Étude d'un déphasage programmé du cadre de lecture chez Escherichia coli

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Replacement of Murine Leukemia Virus Readthrough Mechanism by Human Immunodeficiency Virus Frameshift Allows Synthesis of Viral Proteins and Virus Replication

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model

A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus

Background/Aim : Hepatitis B virus resistance to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Methods : Viral DNA was extracted from sera, PCR amplified with newly designed primers, and cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. Results : We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasi-species isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled to measure variation of drugs susceptibility of different viral quasi-species by comparison of IC50/IC90s with standards. The presence of famciclovir, or lamivudine induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. Conclusion : Our phenotypic assay enables the in vitro characterization of the DNA synthesis capacity and drug susceptibility of HBV quasi-species isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains

Antiviral activity of Bay 41-4109 on hepatitis B virus in humanized Alb-uPA/SCID mice.

Current treatments for HBV chronic carriers using interferon alpha or nucleoside analogues are not effective in all patients and may induce the emergence of HBV resistant strains. Bay 41-4109, a member of the heteroaryldihydropyrimidine family, inhibits HBV replication by destabilizing capsid assembly. The aim of this study was to determine the antiviral effect of Bay 41-4109 in a mouse model with humanized liver and the spread of active HBV. Antiviral assays of Bay 41-4109 on HepG2.2.15 cells constitutively expressing HBV, displayed an IC(50) of about 202 nM with no cell toxicity. Alb-uPA/SCID mice were transplanted with human hepatocytes and infected with HBV. Ten days post-infection, the mice were treated with Bay 41-4109 for five days. During the 30 days of follow-up, the HBV load was evaluated by quantitative PCR. At the end of treatment, decreased HBV viremia of about 1 log(10) copies/ml was observed. By contrast, increased HBV viremia of about 0.5 log(10) copies/ml was measured in the control group. Five days after the end of treatment, a rebound of HBV viremia occurred in the treated group. Furthermore, 15 days after treatment discontinuation, a similar expression of the viral capsid was evidenced in liver biopsies. Our findings demonstrate that Bay 41-4109 displayed antiviral properties against HBV in humanized Alb-uPA/SCID mice and confirm the usefulness of Alb-uPA/SCID mice for the evaluation of pharmaceutical compounds. The administration of Bay 41-4109 may constitute a new strategy for the treatment of patients in escape from standard antiviral therapy

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif

Antiviral activity of Bay 41-4109 on HBV replication in HepG2.2.15 cells.

<p>Cells were treated with Bay 41-4109 (25, 50, 100, 200 and 400 nM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025096#s2" target="_blank">Methods</a> section. HBV DNA in HepG2.2.15 supernatant was quantified by real-time PCR. The data represent the results of three independent experiments, performed in duplicate.</p

Immunohistological analysis of human hepatocytes in mouse livers.

<p>Serial sections of chimeric mouse livers from treated (T529 and T490) and untreated (C371) mice were analyzed by labeling with anti-α1 anti-trypsin (αAT) or with anti-HBc (HBc) antibodies.</p

Antiviral activity of Bay 41-4109 on HBV replication in humanized Alb-uPA/SCID mice.

<p>(A) Human albumin concentrations in sera from treated (full lines) and untreated (dotted lines) animals. (B) The HBV viral load during the course of the experiment was quantified by real-time PCR in sera from treated (full lines) and untreated (dotted lines) animals. (C) Histograms represent the mean HBV load ratio at specific time points in each treated (white) and untreated (black) animal. Data are represented on semilogarithmic graphs.</p