Made available in DSpace on 2015-04-11T13:54:59Z (GMT). No. of bitstreams: 1 soriane.pdf: 3202394 bytes, checksum: 55f87924522756721f3a0349e37b2bac (MD5) Previous issue date: 2013-09-17 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Introduction: Hepatitis C virus (HCV) infection is the major cause of liver disease worldwide. Approximately 170 million more people are infected with the virus . The immune response against HCV leads to secretion of cytokines and activation and proliferation of effector T cells to act in infected hepatocytes and thus initiate the inflammatory process in the liver. Objective: This study aimed to characterize the immune response and to estimate the frequency of polymorphism of TNF - α -308 G / A in patients with chronic hepatitis C treated at the Tropical Medicine Foundation Dr. Heitor Vieira Dourado. Material and Methods: In this study we used samples from 30 patients with hepatitis C virus infection and 28 samples from individuals not infected with HCV . The analysis of T lymphocyte and adhesion molecules were performed by flow cytometry. The cytokine levels were assessed by flow cytometry using the CBA kit. The characterization of TNF- α -308 polymorphism was obtained by PCR-RFLP. The fibrosis was determined after histological analysis of liver biopsy according to the METAVIR score. Results: Our data revealed that percentage of regulatory T lymphocytes (T regs) and cytokines are increased in patients with hepatitis C when compared to the control group. Patients with fibrosis ≥F3 (N = 10) was characterized relevant frequency ( 100%) of individuals with high production of IL-2 and low frequency (50%) of individuals with high production of IL-10 and patients with fibrosis ≤F2 was characterized relevant frequency ( 85%) of individuals with high production of IL-4 and frequency ( 55%) of individuals with high production of IL-10. Conclusion: The effector T cells are regulated by T lymphocytes regs. In the fibrosis ≥F3 was characterized Th1 cytokine profile while in fibrosis ≤F2 ws characterized TH2 cytokine profile. The low IL-10 production was associated an increased fibrogenic activity in patients with hepatitis C. / Introdução: A infecção pelo vírus da hepatite C (VHC) é a principal causa de doença hepática em todo mundo. Aproximadamente 170 milhões de pessoas estão infectadas com o vírus. A resposta imune contra o VHC conduz a secreção de citocinas e ativação e proliferação de células T efetoras para atuarem nos hepatócitos infectados e assim iniciam o processo inflamatório no fígado. Objetivo: o estudo teve como objetivo caracterizar a resposta imune e estimar a frequência do polimorfismo do TNF-α -308 G/A em pacientes com hepatite C crônica atendidos na Fundação de Medicina Tropical Doutor Heitor Vieira Dourado. Material e Métodos: Foram utilizadas amostras de 30 de pacientes com hepatite C e 28 amostras de indivíduos não infectados com o VHC. As análises dos linfócitos T e moléculas de adesão foram realizadas por citometria de fluxo. As citocinas séricas foram analisadas por citometria de fluxo utilizando o kit CBA. A caracterização do polimorfismo de TNF-α- 308 foi obtida por PCR-RFLP. A fibrose foi determinada após análise histológica de biópsia hepática de acordo com o escore METAVIR. Resultados: Os pacientes com hepatite C apresentaram porcentagem de linfócitos T regulatórios (T regs) e citocinas aumentadas quando comparados ao grupo controle. Na fibrose ≥F3 (N=10) foi caracterizada relevante frequência (100%) de indivíduos com alta produção de IL-2 e baixa frequência (50%) de indivíduos com alta produção de IL-10 e na fibrose ≤F2 foi caracterizada relevante frequência (85%) de indivíduos com alta produção de IL-4 e frequência (55%) de indivíduos com alta produção de IL-10. Conclusão: Os linfócitos T efetores são regulados pelos linfócitos T regs. Na fibrose ≥F3 foi caracterizado um perfil de citocinas Th1 enquanto na fibrose ≤F2 um perfil de citocinas Th2. A baixa produção de IL-10 foi associada com uma atividade fibrogênica aumentada em pacientes com hepatite C.

Orientadores: Clarice Weis Arns, Luciana Konecny Kohn / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T21:48:22Z (GMT). No. of bitstreams: 1 Bastos_JulianaCristinaSantiago_M.pdf: 1673040 bytes, checksum: 1234828d961406573d21b2476f1f1800 (MD5) Previous issue date: 2013 / Resumo: O vírus da Hepatite C (família Flaviviridae, gênero Hepacivirus) é causador de infecções crônicas em humanos, que podem evoluir para quadros de cirrose hepática e carcinoma hepatocelular. Até o momento, não há vacina disponível contra essa infecção e o tratamento disponível é caro, tem eficácia limitada e gera uma vasta gama de efeitos secundários, o que dificulta a continuidade do tratamento. Como esse vírus não replica eficientemente em cultura de células e em animais, o vírus da diarréia viral bovina é utilizado como modelo substituto para ensaios de avaliação de atividade antiviral e em ensaios de mecanismo de ação. A partir de invertebrados e micro-organismos marinhos, foram preparados extratos e frações, e algumas substâncias foram isoladas para a avaliação da sua possível atividade antiviral. Dos 422 testados, 5% foram considerados promissores e, destes, 20% mostraram-se ativos apresentando uma proteção de mais de 97% às células frente ao vírus. Os melhores resultados foram obtidos dos extratos produzidos a partir das amostras de esponjas Hyrtios sp. (BA07ES-56: PI=99%, IS=25), Aaptos sp. (BA07ES-59: PI=99%, IS=8,25) e de bactérias Bacillus sp. (555: PI=98%, IS>18; 584: PI=98%, IS=27) isoladas da esponja Petromica citrina. Os extratos e compostos promissores foram capazes de atuar em diversas etapas do ciclo replicativo viral (adsorção, penetração, etapas intracelulares do ciclo replicativo e também inativação da partícula viral), levando à sua interrupção quase completa nas condições analisadas. Desse modo, diversas substâncias presentes nesses organismos estudados são ativas e podem levar ao desenvolvimento de fármacos que garantam uma terapia alternativa para o tratamento da hepatite C / Abstract: The Hepatitis C virus (family Flaviviridae, genus Hepacivirus) causes chronic infections in humans, which can develop to liver cirrhosis and hepatocellular carcinoma. This represents a major public health problem worldwide. To this moment, there is no vaccine available against this infection and the treatment available is expensive, has limited efficacy and generates a wide range of side effects, making it difficult to continue the treatment. All this reflects the need to seek new agents with antiviral action against this virus. As this virus does not replicate efficiently in cell culture and in animals, bovine viral diarrhea virus is used as a surrogate model for screening assays of antiviral activity, and mechanism of action assays. From marine invertebrates and micro-organisms isolated from them, extracts and fractions were prepared, and substances were isolated for assessment of their possible antiviral activity. Of the 422 tested, 5% were considered promising, and of these, 20% were active presenting a protection percentage of more than 97%. The best results were obtained from the extracts produced from the samples of sponge Hyrtios sp. (BA07ES-56: IP=99%, SI=25), Aaptos sp. (BA07ES-59: IP=99%, SI=8,25) and bacteria Bacillus sp. (555: IP=98%, SI>18; 584: IP=98%, SI=27) isolated from the sponge Petromica citrina. The promising extracts and compounds acted in several stages of viral replicative cycle (adsorption, penetration, intracellular steps of the replicative cycle and also inactivation of the viral particle). Thus, various substances are active and may lead to the development of drugs which ensure an alternative therapy for the treatment of hepatitis C / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

La polymérase NS5B du virus de l’hépatite C (VHC) porte une activité ARN polymérase ARN-dépendante essentielle pour la réplication de l'ARN génomique viral. Cette réplication implique la synthèse d'un intermédiaire de réplication de polarité négative. In vitro et probablement in vivo, la NS5B initie la synthèse d'ARN par un mécanisme de novo qui nécessite des interactions spécifiques entre la polymérase virale et des éléments des ARN viraux. Dans une première partie nous avons étudié le rôle du GTP et d’un domaine C-terminal nommé linker de la polymérase. Nos résultats démontrent que des concentrations élevées de GTP sont nécessaires pour la transition de l'initiation à l'élongation de la synthèse de l'ARN. Des mutations dans le linker à la position 556 ne modifient pas la concentration de GTP nécessaire pour la transition. Toutefois, l'initiation de la synthèse d'ARN est augmentée par la mutation S556K. Une analyse structurale menée en parallèle suggère une implication directe du linker dans l'initiation de novo de la synthèse de l'ARN. Dans les deuxièmes et troisièmes parties, nous avons étudié le rôle de motifs ARN dans la traduction et la synthèse de l’'ARN du VHC. Nous avons démontré que la tige boucle SL-E1 formée par la région entre les nt 177 et 222 de l'extrémité 3' de l’ARN (-) est importante pour la synthèse d'ARN in vitro par la NS5B recombinante et dans les cellules Huh7 exprimant le complexe de réplication (RC) du VHC. SL-E1 est impliquée dans l’initiation de la synthèse d’ARN, au moins in vitro. Nous avons également étudié le rôle des tiges boucles SLV et SLVI du gène core. Nos données n'ont pas montré de rôle évident de ces séquences ou de leur complément dans la synthèse de l'ARN in vitro par la NS5B recombinante et en culture cellulaire par le RC du VHC. Nous avons confirmé leur effet négatif sur la traduction IRES dépendante par interaction ARN-ARN longue distance entre SL-VI et le 5'UTR et démontré que le miR122 ne peut pas empêcher cet interaction. Par contre, la présence de SL-VI prévient l’inhibition de la traduction induite par l’interaction entre le domaine III de l’IRES et la tige boucle 5BSL3.2 en 3’ du génome. Ces résultats démontrent la complexité des interactions ARN/ARN et ARN/protéines dans la régulation de la réplication virale. / The hepatitis C virus (HCV) NS5B protein displays a RNA-dependent RNA polymerase activity essential for replication of the viral RNA genome. This replication involves the synthesis of a replication intermediate of negative polarity. In vitro and likely in vivo, the NS5B initiates RNA synthesis by a de novo mechanism which requires specific interactions between the polymerase and viral RNA elements. In the first part of results, we described a combined structural and functional analysis of HCV-NS5B to study the role of a C-terminal segment (termed linker) and of GTP in RNA synthesis. Our results demonstrated that high GTP concentrations are necessary for the transition from the initiation to the elongation of RNA synthesis, and that linker mutations at position S556 did not modify the GTP requirement of NS5B for this transition. However, the initiation of RNA synthesis was greatly enhanced by a S556K mutation. These results together with a structural analysis point to the direct involvement of the linker in the de novo initiation of RNA synthesis. In the second and third parts of results, we studied the role of RNA elements in RNA synthesis. We demonstrated that the SL-E1 stem–loop formed by nucleotides 177–222 from the 3’-end of the HCV (-) RNA is important for RNA synthesis both in vitro by the recombinant NS5B and in Huh7 cells by HCV replication complex (RC). We also showed that SL-E1 is involved in initiation of RNA synthesis, at least in vitro. Then we studied the role of other viral RNA elements in core coding sequences (SLV and SLVI stem loops) and the involvement of the microRNA miR122 in RNA translation and RNA synthesis. For SLV and SLVI, our data did not show any clear role of these core-coding sequences or of their complement in the (-) RNA in RNA synthesis both in vitro by the recombinant NS5B and in cell culture by HCV-RC. We confirmed their negative effect on HCV-IRES translation through long range RNA-RNA interaction between SL-VI sequences and the 5’UTR and demonstrated that miR122 cannot disrupted this interaction and switches the region to an open conformation. Conversely, our data indicated that the SL-VI domain can counteract the negative effect of the interaction between the domain III of IRES and the 5BSL3.2 stem loop localized at the 3’end of the genome. These results point to the complexity of RNA/RNA and RNA/proteins interactions in the HCV replication cycle.

Since the discovery in 1989 of hepatitis C virus (HCV) as the infectious agent responsible for the vast majority of post-transfusion non-A non-B hepatitis, blood transfusions are no longer a source for HCV transmission in Sweden. Anti-HCV testing was implemented for all blood donations in 1992. Since then intravenous drug use (IDU) has become the major route of transmission in the western world. Six genotypes and more than 80 subtypes of HCV have now been identified world-wide. These genotypes and subtypes are determined by genetic divergences between the HCV strains. Subtypes 1a, 1b, 2b, 2c, and 3a have global spread, while the other subtypes have a more limited geographical distribution. Little was known on the prevalence of HCV among blood donors and on which genotypes and subtypes of HCV were circulating in Sweden before the testing of all blood donations was implemented. The prevalence of anti-HCV was therefore investigated in sera sent to the Swedish Institute for Infectious Disease Control (SMI) from 412 patients; 241 were sampled between 1970 and 1991 before the universal screening in 1992, while 171 were sampled between 1992 and 2002. The samples derived from 193 (47%) blood donors, (104 sampled before, and 89 after 1992), and from seven other groups of patients. Two groups had suspected known routes of infection, intravenous drug use (IDU) 33 patients and hemodialysis, 16 patients, while it was unknown for the other patients. Anti-HCV was detected in 120 (29%) samples. The highest frequency was found among IDUs, (91%). Before general screening was implemented, 2.8% of the blood donors were positive for hepatitis C, whereas 28% of those sampled after 1992 were anti-HCV positive. Those latter samples were sent to SMI due to anti-HCV reactivity in a primary test at the blood centre. HCV RNA could be detected by PCR in 56 (47%) of the anti-HCV positive samples, the subtype could be determined by sequencing in 45 (80%) of those. The subtypes found were 1a in 31 %, 1b in 18%, 2b in 22%, and 3a in 27%. One sample was of subtype 2c. There was a tendency of increase of genotype 2 and a decrease in subtype 1a with time. 1a was found in 38% of the samples collected before 1992, while it was only found in 19% of the samples from 1992 or later. On the other hand genotype 2 was found in 17% sera sampled before 1992 and in 37% of the samples collected 1992 or later. It is not known if this genotype has recently been introduced into Sweden. Further analysis on larger series of samples is needed to confirm these preliminary results. / AcknowledgmentsI would like to express my gratitude to several people who have been supportive in different ways throughout this project.First of all, I want to thank my supervisor Helene Norder, for giving me the possibility to do my diploma thesis at the Department of Virology, Swedish Institute for Infectious Disease control (SMI) and for helping me during this study and for the many insightful conversations during the design and development stages of the application, and also for the many helpful comments and suggestions on the text of the thesis.I want to express my appreciation to my laboratory supervisor Regina Wallin, Camilla Jern and Josefine Ederth for helping me during the procedure for this study. Then, I want to thank my examiner Magnus Johansson from the Södertörns university collegefor his advice on writing this paper. Finally, I would like to thank my family and specially my mother Bahar Hamid for always supporting me during my whole life.Last, but not least, I would like to thank my friends Annika Andersson and Yourdons Yemane for being encouraging, understanding and always supportive.

Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

Le virus de l'hépatite C (HCV) est responsable de maladies chroniques du foie et l'une des principales causes de développement du carcinome hépatocellulaire (HCC). Cependant, les mécanismes moléculaires qui permettent le développement d’un HCC suite à une infection chronique par le HCV restent incompris. Les microARN (miR), de petits ARNs non codants qui régulent l'expression des gènes au niveau post-transcriptionnel, sont connus pour jouer un rôle important dans l'homéostasie cellulaire du foie. De plus en plus d’études suggèrent que l'infection par le HCV induit la modification de réseaux intracellulaires impliquant les miRs hépatiques contribuant au développement des lésions du foie, y compris le HCC. En utilisant des techniques d'analyse systématiques, nous avons identifié des miRs qui modulent le cycle viral du HCV mais également des miRs modulés lors de l'infection par le HCV. Cette analyse globale des interactions entre les miRs de l'hôte et le HCV améliore les connaissances actuelles sur les interactions entre le HCV et l’hôte qui contribuent vraisemblablement à la tumorigenèse dans le foie, et ouvre des perspectives pour de potentielles nouvelles approches pour prévenir et/ou traiter le HCC chez les patients infectés par le HCV. / Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms that enable HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRs), small non coding RNAs that regulate gene expression at a post-transcriptional level have been reported to play an important role in cellular homeostasis within the liver. Increasing evidence suggests that HCV infection induces alteration of intrahepatic miR networks and that deregulation of miRs contributes to liver disease including HCC. Using high-throughput screening and RNA sequencing, we identified miRs that modulate the HCV life cycle and miRs that are modulated upon HCV infection. This comprehensive analysis of the HCV-host miR network improves the current knowledge of the HCV-host interactions that likely contribute to tumorigenesis in the liver and opens perspectives for novel potential approaches to prevent and/or treat HCC in HCV-infected patients.

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. / South Africa

No description available.

L’entrée du virus de l’hépatite C (VHC) dans les hépatocytes est un événement multi-étapes complexe, impliquant un certain nombre de facteurs cellulaires. Elle est initiée par la fixation des particules virales sur des structures d’héparanes sulfates (HS) présentes à la surface de l’hépatocyte. Cette étape initiale reste cependant peu comprise. En effet, en raison de l’interaction de la particule virale du VHC avec des lipoprotéines, la contribution exacte des différents composants du virion à cette interaction reste controversée. Au cours de cette thèse, nous avons étudié le rôle potentiel de protéines d'enveloppe du VHC et de l'apolipoprotéine E dans l'étape de liaison aux HS. Nous avons d’abord montré que la délétion de la région hypervariable 1 (HVR1), une région précédemment proposée pour participer à l’interaction avec les HS, n'avait aucun effet sur la liaison du virion aux HS, indiquant que cette région n'est pas impliquée dans cette interaction. Nous avons également utilisé des anticorps monoclonaux neutralisants reconnaissant différentes régions des glycoprotéines d'enveloppe du VHC dans un test de compétition utilisant des billes d’agarose couplées à l’héparine, un homologue structural des HS, pour précipiter le virus. Bien que les glycoprotéines d’enveloppe du VHC dissociées de la particule virale interagissaient avec l'héparine, aucun de ces anticorps n’était capable d'interférer avec l'interaction entre la particule virale et l’héparine, suggérant fortement que les glycoprotéines d'enveloppe du VHC présente à la surface des virions ne sont pas accessibles pour interagir avec les HS. En revanche, nos résultats d’études cinétiques, d’interaction avec l’héparine ainsi que les expériences d'inhibition avec des anticorps anti-apolipoprotéine E indiquent que cette apolipoprotéine joue un rôle majeur dans l'interaction initiale entre le VHC et les HS. Enfin, la caractérisation des déterminants structuraux des HS nécessaires à l'infection par le VHC, à l’aide d’ARN interférents ciblant des enzymes impliquées dans la voie de biosynthèse des HS et par compétition avec des héparines modifiées, indique que la N-sulfatation et la 6-O-sulfatation sont nécessaires pour l’initiation de l'infection par le VHC. Par contre la 2-O-sulfatation n’est pas indispensable pour l’étape d’entrée cellulaire du VHC. Enfin, nous avons également montré que la taille minimale des oligosaccharides d’HS requise pour l'infection par le VHC est un decasaccharide. En conclusion, l’ensemble de ces données indique que le VHC détourne l'apolipoprotéine E pour initier son interaction avec des structures d’HS spécifiques. / Hepatitis C virus (HCV) entry into hepatocytes is a complex multistep process involving a series of cellular factors. HCV entry is initiated by the binding of viral particles to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS-binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction. Neutralizing monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pull-down assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with virion-heparin interaction, strongly suggesting that, at the virion surface HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pull-down and inhibition experiments with anti-apolipoprotein E antibodies indicate that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of HS structural determinants required for HCV infection by silencing enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation are required for HCV infection, and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures.

