Le Virus de l'Hépatite B (VHB) infecte 350 millions d'individus à l'échelle mondiale. Il est responsable d'hépatites aigües pouvant évoluer vers la chronicité puis le carcinome hépatocellulaire. Le génome du VHB est constitué d'un ADN partiellement bicaténaire. De par sa nature, il pourrait être sensible à l'action de certaines nucléases cellulaires qui hydrolysent l'ADN double brin. Nous avons ainsi mis en évidence la capacité de la Désoxyribonuclase I (DNase I) à être incorporée dans les virions du VHB, ce qui permet la dégradation du génome viral et la diminution de son infectivité. La DNAse I est particulièrement surexprimée en hypoxie et pourrait contribuer à l'élimination du virus chez les individus cirrhotiques. Par ailleurs, nous avons montré que la cytidine désaminase APOBEC3DE appartenant à une famille de facteurs de restriction viraux possède un rôle proviral. En effet, son association avec APOBEC3F et APOBEC3G mène à une diminution de l'activité de ces dernières et ceci favorise la réplication du VHB. La formation d'hétérodimères APOBEC3DE/APOBEC3F et APOBEC3DE/APOBEC3G semble génèrer un encombrement stérique ne permettant pas l'encaspidation d'APOBEC3F et APOBEC3G, raison pour laquelle le génome du VHB est moins muté lorsqu'APOBEC3DE est exprimée. / Hepatitis B Virus (HBV) infects 350 millions people worldwilde. It triggers accute hepatitis that can turn into cirrhosis then hepatocellular carcinoma. HBV genome is composed of a partially double-stranded DNA.Thus, it could be targeted by some cellular nucleases that hydrolyze double-stranded DNA. We have highlighted that Deoxyribunuclease I (DNase I) can be incorporated into HBV virions and degrade its genome, leading to a loss of viral infectivity. Moreover, DNase I is upregulated under hypoxia which is a caracteristic of liver cirrhosis. DNase I could be involved in HBV elimination in cirrhotic patients. In an other study, we found that APOBECDE, a cytidine deaminase of the same family than some restriction factors, has a proviral activity. Indeed, association of APOBEC3DE with APOBEC3F or APOBEC3G leads to a loss of cytidine deaminase activity and a better viral replication. When APOBEC3DE is associated with those two proteins, APOBEC3F and APOBEC3G cannot be incorporated into HBV virions. This is the reason why HBV is more infectious when APOBEC3DE is expressed.

Orientadores: Fernando Lopes Gonçales Junior, Neiva Sellan Lopes Gonçales / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T15:36:18Z (GMT). No. of bitstreams: 1 Jardim_RuthNogueiraCordeirodeMoraes_M.pdf: 4267075 bytes, checksum: e1769c6758a1c8847d53c93bc31bd35a (MD5) Previous issue date: 2006 / Resumo: A infecção oculta pelo VHB é caracterizada pela presença do DNA-VHB em indivíduos com o antígeno de superfície (HBsAg) indetectável. A prevalência e significado clínico desta infecção ainda não são totalmente conhecidos. Este trabalho teve por objetivo determinar a prevalência de infecção oculta pelo VHB em dois grupos de pacientes imunossuprimidos (pacientes em tratamento por hemodiálise e pacientes HIV positivos) com anti-HBc positivo co-infectados ou não pelo vírus da hepatite C (VHC). Foi investigada uma possível correlação entre prevalência do DNA-VHB e carga viral, níveis de CD4, fatores de risco e administração de lamivudina no grupo de HIV positivos. O primeiro grupo (G1) foi formado por 34 pacientes hemodialisados que eram HBsAg negativo/anti-HIV negativo. O segundo grupo (G2) formado por 159 pacientes infectados pelo vírus da imunodeficiência humana (anti-HIV positivo) e HBsAg negativo. Foi utilizado como grupo controle (G3), 150 doadores de sangue com marcadores sorológicos negativos para o HBsAg e para o HIV, mas reagentes para o anti-HBc. A pesquisa do DNA do VHB foi realizada pela PCR ?in house? , segundo a técnica de Kaneko et al. (1989), utilizando-se ?primers? específicos da região do core do VHB (limite de detecção 100 cópias/ml). Entre os pacientes hemodialisados, a infecção oculta não esteve presente, talvez devido ao controle da infecção pelo VHB nestes centros, pela adoção das medidas de precauções universais e imunização específica por vacinação contra o VHB que possivelmente, foram eficazes para evitar a presença de infecção oculta pelo VHB. A prevalência de positividade do VHB-DNA entre os pacientes HIV positivos foi de 5,03% (8/159). Este dado mostrou que não houve diferença significativa entre o grupo HIV positivo e o grupo controle (5,03% x 4%). De acordo com estes resultados, provavelmente, a imunossupressão nestes pacientes HIV positivos não foi fator determinante na prevalência de infecção oculta. O DNA-VHB foi observado independente da presença do anti-HBs nos pacientes HIV positivos coinfectados ou não pelo VHC. Esta observação sugere que o anti-HBs não foi capaz de proteger, na totalidade, os pacientes. A prevalência de infecção oculta pelo vírus da hepatite B não foi diferente entre os grupos estudados, HIV positivos e doadores de sangue. Não foi observada associação entre contagem de CD4, carga viral, fator de risco e nem à utilização da lamivudina como parte do tratamento anti-retroviral e ocorrência de hepatite B oculta / Abstract: Occult hepatitis B virus infection is characterized by presence of HBV-DNA in individuals with undetectable hepatitis B surface antigen (HBsAg). The prevalence and clinical significance of this infection remain incompletely defined. The aim of this study was to determinate the prevalence of occult HBV infection between two immunosuppressed populations (maintenance haemodialysis patients and HIV positive patients) with anti-HBc positive, co-infected or not with hepatitis C virus (HCV). Possible correlations were investigated between prevalence of HBV-DNA, viral load, CD4 levels, risk factors and lamivudina administration in the HIV group. The first group (G1) was formed by 34 hemodialysed patients that were HBsAg negative/anti-HIV negative. The second group (G2) formed by 159 human immunodeficiency virus infected patients (anti- HIV positive) and HBsAg negative. Used as a control group (G3), 150 blood donors with serologic markers negative to HBsAg and HIV, but positive for anti-HBc. HBV-DNA testing was performed using ?in house? nested PCR as described by Kaneko et al. and was detected using specific primers derivated from core regions of HBV genome (detection limit 100 copies / ml). Occult hepatitis B virus infection was absent in hemodialysis patients may be due the control of HBV infection in these centers, by adoption universal precautions measures and specific immunization by hepatitis B vaccination perhaps were effective to avoid occult HBV infection. The prevalence of HBV-DNA in HIV positive patients was 5,03% (8/159). This data showed that there was not significant difference between group HIV positive and control (5,03% x 4%). According these results probably the Immunosuppression in this HIV positive patients were not a determinant factor in prevalence of occult infection. The HBV-DNA was observed independent the presence of anti-HBs in HIV positive patients co-infected or not by HCV. This observation suggest that anti-HBs was not able to protect in the totality this patents. The prevalence of occult hepatitis B virus infection was not different among the group studied HIV positive patients and blood donors. It was not observed association between CD4 cell count, viral load, risk factor or treatment antiretroviral with lamivudine and occurrence of occult hepatitis B / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

O presente estudo realizou o sequenciamento de nova geração do vírus da hepatite C genótipo 1, incluindo os subtipos 1a (n=51) e 1b (n=49), e identificou variantes associadas com resistência (RAV) aos antivirais de ação direta em pacientes sem tratamento prévio. No subtipo 1a, foram encontradas RAV para as regiões NS3-4A, NS5A e NS5B em 10%, 22% e 8% dos pacientes, respectivamente. RAV detectadas foram: T54S (2%), V55A (2%), Q80K (4%) e R155K (2%) na protease NS3-4A; Q30H (4%), H58P (10%) e Q30H/R+Y93C/H/N (8%) na região NS5A; e A421V (8%) na polimerase NS5B. As frequências das RAV para o subtipo 1b foram 12%, 53% e 31% para as regiões NS3-4A, NS5A e NS5B, respectivamente. Foram encontradas as RAV F43I (2%), T54S (4%), Q80H (2%), D168E (2%) e M175L (2%) na região NS3-4A; L28M (2%), R30Q (2%), L31M (2%), Q54H (27%), A92T (2%), Y93H (4%), Q54H+A92T (6%), Q54H+Y93H (6%) e A92T+Y93H (2%) na região NS5A e, L159F (2%), C316N (4%), A421V (7%), L159F+C316N (9%) e S556G (9%) na polimerase. Utilizando esta metodologia, um recombinante inter-subtipo 1a/1b foi identificado. / This study performed the next-generation sequencing of the hepatitis C virus genotype 1, including subtypes 1a (n = 51) and 1b (n = 49), and identified resistance-associated variants (RAVs) to direct-acting antivirals in previously untreated patients. In subtype 1a, RAVs were found for NS3-4A, NS5A, and NS5B regions in 10%, 22% and 8% of patients, respectively. RAVs detected were: T54S (2%), V55A (2%), Q80K (4%) and R155K (2%) in NS3-4A protease; Q30H (4%), H58P (10%) and Q30H/R+Y93C/H/N (8%) in NS5A region; and A421V (8%) in NS5B polymerase. Frequencies of RAV for subtype 1b were 12%, 53% and 31% for NS3-4A, NS5A and NS5B regions, respectively. RAVs F43I (2%), T54S (4%), Q80H (2%), D168E (2%) and M175L (2%) were found in NS3-4a region; L28M (2%), R30Q (2%), L31M (2%), Q54H (27%), A92T (2%), Y93H (4%), Q54H+A92T (6%), Q54H+Y93H (6%) and A92T+Y93H (2%) in NS5A region and, L159F (2%), C316N (4%), A421V (7%), L159F+C316N (9%) and S556G (9%) in polymerase. By using this methodology, a recombinant inter-subtype 1a/1b was identified.

