeficiency virus (HIV) and HBV coinfected patients. Following selection of resistant mutants, hepatitis flare or rapid progression to cirrhosis may occur. Treatment of patients with new nucleotide analogues such as adefovir dipivoxil (ADV) or tenofovir disoproxil fumarate (TDF) has shown good efficacy in controlling wild-type or lamivudine-resistant HBV replication.

Characterization of the expression of the hepatitis C virus F protein.

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9.6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products.

Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections.

Background: Dipeptidyl peptidase IV is a transmembrane enzyme widely expressed in many cell types, but also present as a soluble form in biological fluids. Its abnormal activity is sometimes associated with liver disease related pathologies.

Objectives: The aim of this study was to evaluate the clinical relevance of changes in serum DPPIV activity in hepatitis C and other viral infections.

From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity.

RNA viruses are characterized by their high rates of genetic variation. Their genetic diversity is generally studied by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification and nucleotide (nt) sequence determination. The misinterpretation of viral diversity due to copy errors introduced by the enzymes used in this two-step protocol has not yet been assessed systematically.

A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR.

The hepatitis C virus (HCV) 5' untranslated region (UTR) has been extensively studied with regard to its internal ribosomal entry site (IRES) activity. In this work we present results suggesting the existence of a strong promoter activity carried by the DNA sequence corresponding to the HCV 5' UTR. This activity was not detected when the HCV 5' UTR sequence was replaced by HCV 3' UTR or poliovirus 5' UTR sequences.

Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells.

Hepatitis C virus (HCV) is predominantly a hepatotropic virus. Nonetheless, there is mounting evidence that hematopoietic cells may support HCV replication. The HCV 5' untranslated region (5'UTR), responsible for initiation of viral translation, via an internal ribosome entry site (IRES), has been previously described to contain specific nucleotide substitutions when cultured in infected lymphoid cells.

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type.

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity.

Preferential associations of alleles of three distinct genes argue for the existence of two prototype variants of human herpesvirus 7.

We had previously described six distinct alleles of the glycoprotein B (gB) gene of human herpesvirus 7 (HHV-7). The genetic changes corresponding to these alleles did not affect gB gene transcription or translation in in vitro assays. The study of distinct HHV-7-positive human samples showed preferential associations of some gB alleles with some alleles of two other genes, distantly located on the HHV-7 genome, coding for the phosphoprotein p100 (p100) and the major capsid protein (MCP).

Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation.

Yellow fever 5' noncoding region as a potential element to improve hepatitis C virus production through modification of translational control.

The lengthy 5' noncoding region (5' NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak.

Evaluation of molecular strategies to develop a live dengue vaccine.

Background: Millions of individuals are estimated to become infected with dengue virus each year, particularly in tropical and subtropical regions. Mortality is low but infection can lead to a severe form of dengue, characterised by haemorrhage and shock. A safe and effective vaccine against dengue is still not available.

Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C.

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used.

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells.

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174-370 of the polyprotein) or with the capsid protein gene (residues 1-370).

Growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the RNA genome.

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure.

Processing of dengue type 4 and other flavivirus nonstructural proteins.

Dengue type 4 (DEN4) and other flaviviruses employ host and viral proteases for polyprotein processing. Most proteolytic cleavages in the DEN4 nonstructural protein (NS) region are mediated by the viral NS2B-NS3 protease. The N-terminal third of NS3, containing sequences homologous to serine protease active sites, is the protease domain.

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation.