Search

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or “G4” that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3′end of the HCV (−) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy’ of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization. Using surface plasmon resonance, we show that mutations within the SL2 bulge impact dimerization in vitro. Transfection of Huh7 cells with luciferase-encoding full-length genomes containing SL2 mutations abolishes viral replication. Luciferase expression indicates that viral translation is not or slightly affected and that the viral RNA is properly encapsidated. However, RT-qPCR analysis demonstrates that viral RNA synthesis is drastically decreased. In vitro synthesis experiments using the viral recombinant polymerase show that modifications of intra-molecular interactions have no effect on RNA synthesis, while impairing inter-molecular interactions decreases polymerase activity. This confirms that dimeric templates are preferentially replicated by the viral polymerase. Altogether, these results indicate that the dimerization of the HCV genomic RNA is a crucial step for the viral life cycle especially for RNA replication. RNA dimerization could explain the existence of HCV recombinants in cell culture and patients reported recently in other studies.

Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3′ end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5′ untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5′UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.

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. The strategy is based on the delivery of a non-coding (-)RNA carrying the structures required for the binding of the viral replication complex and the complementary sequence of an antiviral gene. The viral replication complex replicates the (-)RNA similarly to the viral genome to give a coding (+)RNA from which the antiviral protein will be expressed. As non-infected cells do not express the replication complex, this specific machinery can be used to target virus-infected cells without affecting healthy cells. We show that this approach can be successfully applied to the hepatitis C virus. In both replicon-harboring cells (genotype 1b) and JFH-1 infected cells (genotype 2a), nrRNAs induced a strong decrease in genomic RNA and viral protein NS5A. These effects were correlated with a strong activation of several interferon-stimulating genes.

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. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.

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. Among the selected molecules, two were able to inhibit in vitro the polymerase activity of the HCV NS5B. These aptamers appeared to be specific for HCV polymerase, as no inhibition of poliovirus 3D polymerase activity was observed. The binding and inhibitory potential of one aptamer (27v) was associated with the 35 nt-long variable region. This oligonucleotide displayed an apparent dissociation constant (K(d)) in the nanomolar range. Our results showed that it was able to compete with RNA templates corresponding to the 3'-ends of the (+) and the (-) HCV RNA for binding to the polymerase. The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.

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 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2′- O -methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3′-end of the minus-strand RNA.

The aim of this study was to examine whether hepatitis C virus (HCV) pretreatment quasispecies complexity was linked to virological response or other clinical and biological parameters, in human immunodeficiency virus (HIV)-coinfected patients undergoing anti-HCV treatment. In addition, HCV quasispecies composition is described longitudinally in these patients before, during, and after treatment. The 52 HIV-coinfected patients were included in a randomized therapeutic trial. At inclusion, they had CD4+ counts of >250/μl, HIV plasma load of

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I 33 ) that bound tightly to the enzyme. I 33 inhibited both in vitro IN activities, i.e. 3′ end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I 33 . Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an α-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.

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. These RNA templates contained different regions of the 3' end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3' UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3' end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3' end of the minus-strand RNA: (a) the presence of a C residue as the 3' terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3' end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.

In this overview, the antiviral properties of the Curie-pyridinone compounds, a new class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) developed as anti-HIV agents, are described. These compounds are hybrids between hydroxyethoxymethyl-phenylthiothymine (HEPT) and Merck pyridinones. Several structure-activity relationships (SAR) studies between HIV-1 reverse transcriptase (RT) and the Curie-pyridinones are described. The Curie-pyridinones are potent inhibitors of both HIV-1 replication in cell culture and of HIV-1 RT activity in vitro. They are specific to HIV-1 and do not inhibit the replication of HIV-2. The mechanism of inhibition is non-competitive with respect to the natural substrate dGTP. For these reasons, the Curie-pyridinones can be considered as non-nucleoside inhibitors of HIV-1 RT. Moreover, they have the unusual ability to reach the reverse transcription complex inside the extracellular virions and may therefore be useful as retrovirucides. This might lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

The untranslated 5' leader region of the human immunodeficiency virus type 1 (HIV-1) RNA plays an essential role in retroviral replication. It is the first retrotranscribed RNA region, primed from a cellular tRNALys3 partially annealed to the HIV-1 primer binding site (PBS). The structural and functional features of the HIV-1 reverse transcription initiation complex have been thoroughly studied. In this work, we used chemically modified antisense oligonucleotides (AS-ODN) as competitors of the natural tRNALys3 primer for the PBS region. Modified 2'-O-methyl AS-ODN were able to inhibit in vitro HIV-1 reverse transcription and displace the tRNALys3 previously annealed to the PBS. The destabilization of the initiation complex by 2'-O-methyl ODN was a sequence-specific process. We further demonstrated the importance of an anchor region contiguous to the PBS in the annealing of the antisense molecule, allowing the displacement of tRNALys3. The 20-mer 2'-O-methyl molecules were also able to inhibit viral replication in HIV-1-human infected cells, either by blocking cDNA synthesis during the early phase or by interfering with the annealing of the tRNALys3 primer to the PBS during the late phase of the viral cycle. Thus, the highly conserved retroviral initiation complex was shown to be a promising target when using the antisense strategy.

The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.

Pathogenic enteric viruses can be introduced into the environment as a result of human activities. Enteroviruses are regularly detected in environmental waters or shellfish and can provoke potentially serious diseases. Some authors believe that enteroviruses could represent an interesting indicator of viral contamination in the environment. Since molecular approaches seem to be promising for the detection of these viruses, we developed a simple qualitative RT-PCR procedure for enteroviruses, together with a quantitative RT-PCR assay using RNA internal standard. After one-tube-RT-PCR, this standard and wild enterovirus RNA were detected by differential hybridization with specific probes and a fluorimetric reaction. The quantification of enteroviruses, conducted in a sewage treatment plant, showed a decreasing number of genomic copies from the entrance to the exit (from 3.8 x 10(5) to 5.4 x 10(4) RNA copies/mL) but indicated the presence of enterovirus RNA in the neighboring river (2.2 x 10(3) RNA copies/mL). In bathing areas, enterovirus RNA was detected in 16 out of 226 samples, with copies numbers ranging from 3.7 x 10(2) RNA copies/mL to 7 x 10(4) RNA copies/mL.

Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-l activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC(50) values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT. Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to compound 27. Although cross resistance is frequently observed between non-nucleoside reverse transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevirapine with an IC(50) of 40 nM.

International audience; Higher eukaryotic organisms have a variety of specific and nonspecific defense mechanisms against viral invaders. In animal cells, viral replication may be limited through the decrease in translation. Some viruses, however, have evolved mechanisms that counteract the response of the host. We report that infection by HIV-1 triggers acute decrease in translation. The human protein kinase GCN2 (eIF2AK4) is activated by phosphorylation upon HIV-1 infection in the hours following infection. Thus, infection by HIV-1 constitutes a stress that leads to the activation of GCN2 with a resulting decrease in protein synthesis. We have shown that GCN2 interacts with HIV-1 integrase (IN). Transfection of IN in amino acid-starved cells, where GCN2 is activated, increases the protein synthesis level. These results point to an as yet unknown role of GCN2 as an early mediator in the cellular response to HIV-1 infection, and suggest that the virus is able to overcome the involvement of GCN2 in the cellular response by eliciting methods to maintain protein synthesis.

International audience; We evaluated the impact of cytomegalovirus (CMV)-induced immune responses, autoimmune-induced immune responses, and microbial translocation on immune activation in 191 human immunodeficiency virus type 1-infected patients from the ANRS CO3 Aquitaine Cohort. All enrolled subjects had achieved long-term virological suppression during receipt of combination antiretroviral therapy (cART). HLA-DR(+)/CD38(+) expression was 16.8% among CD8(+) T cells. Independent of age, CD4(+) T-cell count, 16S ribosomal DNA load, and regulatory T-cell count, positive results of Quantiferon CMV analysis (P = .02), positive results of CMV-pp65 enzyme-linked immunosorbent spot analysis (P = .01), positive results of CMV-pp65-specific CD8(+) T-cell analysis (P = .05), and CMV seropositivity (P = .01) were associated with a higher percentage of CD8+ T cells that expressed HLA-DR+/CD38+. Autoimmune response and microbial translocation were not associated with immune activation. Therefore, the CMV-induced immune response seems to be associated with chronic immune activation in cART recipients with sustained virological suppression.