A Síndrome de Gilbert é caracterizada por uma hiperbilirrubinemia indireta benigna que ocorre na ausência de hemólise ou doença estrutural do fígado. Manifesta-se por episódios intermitentes de icterícia, desencadeados por exposição a estressores físicos, baixa ingesta calórica, entre outros. A base genética da redução da atividade da enzima UDP - Glucoroniltransferase foi descoberta em 1995: em uma população caucasiana. Todos os pacientes estudados apresentaram uma adição dos nucleotídeos Timina-Adenina (TA) na região TATA box presente no promotor do gene UGT1A1, em ambos os alelos. Embora considerada uma condição benigna, a síndrome de Gilbert tem sido recentemente associada à hiperbilirrubinemia e a outros efeitos colaterais na utilização de algumas drogas como o Indinavir e Irinotecan. Outro ponto importante diz respeito ao nível de bilirrubina sérica como um indicador da severidade do acometimento de hepatopatas. A presença de mutação no gene UGT1A1 em pacientes hepatopatas pode levar ao aumento da bilirrubina sérica, supervalorizando o acometimento hepático da condição patológica. O objetivo deste estudo foi verificar a frequência do alelo UGT1A1*28 em doadores de sangue da Fundação Pró-sangue Hemocentro de São Paulo HC-FMUSP e em pacientes portadores de hepatite crônica C atendidos no ambulatório de Gastroenterologia Clínica da FMUSP. Relacionar o genótipo TA7/7 com o aumento de bilirrubina sérica nos pacientes com hepatite crônica C e avaliar a técnica de análise de fragmento no rastreamento e genotipagem da Síndrome de Gilbert. A frequência encontrada para o genótipo TA7/7 no grupo doador foi de 9% (30/313) e no grupo de pacientes VHC, de 10% (51/494). O genótipo TA7/7 parece estar relacionado com o aumento de bilirrubina. A técnica de análise de fragmentos mostrou-se rápida, sendo possível para fazer uma análise em grande escala. A herança genética da população brasileira é muito heterogênea. É constituída de caucasianos, africanos, indios, orientais e outros. Os dados sugerem que a variação genética da região promotora do gene UGT1A1 é alta entre pacientes com bilirrubina maior que 1mg/dL, e que a genotipagem para UGT1A1*28 deve ser considerada na avaliação dos pacientes com hepatite C crônica com hiperbilirrubinemia. / Gilberts syndrome is a benign condition characterized by unconjugated hiperbilurubinemia that occurs in the absence of hemolysis or liver chronic disease. It is clinically manifested by intermittently jaundice, triggered by exposition to physical stress, low calory diet, among others. The genetic base is the reduction of the activity of UDP-glucuronosyltransferase enzyme described in 1995: in a Caucasian population, all patients studied presented a Thymine Adenine (TA) addition in the TATA box region in both alleles of the UGT1A1 gene promoter. Although, Gilberts syndrome has been considered a benign condition, recently it has been associated to hiperbilirrubinemia and other adverse events during the utilization of some drugs such as Indinavir and Irinotecan. Another important issue to consider is that bilirubin is used to evaluate the severity of liver dysfunction in chronic liver diseases. The presence of this mutation in those patients could increase bilirubin levels, overestimating liver damage. The aim of this study were: 1) to verify the frequency of the genotype UGT1A1*28 (TA7/7) in blood donors and in chronic hepatitis C patients from the Gastroenterology outpatients clinics of the University of São Paulo School of Medicine; 2) to establish a relationship with TA7/7 genotype and bilirubin elevation in chronic hepatitis C patients and 3) to evaluate the fragment analysis technique to screening and genotyping the Gilbert syndrome. The frequencies of TA7/7 genotype found in blood donors group were 9.6% (30/313) and in the chronic hepatitis C group were 10% (51/494). The TA7/7 genotype seems to be related with increase of bilirubin. The fragment analysis technique is fast and able to a large scale screening approach. The genetic background of Brazilian population is highly heterogeneous. It is comprised of Caucasians, Africans, Indians, Orientals and others. The data suggests that genetic variation of promoter region of UGT1A1 gene is high among patients with bilirubin levels greater than 1 mg/dl, and UGT1A1*28 genotypes should be considered when evaluating chronic hepatitis C patients with hiperbilirubinemia.

