Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405.

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5B initiates RNA synthesis by a de novo mechanism and then processively copies the whole RNA template. Dissections of de novo RNA synthesis by genotype 1 NS5B proteins previously established that there are two successive crucial steps in de novo initiation.

Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis.

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "β-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor.

Hijacking hepatitis C viral replication with a non-coding replicative RNA.

The current treatments used against RNA viruses have a limited efficacy and are often hampered by the induction of side-effects. The specific delivery of antiviral proteins in infected cells should increase their efficiency and reduce their impact on healthy cells. Here, we describe the development of a new approach which takes advantage of the viral replication machinery to specifically target the antiviral protein expression to the infected cells.

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis.

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements.

Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells.

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events.

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.

The hepatitis C virus minigenome: a new cellular model for studying viral replication.

The cellular models used usually to study hepatitis C virus replication involve coupling between translation and replication. Because this linkage makes detailed analyses difficult a new cellular model was developed where replication is rendered independent of translation. The RNA replication was studied using RNA minigenomes where the reporter gene was flanked by the two untranslated regions of HCV.

Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA.

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment.

Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro.

The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 81 nucleotide (nt)-long synthetic DNA containing 35 random nucleotides by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Thirty ligands selected for their binding affinity to the NS5B were classified into four groups on the basis of their sequence homologies.

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase.

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC(50) values lower than 1 microM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template.

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.

HCV RNA-dependent RNA polymerase replicates in vitro the 3' terminal region of the minus-strand viral RNA more efficiently than the 3' terminal region of the plus RNA.

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B.

One-step chromatographic purification procedure of a His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein overexpressed in Escherichia coli as a secreted form.

A His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein was overexpressed in E. coli as a secreted form in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several hundred milliliters of culture, a centrifugation was used to eliminate the cells.

Sequence variations in the amino- and carboxy-terminal parts of the surface envelope glycoprotein of HTLV type 1 induce specific neutralizing antibodies.

The surface envelope glycoprotein gp46 of the human T cell leukemia virus type 1 elicits a strong immune response. Its protective role against HTLV-1 infection in animal models is well established, suggesting that recombinant envelope glycoproteins or synthetic peptides could be used as an effective vaccine. However, reports have indicated that some variations in envelope sequences may induce incomplete cross-neutralization between HTLV-1 strains.

Expression, purification and biological properties of the carboxyl half part of the HTLV-I surface envelope glycoprotein.

The carboxyl half of the surface envelope protein of HTLV-I contains the major immunodominant and neutralizable domains. Using two affinity chromatography steps and a combination of high salt concentration and non-ionic detergent, we purified this part of the envelope protein from Escherichia coli. Analysis of some immmunological and biological properties of this protein indicated that it was folded in a way that preserved the correct structure of this domain of the HTLV-I envelope protein.

Competitive polymerase chain reaction to quantify tumor cells in peripheral blood of patients with T(14;18)-bearing follicular non-Hodgkin's lymphoma: an exploratory study in 8 patients.

Detection of residual disease in follicular lymphoma is hampered by the observation of t(14;18)-bearing cells in the blood of healthy adult humans. To overcome this problem, we decided to validate a quantification method of t(14;18)-bearing cells and test it in t(14;18)-bearing follicular lymphomas (FL). We designed a competitive PCR method to quantify t(14;18)-bearing cells in peripheral blood.

Evolution of BCL-2/IgH hybrid gene RNA expression during treatment of T(14;18)-bearing follicular lymphomas.

Bcl-2, the gene over-expressed in follicular lymphomas (FL), is able to block chemotherapy-induced apoptosis. Consequently, we wondered whether bcl-2/IgH expression variations during treatment of FL could predict the outcome of patients with t(14;18)-bearing FL. For this purpose, we used a reverse transcription polymerase chain reaction (RT-PCR) assay to analyse 180 serial peripheral blood samples (PBS) during 34 treatment phases in 25 patients with t(14;18)-bearing FL.

Amino acid changes at positions 173 and 187 in the human T-cell leukemia virus type 1 surface glycoprotein induce specific neutralizing antibodies.