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. The first is dinucleotide formation, which requires a closed conformation, and the second is the transition to elongation, which requires an opening of NS5B. We also recently published a combined structural and functional analysis of genotype 2 HCV-NS5B proteins (of strains JFH1 and J6) that established residue 405 as a key element in de novo RNA synthesis (P. Simister et al., J. Virol. 83:11926–11939, 2009; M. Schmitt et al., J. Virol 85:2565–2581, 2011). We hypothesized that this residue stabilizes a particularly closed conformation conducive to dinucleotide formation. Here we report similar in vitro dissections of de novo synthesis for J6 and JFH1 NS5B proteins, as well as for mutants at position 405 of several genotype 1 and 2 strains. Our results show that an isoleucine at position 405 can promote both dinucleotide formation and the transition to elongation. New structural results highlight a molecular switch of position 405 with long-range effects, resolving the implied paradox of how the same residue can successively favor both the closed conformation of the dinucleotide formation step and the opening necessary to the transition step.

Background We have previously identified the guaninerich oligonucleotide (ODN) 93del as a potent inhibitor in vitro of HIV-1 integrase. Moreover, low nanomolar concentrations of ODN 93del have been shown to inhibit HIV-1 replication in infected cells. Methods To investigate the ex vivo mechanism of ODN 93del inhibition, we analysed its antiviral effects on the early steps of HIV-1 replication such as viral entry, reverse transcription and integration using quantitative PCR. Results In addition to the effect on viral entry previously described for other guanine-quadruplex ODNs, transfection experiments showed that ODN 93del severely affects the proviral integration step independently of the effect on viral entry. Moreover, incubation of viral particles with ODN 93del revealed a potential microbicide activity of the aptamer. Conclusions Our data point to an original multimodal inhibition of HIV-1 replication by ODN 93del, strongly suggesting that targets of guanine-quartet-forming ODNs involve entry as well as other intracellular early steps of HIV-1 replication.

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. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic 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. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

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. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

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. It was shown that these RNA minigenomes could be stably replicated into Huh7 cells expressing the HCV replication complex. This was obtained either by constitutively expressing the non-structural proteins into Huh7 hepatoma cells or by using Huh7 cells harboring replicons.

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. These results were further confirmed by using bicistronic constructions. We demonstrated the presence of an mRNA initiated in this 5' UTR sequence and located the initiation site by the 5' RACE method at nucleotide 67. Furthermore, northern experiments and flow cytometry analysis showed the unambiguous activity of such a promoter sequence in stably transfected cells. Our results strongly suggest that the data obtained using bicistronic DNA constructs carrying the HCV 5' UTR should be analyzed not only at the translational but also at the transcriptional level.

Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-l activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC50 values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT. Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to compound 27. Although cross resistance is frequently observed between non-nucleoside reverse transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevira...

Reverse transcription takes place in the cytoplasm of infected cells, although it has been demonstrated that retroviruses can also initiate reverse transcription prior to infection of target cells. In addition to partial reverse transcripts, full-length proviral molecules have been detected in the plasma and seminal fluid of HIV-1 seropositive patients. Intravirion endogenous reverse transcription appears to be directly correlated with an increased level of infectivity. Therefore, the ability of an inhibitor to reach and inhibit the replication complex in the core of the free-virion may constitute an important part of its capacity to suppress viral infection. In this work we tested the ability of some reverse transcriptase inhibitors to decrease viral infectivity in pretreated highly purified virions. Our results showed that Curie pyridinone [Dolle et al. (1995), J Med Chem 38: 4 679–4 686], a non nucleoside RT inhibitor, strongly inhibited the infectivity of extracellular HIV-1 particles. Other non nucleoside inhibitors (TIBO R82913, HEPT, nevirapine) tested in these conditions were unable to do so. Our data indicate that the effect of Curie pyridinone on intact virions may be related to its capacity to tightly bind the target RT. This approach may lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

A computer programme is described for the analysis of radioimmunoassays assuming a general sigmoid shape. The mathematical treatment uses the results obtained from a sigmoid equation to perform a limited operational search in order to optimize the fitness of the curve. The programme can be installed in most desk top microcomputers and has been tested for the calculation of Ig concentrations in biological fluids.