Lors d’une infection chronique, le virus de l'hépatite C (HCV) circule sous forme de lipoviroparticule (LVP) : particules hybrides associant des composants viraux (ARN, les protéines structurelles) et des composants cellulaires (apolipoprotéines, cholestérol). Au cours de ma thèse, nous nous sommes intéressés à identifier la plateforme d'assemblage du HCV, et le rôle du rassemblement des protéines virales par NS2 dans sa formation. Nous avons montré que des interactions de natures différentes sur la membrane du RE sont impliquées dans cette association protéique. Nos résultats suggèrent que des interactions complexes (directes ou via des « membranes résistantes aux détergents » (DRM)) entre les protéines complexées par NS2, peuvent immédiatement précéder la formation LVP. Nous avons également démontré que l’hétérodimère E1E2, les apolipoprotéines B et E (ApoB, ApoE) s’associent en un complexe de protéines dans le réticulum endoplasmique (RE) lorsqu'elles sont exprimées ensembles. Ce complexe se forme au début de l'assemblage du HCV, quelle que soit l'expression des autres protéines virales, et est conservée sur les LVP sécrétées. Basé sur ces données, nous avons proposé un mécanisme expliquant l’initiation de la morphogenèse des LVP. Ensuite, nous avons évalué l'importance de l'association E1E2/ApoE pour le cycle de vie du virus. Nous avons initié une étude pour identifier les acides aminés E1E2 impliqués dans l'interaction avec les apolipoprotéines. Avec ces données, nous souhaitons proposer une meilleure compréhension des mécanismes de la morphogenèse du HCV. / In chronic infection, the hepatitis C virus (HCV) circulates as lipoviral particles (LVP): hybrid particles associating viral (RNA, structural proteins) and cellular components (apolipoproteins, cholesterol). During my PhD, we were interested in identifying the HCV assembly platform, and the role of the association of the viral proteins by NS2 during its formation. We showed that different natures of interactions on the ER membrane are involved in this proteic association. Our results suggest that a complex interplay between proteins of the complex formed by NS2, directly or through “detergent resistant membranes” (DRMs) may be immediately followed by LVPs formation. We also demonstrated that E1E2 heterodimer, apolipoproteins B and E (ApoB, ApoE) associate as a protein complex in the endoplasmic reticulum (ER) when expressed together. This complex is formed early in HCV assembly, regardless the expression of other viral proteins, and is conserved on the secreted LVPs. Based on these data, we proposed a mechanism explaining LVP morphogenesis initiation. Then we assessed the importance of E1E2/ApoE association for viral life cycle. We initiate a study to identify the E1E2 amino acids involved in the interaction with apolipoproteins. With these data, we wished to provide a better understanding of the mechanisms of the HCV morphogenesis.

Philosophiae Doctor - PhD / To ameliorate Hepatitis C Virus (HCV) therapeutic and diagnostic challenges requires robust intervention strategies, including approaches that leverage the plethora of rich data published in biomedical literature to gain greater understanding of HCV pathobiological mechanisms. The multitudes of metadata originating from HCV clinical trials as well as low and high-throughput experiments embedded in text corpora can be mined as data sources for the implementation of HCV-specific resources. HCV-customized resources may support the generation of worthy and testable hypothesis and reveal potential research clues to augment the pursuit of efficient diagnostic biomarkers and therapeutic targets. This research thesis report the development of two freely available HCV-specific web-based resources: (i) Dragon Exploratory System on Hepatitis C Virus (DESHCV) accessible via http://apps.sanbi.ac.za/DESHCV/ or http://cbrc.kaust.edu.sa/deshcv/ and(ii) Hepatitis C Virus Protein Interaction Database (HCVpro) accessible via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/.DESHCV is a text mining system implemented using named concept recognition and cooccurrence based approaches to computationally analyze about 32, 000 HCV related abstracts obtained from PubMed. As part of DESHCV development, the pre-constructed dictionaries of the Dragon Exploratory System (DES) were enriched with HCV biomedical concepts, including HCV proteins, name variants and symbols to enable HCV knowledge specific exploration. The DESHCV query inputs consist of user-defined keywords, phrases and concepts. DESHCV is therefore an information extraction tool that enables users to computationally generate association between concepts and support the prediction of potential hypothesis with diagnostic and therapeutic relevance.Additionally, users can retrieve a list of abstracts containing tagged concepts that can be used to overcome the herculean task of manual biocuration. DESHCV has been used to simulate previously reported thalidomide-chronic hepatitis C hypothesis and also to model a potentially novel thalidomide-amantadine hypothesis.HCVpro is a relational knowledgebase dedicated to housing experimentally detected HCV-HCV and HCV-human protein interaction information obtained from other databases and curated from biomedical journal articles. Additionally, the database contains consolidated biological information consisting of hepatocellular carcinoma(HCC) related genes, comprehensive reviews on HCV biology and drug development,functional genomics and molecular biology data, and cross-referenced links to canonical pathways and other essential biomedical databases. Users can retrieve enriched information including interaction metadata from HCVpro by using protein identifiers,gene chromosomal locations, experiment types used in detecting the interactions, PubMed IDs of journal articles reporting the interactions, annotated protein interaction IDs from external databases, and via “string searches”. The utility of HCVpro has been demonstrated by harnessing integrated data to suggest putative baseline clues that seem to support current diagnostic exploratory efforts directed towards vimentin. Furthermore,eight genes comprising of ACLY, AZGP1, DDX3X, FGG, H19, SIAH1, SERPING1 and THBS1 have been recommended for possible investigation to evaluate their diagnostic potential. The data archived in HCVpro can be utilized to support protein-protein interaction network-based candidate HCC gene prioritization for possible validation by experimental biologists.

