APOBEC3G genetic variants and their influence on the progression to AIDS.

The cytosine deaminase APOBEC3G, in the absence of the human immunodeficiency virus type 1 (HIV-1) accessory gene HIV-1 viral infectivity factor (vif), inhibits viral replication by introducing G-->A hypermutation in the newly synthesized HIV-1 DNA negative strand. We tested the hypothesis that genetic variants of APOBEC3G may modify HIV-1 transmission and disease progression. Single nucleotide polymorphisms were identified in the promoter region (three), introns (two), and exons (two). Genotypes were determined for 3,073 study participants enrolled in six HIV-AIDS prospective cohorts. One codon-changing variant, H186R in exon 4, was polymorphic in African Americans (AA) (f = 37%) and rare in European Americans (f < 3%) or Europeans (f = 5%). For AA, the variant allele 186R was strongly associated with decline in CD4 T cells (CD4 slope on square root scale: -1.86, P = 0.009), The 186R allele was also associated with accelerated progression to AIDS-defining conditions in AA. The in vitro antiviral activity of the 186R enzyme was not inferior to that of the common H186 variant. These studies suggest that there may be a modifying role of variants of APOBEC3G on HIV-1 disease progression that warrants further investigation.

Replicating adenoviruses targeting tumours with constitutive activation of the Wnt signalling pathway

Abstract Activation of the Wnt pathway through mutation of the adenomatous polyposis coli and 13-catenin genes is a hallmark of colon cancer. These mutations lead to constitutive activation of transcription from promoters containing binding sites for Tcf/LEF transcription factors. Tumour-selective replicating oncolytic viruses are promising agents for cancer therapy. They can in principle spread throughout a tumour mass until all the cancerous cells are killed, and clinical trials have shown that they are safe except at very high doses. Adenoviruses are interesting candidates for virotherapy because their biology is well understood and their small genome can be rapidly mutated. Adenoviruses with Tcf binding sites in the E2 early promoter replicate selectively in cells with an active Wnt pathway. Although these viruses can replicate in a broad panel of colon cancer cell lines, some colorectal cancer cells are only semi-permissive for Tcf-virus replication. The aim of my thesis was to increase the safety and the efficacy of Tcf-viruses for colon cancer virotherapy. I replaced the endogenous ElA viral promoter by four Tcf binding sites and showed that transcription from the mutant promoter was specifically activated by the Wnt pathway. A virus with Tcf binding sites in the ElA and E4 promoters was more selective for the Wnt pathway than the former Tcf-E2 viruses. Moreover, insertion of Tcf binding sites into all early promoters further increased viral selectivity, but reduced viral activity. I showed that Tcf-dependent transcription was inhibited by the interaction between ElA and p300, but deletion of the p300-binding site of ElA generally led to viral attenuation. In the semi-permissive cell lines, replication of Tcf-viruses remained lower than that of the wild-type virus. The E2 promoter was the most sensitive to the cell type, but I was unable to improve its activity by targeted mutagenesis. To increase the toxicity of the Tcf-E1A/E4 virus, I decided to express a suicide gene, yeast cytosine deaminase (yCD), late during infection. This enzyme converts the prodrug 5-FC to the cytotoxic agent 5-FU. yCD was expressed in a DNA replication-dependent manner and increased viral toxicity in presence of 5-FC. Tcf-ElA and yCD adenoviruses are potentially useful vectors for the treatment of liver metastases from colorectal tumours. Résumé Dans la quasi-totalité des cancers du côlon, la voie Wnt est activée par des mutations dans les gènes codant pour APC ou pour la (3-caténine. Ces mutations activent de façon constitutive la transcription de promoteurs contenant des sites de liaison pour les facteurs de transcription Tcf/LEF. Les virus réplicatifs spécifiques aux tumeurs sont des agents prometteurs pour la thérapie cancéreuse. En principe, ces vecteurs peuvent se propager dans une masse tumorale jusqu'à destruction de toutes les cellules cancéreuses, et des études cliniques ont démontré que de tels vecteurs n'étaient pas toxiques, sauf à de très hautes doses. Les adénovirus sont des candidats intéressants pour la thérapie virale car leur biologie est bien définie et leur petit génome peut être rapidement modifié. Des adénovirus comportant des sites de liaison à Tcf dans leur promoteur précoce E2 se répliquent sélectivement dans les cellules qui possèdent une voie Wnt active. Ces virus sont capables de se répliquer dans un grand nombre de cellules cancéreuses du côlon, bien que certaines de ces cellules ne soient que semi-permissives pour la réplication des virus Tcf. Le but de ma thèse était d'augmenter la sécurité et l'efficacité des virus Tcf. Le promoteur viral endogène ElA a été remplacé par quatre sites de liaison à Tcf, ce qui a rendu son activation dépendante de la voie Wnt. Un virus comportant des sites de liaison pour Tcf dans les promoteurs ElA et E4 était plus sélectif pour la voie Wnt que les précédents virus Tcf-E2, et un virus comportant des sites Tcf dans tous les promoteurs précoces était encore plus sélectif, mais moins actif. J'ai montré que l'interaction entre ElA et p300 inhibait la transcription dépendante de Tcf, mais la délétion du domaine concerné dans ElA a eu pour effet d'atténuer les virus. Dans les cellules semi-permissives, la réplication des virus Tcf était toujours plus basse que celle du virus sauvage. J'ai identifié le promoteur E2 comme étant le plus sensible au type cellulaire, mais n'ai pas pu augmenter son activité par mutagenèse. Pour augmenter la toxicité du virus Tcf-E1A/E4, j'ai décidé d'exprimer un gène suicide, la cytosine déaminase (yCD), pendant la phase tardive de l'infection. Cette enzyme transforme la procirogue 5-FC en l'agent cytotoxique 5-FU. yCD était exprimée après réplication de l'ADN viral et augmentait la toxicité virale en présence de 5-FC. Les virus Tcf-ElA et yCD sont des vecteurs potentiellement utiles pour le traitement des métastases hépatiques de cancers colorectaux.

Basal ganglia-cortical structural connectivity in Huntington's disease.

Huntington's disease is an incurable neurodegenerative disease caused by inheritance of an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat within the Huntingtin gene. Extensive volume loss and altered diffusion metrics in the basal ganglia, cortex and white matter are seen when patients with Huntington's disease (HD) undergo structural imaging, suggesting that changes in basal ganglia-cortical structural connectivity occur. The aims of this study were to characterise altered patterns of basal ganglia-cortical structural connectivity with high anatomical precision in premanifest and early manifest HD, and to identify associations between structural connectivity and genetic or clinical markers of HD. 3-Tesla diffusion tensor magnetic resonance images were acquired from 14 early manifest HD subjects, 17 premanifest HD subjects and 18 controls. Voxel-based analyses of probabilistic tractography were used to quantify basal ganglia-cortical structural connections. Canonical variate analysis was used to demonstrate disease-associated patterns of altered connectivity and to test for associations between connectivity and genetic and clinical markers of HD; this is the first study in which such analyses have been used. Widespread changes were seen in basal ganglia-cortical structural connectivity in early manifest HD subjects; this has relevance for development of therapies targeting the striatum. Premanifest HD subjects had a pattern of connectivity more similar to that of controls, suggesting progressive change in connections over time. Associations between structural connectivity patterns and motor and cognitive markers of disease severity were present in early manifest subjects. Our data suggest the clinical phenotype in manifest HD may be at least partly a result of altered connectivity. Hum Brain Mapp 36:1728-1740, 2015. © 2015 Wiley Periodicals, Inc.

Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.