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 K D antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.

Generation of Ig-isotype diversity was studied in hemolytic plaque-forming cells (PFC) of mice immunized with sheep erythrocytes (SRBC). Spleen cells were taken at various days after immunization, cultured in vitro for 24 hr in the presence of bacterial lipopolysaccharide (LPS), and PFC occurring in these mass cultures were micromanipulated and individually cultured for 48 hr. Daughter PFC were revealed by direct or indirect procedures in order to study the Ig-isotype they produced. Parental PFC of all three Ig isotypes studied, IgM, IgG, and IgA, generated daughter PFC, producing the same isotype but with maximum yields on different days: postimmunization day 3 for IgM PFC, day 4 for IgG PFC, and day 5 for IgA PFC. A proportion, but not all, of parental IgM PFC generated daughter IgG producers only during the first 3 days after immunization and IgA producers during the 4th to 6th post-immunization days. Parental, especially 4th day IgG PFC also generated daughter IgA producers, thus showing that IgA producers could stem from both IgM and IgG precursors in addition to IgA-producing precursors. It was also found that IgG PFC could present a reverse switching and generate IgM producers. This suggests that the overall orderly IgM, IgG, IgA conversion is possibly not strictly programmed by an internal control of B cells but submitted to some extent to microenvironmental influences.

Hemolytic plaque-forming cells (PFC) simultaneously lysing two different hapten-red cell indicators (double PFC) appear regularly along with PFC lysing only one indicator, on the 4th to 5th day of secondary response in mice primed 2 to 4 months before with hapten-protein conjugates mixed in Freund's complete adjuvant. Addition of soluble-specific inhibitors, i.e., the same haptens conjugated to different protein-carriers, results in an inhibition of the corresponding specific lytic activity and the disappearance of the double PFC. Micromanipulation of these cells from the initial medium into a second medium, containing both indicator RBC and one specific inhibitor, inhibited the corresponding specific lysis in 80% of double cells without affecting the unrelated lytic activity. Subsequent micromanipulation into a third inhibitor-free medium resulted in a recovery of the double lysis in more than 50% of them. This evidence for the synthesis of two different antibody molecules by double PFC was further strengthened by individual cell culture experiments; most of these cells divided and engendered daughter PFC-secreting antibody of only one of the two specificities, thus showing first, the transient nature of double production, and second, that each lytic activity can be inherited separately. Individual cell cultures also showed occurrence of clonal variation in progeny of single PFC. Among PFC from mice immunized with two antigens to progeny variation occurred in 24 out of 46 (0.656) parental cells as against only 1 out of 40 (0.025) observed among individual PFC from animals immunized with one antigen. Moreover, this variation was antigen driven since absence for 48 hr of the one antigen resulted in discontinuance of generation of daughter PFC of the corresponding specificity. A similar degree of antigen-driven variation was observed when spontaneous PFC or PFC arising early after immunization (2nd or 3rd day) with one antigen were individually cultured for 48 hr with a different antigen; one part of parental PFC (0.545) yielded variant daughters of the specificity of the latter antigen, whereas the other part (0.455) yielded progeny of the same as the parental PFC specificity in a clonal manner. These results suggest that differentiation of B cells during the early phase after antigenic stimulation would involve a process of intracellular selection and/or rearrangement of V genes.