Le virus de l'hépatite Delta (HDV) est un agent infectieux transmissible satellite du virus de l'hépatite B (HBV), induisant des maladies du foie plus sévères que la mono–infection par le HBV. Aucun traitement totalement efficace n'est disponible contre l'HDV et les 15 millions de personnes infectées par le HDV dans le monde sont exposées a un risque élevé de cirrhose et de carcinome hépatocellulaire. HDV est un virus unique qui ne code pas pour une polymérase virale contrairement aux autres virus a ARN. La réplication de l'ARN HDV s'effectue par un double mécanisme de cercle roulant générant des brins d'ARN de longueur génomique ou antigénomiques unitaires. La synthèse de l'ARN génomique est sensible à de faibles concentrations d'alpha–amanitine, ce qui suggère qu'elle soit médiée par l'ARN polymérase II (ARN Pol II) classiquement ADN dépendante. Ce processus repose sur la petite protéine du HDV (S–HDAg), qui doit être acétylée sur l'acide amine K72 pour activer la synthèse de l'ARN génomique. Nous avons récemment identifié la protéine BAZ2B (Bromodomain Associated Zinc finger protein 2B) comme un interactant majeur de S–HDAg par capture par affinité, couplée à la spectrométrie de masse à partir de l'expression de S– HDAg étiqueté par un double motif Strep–TagR dans les cellules HepaRG différentiées. La fonction biologique de BAZ2B est inconnue. Cependant, en comparant avec des protéines apparentées BAZ (BAZ–1A/1B/2A), on postule que BAZ2B représente la sous–unité accessoire d'un nouveau complexe de remodelage de chromatine de type ISWI, qui régule le positionnement des nucléosomes par hydrolyse de l'ATP. Des études récentes ont révélé que le bromodomaine de BAZ2B (BRD) reconnait la signature épigénétique spécifique K14ac–X–X–R sur l'histone H3. Cela pourrait impliquer le mode d'action du complexe de remodelage de la chromatine dont BAZ2B représente l'unité régulatrice reconnaissant des marques spécifiques d'acétylation des histones propagées séquentiellement modifiant la dynamique de la chromatine et favorisant le recrutement de l'ARN Pol II pour activer la transcription. Nous émettons l'hypothèse que l'acétylation, médiée par p300, du motif K72–X–X–R conserve dans les S–HDAg interagissant avec l'ARN antigénomique pseudo double brin, mimerait l'acétylation des histone H3 en K14 permettant de recruter le complexe de remodelage de la chromatine BAZ2B associée et de lancer la réplication HDV. Brièvement, pour confirmer la pertinence fonctionnelle du recrutement BAZ2B pour la réplication HDV, nous avons transfecté des lignées cellulaire Huh–7 exprimant de façon stable, soit la protéine sauvage S–HDAg ou le mutant R75A pour étudier la réplication HDV à partir plasmide pSVLD2m défectif pour l'expression de S–HDAg. Nos résultats indiquent que la synthèse de l'ARN génomique est fortement réduite dans les cellules exprimant le mutant R75A S–HDAg par rapport aux cellules exprimant le type sauvage S–HDAg, alors que la quantité d'ARN antigénomique est restée le même dans les deux cas. Des expériences de co–cristallisation et de siRNA sont actuellement menées afin de mieux caractériser au niveau moléculaire l'association entre BAZ2B BRD et des peptides dérivés de la séquence de S–HDAg et d'étudier les conséquences de l'inhibition par siRNA de BAZ2B. L'implication des BAZ2B dans la réplication de HDV pourra ouvrir des possibilités de développement de médicaments anti–HDV, basées sur l'optimisation des inhibiteurs émergents de BAZ2B–BRD / Hepatitis Delta Virus (HDV) is a satellite of Hepatitis B Virus (HBV), leading to more severe life threatening liver diseases than HBV mono–infection. No efficient therapy is available against HDV and the estimated 15 million HDV infected individuals worldwide are at a high risk of cirrhosis and hepatocellular carcinoma. HDV is a unique RNA virus as it does not encode a viral polymerase. HDV RNA replication occurs via a double rolling circle mechanism generating unit–length genomic or antigenomic RNA strands. The synthesis of the genomic RNA is sensitive to low concentrations of α–amanitin, suggesting that the RNA–dependent RNA synthesis is mediated by DNA–dependent RNA polymerase II (RNA Pol II). This process relies on the HDV encoded Small Hepatitis Delta antigen (S–HDAg), which must be acetylated at K72 to activate the synthesis of the genomic RNA. We recently identified BAZ2B (Bromodomain Associated to Zinc finger protein 2B) as a major interactant of S–HDAg by affinity capture coupled to mass spectrometry in differentiated HepaRG cells. The biological function of BAZ2B is however unknown. In comparison with related BAZ proteins (BAZ–1A/1B/2A), it is postulated that BAZ2B is the accessory subunit