The nucleotide sequence of human T-cell leukemia virus type 1 (HTLV-1) is highly conserved, most strains sharing at least 95% sequence identity. This sequence conservation is also found in the viral env gene, which codes for the two envelope glycoproteins that play a major role in the induction of a protective immune response against the virus. However, recent reports have indicated that some variations in env sequences may induce incomplete cross-reactivity between HTLV-1 strains.

Influence of amino acid substitutions on antigenicity of immunodominant regions of the HTLV type I envelope surface gylcoprotein: a study using monoclonal antibodies raised against relevant peptides.

By the use of sera of human T cell leukemia virus type I (HTVL-I)-infected individuals it was shown that amino acid substitutions at positions 192 (proline to serine) and 250 (serine to proline) in major immunodominant regions (175-199 and 239-261) of the surface envelope glycoprotein (gp46) of the virus may influence the humoral response. Since human sera are polyclonal in nature, one cannot readily discriminate between an immunoglobulin-specific recognition and multiple bindings of diverse antibodies. To overcome this difficulty we generated murine monoclonal antibodies to synthetic peptides mimicking all or portions of these gp46 regions.

Different HTLV-I neutralization patterns among sera of patients infected with cosmopolitan HTLV-I.

To determine if sequence variations observed in cosmopolitan HTLV-I interfered with viral recognition by neutralizing antibodies, we evaluated the neutralization potential of sera from persons infected by HTLV-I of this clade selected for amino acid changes in their eny glycoproteins. Each serum was used to neutralize three previously described HTLV-I isolates, 2060, 2072, and 1010, that possess amino acid env sequences differing at several positions, one of them being located in the immunodominant and neutralizable domain (aa 187-199). The results obtained in syncytia and/or reporter gene inhibition assays showed that the neutralization pattern of the sera clearly differed and could be classified in three categories.

Neutralizing activity and antibody reactivity toward immunogenic regions of the human T cell leukemia virus type I surface glycoprotein in sera of infected patients with different clinical states.

The induction of specific neutralizing antibodies is an important part of vaccine strategy against human T cell leukemia virus type I (HTLV-I). A recently developed reporter gene induction assay was used to detect and quantify neutralizing antibodies in sera of HTLV-I-infected patients with different clinical states: Most sera (73/89) displayed an inhibitory activity. Neutralizing antibodies were more frequently detected in sera of patients with tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) or sicca syndrome (SS) (100%) than in sera of patients with adult T cell leukemia (ATL; 50%) or of asymptomatic carriers (AS; 83%).

Identification of HTLV-I- or HTLV-II-producing cells by cocultivation with BHK-21 cells stably transfected with a LTR-lacZ gene construct.

The Syrian Hamster kidney cell line (BHK-21) was stably transfected with a plasmid vector containing the lacZ bacterial gene under the control of a HTLV-I-LTR promoter. In these cells termed pA18G-BHK-21, this lacZ construct is inducible by the tax protein produced by a tax expression vector. It was also shown that beta-galactosidase synthesis was detected within 48 h after cocultivation of pA18G-BHK-21 cells with HTLV-I (HUT-102, MT2, C91/PL, 2060) or HTLV-II (MoT strain) -producing cells.

A RadLV-induced gamma delta T cell lymphoma displaying an antitumoral cytotoxicity.

We described previously the induction by RadLV infection of a lymphoma (NS8) expressing a cytolytic activity against an MCA-induced fibrosarcoma. We report here that the cytolytic activity of these immortalized CD3+, CD8+ T cells is non-MHC-restricted. We then determined the structure and expression of the TCR chains expressed by these cells.

Characterization of monoclonal antibodies directed against the gp46 of human T-cell leukemia virus type I.

Essential HTLV-I biological functions depend on the structural motives of the surface glycoprotein (gp46). Monoclonal antibodies (mAbs) have been generated in order to identify functional regions of gp46. We obtained three monoclonal antibodies (3F3F10, 4F5F6 and 7G5D8) by immunizing Balb/c mice with beta-propiolactone inactivated HTLV-I producing cells and partially purified gp46.