We have previously described how a 16 nucleotides ODN (termed 93del) is capable of inhibiting the activity of recombinant integrase in a cell-free system as well as HIV-1 replication in human-infected cells with IC(50) in the low nanomolar range. Intracellular HIV-1 replication was inhibited when the ODN was added at the onset of infection. These results raise several questions. Is a naked ODN able to enter the cell? Does the virus play a role in ODN entry? The uptake of several ODNs (93del, 60del(sc), TBA, T30923) was evaluated and then tracked by labeling the ODN with a fluorescent dye and assessing its intracellular localization by confocal microscopy. A significant level of cellular uptake of free ODN was observed in several cell lines: HeLa epithelial cells, Huh7 hepatic cells, and H9 lymphocytes, and was detected for all ODNs tested except for TBA. Striking differences were observed when naked ODNs were added to cell in the presence or absence of the virus. When HIV-1 virions were present a sharp increase in cellular fluorescence was observed. These results strongly suggest a role for HIV-1 virions in the uptake of certain ODNs.

The hepatitis C virus genome consists of a linear positive sens (+) single-stranded RNA. RNA-RNA interactions play an essential role in the regulation of the viral cycle.RNA synthesis is performed by the RNA-dependent RNA-polymerase (RdRp) encoded by the virus. It would be initiated at the 3 'end of the molecule to be replicated. A complementary genomic RNA of negative polarity (-) is first synthesized. It then serves as a matrix for the production of genomic strands. The mechanisms that govern the recruitment of the polymerase and the initiation of RNA synthesis remain poorly understood today.The RNA structures found at the 3 'ends and their roles have therefore been studied during the work of this thesis. At the 3 'end of the (+) RNA, dimerization was shown to be essential for replication of the virus in cellulo. This work made it possible to characterize by gel shift assay and cryo-microscopy the dimerization of the genomic RNAs in solution. At the 3 'end of (-) RNA, the dimerization of two molecules could also be characterized by biochemical and biophysical approaches. Moreover, the presence of a G-quadruplex introduces a conformational reshuffle which proves to be indispensable for an efficient RNA synthesis. Similarly to the genomic strand, the resulting structural dynamics of these interactions appear to be necessary for efficient RNA replication by the RdRp.The results obtained here underline the importance of dimerization and conformational variations at the 3 'ends for RNA replication. These data then open the way to new perspectives on understanding the mechanisms that govern the functioning of HCV polymerase.; Le génome du virus de l’Hépatite C est constitué d’un ARN monocaténaire linéaire de polarité positive (+). Les interactions ARN-ARN prennent une place essentielle dans la régulation du cycle viral.La synthèse de l’ARN est réalisée par l’ARN-polymérase ARN-dépendante (RdRp) codée par le virus. Elle serait initiée à l’extrémité 3’ des molécules à répliquer. Un ARN génomique complémentaire de polarité négative (-) est d’abord synthétisé. Il sert ensuite de matrice pour la production des brins génomiques. Les mécanismes qui président au recrutement de la polymérase et à l’initiation de la synthèse d’ARN restent, aujourd’hui, mal connus.Les structures ARN présentes aux extrémités 3’ et leurs rôles ont donc étés étudiés au cours des travaux de cette thèse. Au niveau de l’extrémité 3’ de l’ARN (+), la dimérisation a été montrée indispensable à la réplication du virus in cellulo. Ces travaux ont par la suite permis de caractériser par gel retard et cryo-microscopie la dimérisation des ARN génomiques en solution. Au niveau de l’extrémité 3’ de l’ARN (-), la dimérisation de deux molécules a également pu être caractérisée par des approches biochimiques et biophysiques. Par ailleurs la présence d’un G-quadruplex introduit un remaniement conformationnel qui se révèle indispensable à une synthèse performante de l’ARN. De manière similaire au brin génomique, la dynamique structurale résultante de ces interactions semble donc nécessaire à une réplication efficace de l’ARN par la RdRp.Les résultats obtenus soulignent l’importance de la dimérisation et des variations conformationnelles prisent aux extrémités 3’ pour la réplication de l’ARN. Ces données ouvrent alors la voie vers de nouvelles perspectives quant à la compréhension des mécanismes qui président au fonctionnement de la polymérase du VHC.