of a new chromatin remodeling complex of ISWI–type, which regulates nucleosome positioning through ATP hydrolysis. Recent studies revealed that the BAZ2B bromodomain (BRD) recognizes the distinct epigenetic signature K14ac–X–X–R on histone H3. This suggests that the mode of action of BAZ2B associated chromatin remodeling complex involves recognizing propagated specific histone acetylation marks to subsequently alter the chromatin dynamic and recruit the RNA Pol II for transcriptional activation. We hypothesized that the p300–mediated acetylation of the conserved K72–X–X–R motif in S–HDAg mimics acetylated histones on the pseudo–double stranded antigenomic RNA, to recruit the BAZ2B associated chromatin remodeling complex to initiate RNA Pol II mediated synthesis of HDV genome. To confirm the functional relevance of BAZ2B recruitment for HDV replication, we transfected Huh 7 cells stably expressing either wild–type S–HDAg or R75A mutant S–HDAg with the HDV replication defective plasmid pSVLD2m. Our results indicate that the synthesis of genomic RNA was greatly reduced in cells expressing the R75A mutant S–HDAg in comparison to cells expressing wild–type S–HDAg, whereas the amount of antigenomic RNA remained the same in both cases. Co–crystallization experiments are currently being carried out to better characterize at the molecular level the association between BAZ2B BRD and S–HDAg derived peptides. Furthermore, siRNA experiments directed against the BAZ2B gene are expected to reveal the consequences of BAZ2B inhibition on HDV viral replication. The involvement of BAZ2B in HDV replication may open anti–HDV drug development opportunities, based on the optimization of emerging BAZ2B–BRD inhibitors

L'hépatite C chronique est une des étiologies principales du carcinome hépatocellulaire. En revanche, les mécanismes de tumorigenèse sont peu connus. Récemment, plusieurs modifications du métabolisme du glucose ont été décrites dans les cellules infectées par le HCV. Celles-ci évoquent les reprogrammations métaboliques mises en place dans les cellules cancéreuses. L'effet Warburg, ou glycolyse aérobie, est une des caractéristiques principales des cellules tumorales. Ce phénomène permet d'assurer une production énergétique ainsi qu'un stock en précurseurs de macromolécules suffisants pour permettre la prolifération. Par ailleurs, en complément de l'utilisation de glucose, les cellules tumorales deviennent dépendantes de la métabolisation de la glutamine pour alimenter leur métabolisme énergétique et les différentes voies de biosynthèse. Mes travaux de thèse ont porté sur l'étude des changements métaboliques caractéristiques des cellules cancéreuses, et plus précisément sur le métabolisme de la glutamine, dans les cellules infectées par le HCV. Dans le modèle de culture cellulaire HCVcc, une activation de l'utilisation de la glutamine par le virus a pu être mise en évidence. L'infection par HCV entraine une augmentation du facteur de transcription MYC, de plusieurs transporteurs de glutamine ainsi que de la glutaminase, l'enzyme limitante de la glutaminolyse. De façon intéressante, ces changements semblent survenir également chez les personnes chroniquement infectés par le virus, comme le suggère l'analyse des biopsies de patients. Ces altérations métaboliques pourraient participer à la mise en place d'un environnement favorable au développement tumoral / Chronic infection with hepatitis C virus (HCV) is one of the main etiologies of hepatocellular carcinoma (HCC). However, mechanisms of HCV-related tumorigenesis are ill-defined. Recent literature data suggest that HCV infection may reprogram glucose metabolism in a cancerlike fashion. The Warburg effect, or aerobic glycolysis, is a hallmark of cancer. Activation of this pathway allows tumor cells to sustain high rates of energy production and provide sufficient biosynthetic precursors for proliferation. Likewise, the induction of similar metabolic alterations may favor HCV multiplication through the rapid production of nucleotides, amino acids and lipids. To complement aerobic glycolysis, tumor cells become frequently dependent on glutamine. The partial oxidation of glutamine through the glutaminolytic pathway can fuel their energy metabolism and several anabolic pathways. However, the role of glutamine metabolism in HCV life cycle has not been documented so far. I focused my PhD research project on the characterization of metabolic alterations triggered by HCV. In particular, I evaluated the occurrence of distinctive features of tumor cell metabolism in HCVinfected cells, with a specific attention on glutamine utilization. In the HCVcc cell culture model, I report the induction of a