L’entrée cellulaire du virus de l’Hépatite C (VHC) est un processus complexe qui met en jeu plusieurs facteurs cellulaires. L’un d’eux est un récepteur des lipoprotéines, le « Scavenger Récepteur B1 » (SR-BI). SR-BI a initialement été proposé comme récepteur viral de par son interaction avec la glycoprotéine (GP) E2 du VHC. L’importance de SR-BI pour l’entrée cellulaire du VHC n’était, jusqu’alors suggérée que par des preuves indirectes. En outre, les mécanismes par lesquels SR-BI permet l’entrée du VHC restent peu connus. Néanmoins, grâce à l’identification de cellules hépatiques présentant un très faible niveau d’expression - non détectable - de ce récepteur et devenant susceptibles à l’infection par le VHC par l’expression ectopique de SR-BI, nous avons clairement démontré que SR-BI est indispensable pour l’entrée du VHC. Afin d’étudier le rôle de SR-BI dans l’entrée cellulaire du VHC, qui présente une singulière hétérogénéité en terme de propriétés biochimiques et de composition en protéines cellulaires intégrées à la surface des particules virales, celles-ci ont été séparées par centrifugation analytique en gradient de densité. Nous avons ainsi défini trois fonctions de SR-BI permettant l’entrée de sous-populations du VHC. D’un part, une fonction d’attachement, correspondant à la capture des particules de densités intermédiaires à la surface cellulaire. Cet attachement résulte de l’interaction entre SR-BI et des composants des lipoprotéines présents à la surface des particules virales, sans mettre en jeu d’interaction avec les GP virales. D’autre part, nous avons défini une fonction d’accès, requise pour toutes les sous-populations du VHC. Cette fonction, elle aussi indépendante de l’interaction directe entre la GP E2 et SR-BI, requiert la fonction physiologique de transfert de lipides de SR-BI. En effet, le blocage de cette fonction de transfert à l’aide de molécules inhibitrices ou par insertion de mutations invalidantes de SR-BI réduit significativement l’entrée du VHC. Enfin, nous avons mis en évidence une troisième fonction, appelée fonction de stimulation, nécessitant l’interaction de la GP E2 et SR-BI, ainsi que la fonction de transfert lipidique deSR-BI et qui, par ailleurs, est régulée par des composants des lipoprotéines. Par l’analyse fonctionnelle de mutants, nous avons défini des déterminants viraux, dans la région HVR1 à l’extrémité N-terminale de la GP E2, et cellulaires, dans la partie N-terminale de SR-BI, critiques pour l’interaction entre E2 et SR-BI et, par conséquent, régulent la fonction destimulation. En conclusion, nous avons démontré que le VHC exploite SR-BI de diverses façons et via des interactions à la fois avec des composants viraux et cellulaires incorporés dans les particules du VHC et ainsi permet l’entrée de particules virales très hétérogènes. / Hepatitis C virus (HCV), which is characterised by its highly heterogeneous biophysical properties, is thought to enter the cell in a slow and multistep manner involving several cell surface molecules. One of these cellular molecules is the Scavenger Receptor B1 (SR-BI), which was identified as an HCV receptor due to its interaction with the HCV glycoprotein E2.Until now, the exact usage of SR-BI by HCV and SR-BI mediated mechanism during cell entry remained unknown. In order to understand SR-BI functions during HCV cell entry, we wanted to study (1) the relevance of HCV E2 binding to SR-BI, (2) the implication of the physiological function of SR-BI itself and (3) how SR-BI mediates cell entry of heterogeneous HCV particles. Owing to the identification of two cell lines that express very low levels of endogenous SRBI, receptor complementation assays revealed, that the ectopic expression of SR-BI is indispensible for HCV entry. Accordingly, we showed for the first time, that SR-BI is an essential HCV entry factor. In order to study HCVcc populations that differ in biophysical properties and host protein composition, we separated them by density gradient analysis and assigned three different SR-BI functions to entry of particular HCV sub-populations. First, an attachment function, that leads to the initial capture of HCV particles of intermediate densities to the cell surface. This attachment function is mediated by an interaction between SR-BI and lipoprotein components on the viral particles but not by the viral glycoproteins. Second, we defined an access function, which is important for all different HCV sub-populations. This access function is also not dependent on the interaction between HCV E2 and SR-BI but involves the physiological function of the receptor. Blocking the lipid transfer function of SRBI upon either mutation or by a specific inhibitor abrogated strongly HCV entry. Finally we defined a third function of SR-BI, that we call enhancement function. This function is triggered upon E2-SR-BI interaction, is dependent on lipoprotein components and involves the lipid transfer function of SR-BI. Upon functional mutagenesis studies, we identified as critical determinants HVR1 (Hypervariable Region 1) residues in E2 and the N-terminus of SR-BI, allowing E2-SR-BI interaction and consequently the implementation of the enhancement function. In conclusion we demonstrate that SR-BI is an unparalled virus entry factor. Its usage by HCV to enter the cell is manifold and intriguingly, owing to the heterogeneous nature of HCV particles, involves different viral components exploiting different aspects of SR-BI.