metabolic reprogramming towards higher rates of glutaminolysis upon HCV infection. HCV-induced transcriptional activation of MYC, along with several glutamine transporters and glutaminase, is likely to be responsible for this metabolic shift. Interestingly, increases in transcript levels of these factors in liver biopsies of patients with chronic hepatitis C suggest that this metabolic reprogramming may be relevant in vivo. Moreover, these metabolic changes may expose new drug targets against HCV as suggested by the inhibition of the virus replication upon suppression of glutaminolysis via different strategies. Altogether, these findings uncover a potential link between chronic hepatitis C and HCC through the installation of a favorable metabolic environment for tumor development

Le but de notre travail était d'étudier comment des interactions impliquant des acides nucléiques pouvaient réguler le cycle viral du virus de l'hépatite C (VHC). L'ARN génomique du VHC est très structuré au niveau de ses extrémités 5' et 3'. La tige-boucle 5BSL3.2 a été décrite comme interagissant avec la SLIIId, la Seq9110 et la SL2 par génétique inverse et expériences de complémentation de mutations. La résonance plasmonique de surface (SPR) a été utilisée afin de caractériser ces interactions. Cela nous a conduit à développer des méthodes permettant d’étendre l'utilisation de la SPR. Nous avons pu observer, in vitro, une interaction entre la 5BSL3.2 et miR-122, un microARN fortement exprimé dans les hépatocytes. Nos résultats ont également montré que la SL2 était une séquence ARN pouvant former au moins deux conformations. L'une étant capable d'interagir avec la 5BSL3.2 et l'autre pouvant s'auto-associer. L'étude de cette dimérisation sur modèle cellulaire a montré qu'elle était impliquée dans les mécanismes de la réplication virale. Le développement d'une méthode d'analyse des complexes ternaires a permis la caractérisation simultanée du complexe formé entre la boucle interne de la 5BSL3.2 et ses partenaires (Seq9110 et SLIIId) ainsi que du complexe formé entre la boucle apicale et ses partenaires (SL2 et miR-122). Ces résultats ont montré que les deux sites de liaison de la 5BSL3.2 étaient structurellement indépendants. Ces interactions pourraient donc coexister dans un contexte physiologique. Les résultats montrent que la SLIIId et la Seq9110 sont en compétition pour interagir avec la boucle interne de la 5BSL3.2 tandis que la tige-boucle SL2 et miR-122 sont en compétition pour interagir avec la boucle apicale de la 5BSL3.2. La 5BSL3.2 est donc un motif ARN structuré pouvant agir comme une plaque tournante, capable d'interagir avec d'autres régions de l'ARN génomique tandis que la SL2 a montré que les deux interactions qu'elle pouvait former, mutuellement exclusives, étaient importantes pour la prolifération virale. Les mécanismes de compétition observés pourraient être impliqués dans la commutation entre les différentes étapes du cycle viral. / We are interested in understanding how interactions involving nucleic acids regulate the life cycle of the Hepatitis C virus (HCV). The HCV genomic RNA is highly structured at the 5' and 3' ends. The stem-loop 5BSL3.2 was described to interact with the SLIIId, the Seq9110 and the SL2 by reverse genetics and complementation mutation experiments. Surface plasmon resonance (SPR) was used to characterize these interactions. This led us to develop methods to expand the use of this technique. We described in vitro evidences for an interaction between 5BSL3.2 and miR-122, a microRNA highly expressed in hepatocytes. As shown by our results, SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: one is able to hybridize with 5BSL3.2 and the other one is capable of self-associating. The study of this dimerization in living cells has shown an implication of this phenomenon in viral replication processes. The development of a ternary complex analysis method allowed the characterization of the Seq9110 (or SLIIId) and SL2 (or miR-122) interacting with 5BSL3.2. Our results shown that the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent, suggesting that these interactions may coexist in a physiological context. SLIIId and Seq9110 were shown to compete to interact with 5BSL3.2 internal loop while SL2 and miR-122 were shown to compete to interact with 5BSL3.2 apical loop. In conclusion, 5BSL3.2 is a structured RNA motif that could act as a molecular hub capable of interacting with other genomic RNA regions while the two interactions of the SL2, mutually exclusives, were shown to be crucial for viral proliferation. The competition mechanisms observed could be involved in the commutation between viral cycle steps.

Les dernières décennies ont été marquées par l’émergence ou la réémergence d’un nombre croissant de virus pathogènes. Malheureusement, les antiviraux actuellement sur le marché ciblent un nombre restreint de virus ; il y a donc un besoin urgent de développer des antiviraux à large spectre. Les cyclophilines sont des protéines cellulaires impliquées dans un grand nombre de processus biologiques, qui possèdent une activité enzymatique peptidyl-prolyl cis-trans isomérase (PPIase). Elles sont également impliquées dans la réplication de virus appartenant à des familles éloignées et constituent donc une cible de choix pour le développement d'antiviraux à large spectre. Toutefois, les inhibiteurs de cyclophilines disponibles possèdent de nombreux inconvénients qui rendent leur utilisation clinique difficile.Par une stratégie de "fragment-based drug design", nous avons généré une nouvelle famille d'inhibiteurs de cyclophilines, les SMCypI ("Small-Molecule Cyclophilin Inhibitors"), complètement différents de tous les inhibiteurs de cyclophilines existants. La cristallographie de ces composés a montré qu'ils se fixaient dans les deux poches voisines du site actif des cyclophilines et qu'ils inhibaient leur activité PPIase. Ces composés n’étaient pas immunosuppressifs et bloquaient in vitro l'infection par le VIH, le VHC et les Coronavirus.L'activité anti-VHC du C31, composé le plus actif sur l'activité PPIase des cyclophilines, a été caractérisée. Le C31 était un inhibiteur pan-génotypique du VHC, doté d’une haute barrière contre la résistance et présentant une activité additive avec les inhibiteurs du VHC approuvés. Nous avons montré que le C31 bloquait l'infection par le VHC en rompant l'interaction entre la protéine virale NS5A et la cyclophiline A de façon PPIase-dépendante. Enfin, le C31 était actif sur la réplication des virus zika, de la dengue, de la fièvre jaune et du Nil Occidental.L'activité des SMCypI a été caractérisée sur l'infection par le Coronavirus 229E. De manière intéressante, l’inhibition de l’activité PPIase était nécessaire, mais pas suffisante pour l’activité antivirale. Une étude de la relation structure-activité des composés a révélé qu'un groupement chimique situé à l'interface entre les deux poches du site actif des cyclophilines jouait un rôle clé dans l'effet anti-coronavirus. Le F836 a été identifié comme le composé le plus actif, qui bloquait l'effet cytopathique et la quantité d'ARN du HCoV-229E avec la même efficacité que l'alisporivir, sans toxicité associée. Ce composé bloquait l'entrée du HCoV-229E après l'attachement du virus à la surface cellulaire, et était également actif sur l'entrée des HCoV-OC43 et du MERS-CoV. Nous avons par la suite démontré l’association de la cyclophiline A avec les particules virales. Par l'utilisation de la technologie CRISPR-Cas9, des cellules invalidées pour la cyclophiline A ont été générées. La cyclophiline A apparaissait nécessaire pour l'infection par HCoV-229E et la cible de l'effet antiviral du F836.Les SMCypI constituent un outil pour la compréhension des mécanismes par lesquels les cyclophilines modulent les infections virales et représentent des candidats crédibles pour le développement futur d'antiviraux à large spectre. / Over the past decades, an increasing number of viruses has emerged or re-emerged in humans. Unfortunately, currently approved antiviral drugs target a small set of viruses. Thus, there is an urgent need for the development of broad-spectrum antiviral drugs.Cyclophilins are cellular proteins involved in a large number of biological processes, and in different viral lifecycles from unrelated families. They appear as a potential target for the development of broad-spectrum antiviral approaches. However, currently available cyclophilin inhibitors have drawbacks which limit their clinical use.By means of "fragment-based drug design", we generated a new class of small-molecule cyclophilin inhibitors (SMCypI), unrelated with those already available. Cristallographic studies revealed that the SMCypIs bind to two close pockets of the active site and inhibit cyclophilin PPIase activity. These compounds do not bear immunosuppressive properties and inhibit the replication of HIV, HCV and coronaviruses in vitro.We characterized the anti-HCV activity of C31, the most potent inhibitor of cyclophilin PPIase activity. C31 had pan-genotypic HCV inhibitor properties, with a high barrier to resistance and additive effects with currently approved anti-HCV agents. C31 blocked HCV replication by disrupting the interaction between the nonstructural viral protein NS5A and cyclophilin A in a PPIase-dependent manner. Finally, C31 was active on zika, yellow fever, dengue and West-Nile virus infections.The antiviral activity of the SMCypIs has then been characterized on HCoV-229E infection. Interestingly, PPIase inhibition was necessary, but not sufficient for antiviral effect. A structure-activity relationship study identified a key moiety in the SMCypIs at the interface between the two cyclophilin pockets. F836 has been identified as the most potent compound which inhibited both the cytopathic effect and the intracellular RNA of HCoV-229E without associated cytotoxicity and as potently as alisporivir. This compound targeted HCoV-229E entry at a post-attachment step and was also active on HCoV-OC43 and MERS-CoV strains. We then demonstrated that cyclophilin A was associated with viral particles. By means of CRISPR-Cas9, cell lines depleted for cyclophilin A were generated. Cyclophilin A was identified as a proviral factor for HCoV-229E and was partially involved in F836 antiviral effect. Cyclophilin A expression level was drastically decreased by infection.SMCypIs represent a unique tool to decipher the cellular and molecular mechanisms by which cyclophilins interfere with viral lifecycles, as well as drugable compounds that could find an indication as broad-spectrum antiviral drugs.