Analyse du mécanisme d'entrée du virus de l'hépatite B : Identification d'un nouveau déterminant de l'infectivité

The hepatitis B virus is an extremely contagious human pathogen responsible for severe hepatic diseases like cirrhosis or hepatocellular carcinoma. Even though infection can be prevented by immunization with an efficient vaccine, about 2 billion people have been infected worldwide, resulting in 350 million chronic carriers that are prone to develop liver diseases. Current treatments consist either in the use of interferon, which modulates antiviral defenses and controls infection in 30 to 40% of cases, or in the use of viral polymerase inhibitors that allow a stronger response to treatment but require long-term utilization and frequently lead to the outcome of resistant viruses. A better understanding of the virus life cycle, and particularly of the mechanism by which the virus enters the cell, could provide background for therapeutics that inhibit the early steps of infection. Then, the objective of my PhD work was to study the mechanism of HBV entry. One approach to decipher viral entry is to interfere with the function of envelope proteins. Therefore, we introduced mutations in HBV surface proteins to identify new motives necessary for viral infectivity. This strategy highlighted the role of a new infectivity determinant, in the HBV large envelope protein, which is probably implicated in a fusion process allowing the release of nucleocapsids into the cytosol of infected cells.Le virus de l'hépatite B (VHB) est un agent pathogène humain, très contagieux, responsable de pathologies hépatiques telles que la cirrhose ou le carcinome hépatocellulaire. A l'heure actuelle, on estime que 2 milliards de personnes ont été infectées par ce virus dans le monde, parmi lesquelles on recense 350 millions de porteurs chroniques. Malgré l'existence d'un vaccin efficace, le nombre de personnes atteintes par la maladie reste élevé. Pour ces dernières, des traitements puissants existent consistant en l'utilisation d'interférons, efficaces dans 30 à 40% des cas, ou d'antiviraux dont l'inconvénient majeur est la sélection de virus résistants au traitement. Le mécanisme d'entrée du VHB dans les hépatocytes est encore inconnu. Dans ce contexte, l'objectif de ma thèse a été d'étudier ce mécanisme afin, notamment, de contribuer au développement de nouvelles thérapeutiques empêchant l'entrée virale. Une des approches utilisées pour étudier le processus d'entrée d'un virus consiste à étudier l'implication de ses protéines de surface dans ce phénomène. Ainsi, nous avons recherché la présence de motifs indispensables au processus d'infection dans ces protéines. Pour cela, nous y avons introduit des mutations puis nous avons analysé leur impact sur la capacité des virus à infecter des cellules saines. Cette stratégie nous a permis d'identifier, au sein de la grande protéine de surface du VHB, un nouveau déterminant de l'infectivité possiblement impliqué dans un processus de fusion permettant à l'enveloppe lipoprotéique virale de fusionner avec une membrane cellulaire afin qu'elle libère sa nucléocapside, contenant l'ADN viral, dans le cytoplasme de la cellule infectée

Entrée du virus de l'hépatite B

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The Translocation Motif of Hepatitis B Virus Envelope Proteins Is Dispensable for Infectivity▿

The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity

The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity▿

The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes

Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle.

International audienceHepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems. Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research. At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones. In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named 'replicon'. This system allowed intensive research into replication mechanisms and drug discovery. Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process. It was only in 2005 that infectious viruses were produced in vitro, enabling intensive investigations into the entire life cycle of the hepatitis C virus. This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications

Entry of pseudotyped hepatitis C virus into primary human hepatocytes depends on the scavenger class B type I receptor.

International audienceEntry of the hepatitis C virus (HCV) into the cell seems to be a complex multi-step process involving several cellular factors such as the scavenger class B type I receptor (SRBI). Until now, all investigations conducted to assess the involvement of SRBI have been based on in vitro infection models which use human hepatoma-derived cell lines. However, the HCV entry pathway may be altered in these types of cells because of the impairment of some hepatic characteristics. In this study, we showed that SRBI also plays an essential role in HCV entry into primary human hepatocytes with two distinct approaches: gene extinction and antibodies neutralization assays

Prise en charge de l’hypercalcémie : à propos d’un cas d'hypercalcémie secondaire à un syndrome de Richter hodgkinoïde

L'hypercalcémie est un trouble ionique qu'il ne faut pas méconnaître, et dont la prise en charge est urgente lorsqu'elle est sévère. Nous rapportons le cas d'un patient âgé de 66 ans, présentant une hypercalcémie avec symptômes neurologiques et répercussions cardiaques. Le bilan a mis en évidence un syndrome de Richter hodgkinoïde responsable d’une production excessive de calcitriol. Après traitement de l’hypercalcémie et chimiothérapie, l’évolution a été favorable avec une disparition des symptômes neurologiques et des modifications de l’électrocardiogramme. A la lumière de ce cas, nous rappelons les principales étiologies de l'hypercalcémie, les manifestations cliniques ainsi que les principes de la prise en charge.[A rare cause of hypercalcemia] Hypercalcemia is a condition that must not be mistaken, because it can be life-threatening. Its severe form must be rapidly treated. We present the case of 66-year-old man presenting with neurologic symptoms and cardiac alterations secondary to severe hypercalcemia. The diagnostic work-up revealed excessive production of calcitriol secondary to Richter’s syndrome. After treatment of hypercalcemia and chemotherapy, the neurological symptoms and electrocardiographic changes completely disappeared. The aim of this article is to discuss the main etiologies of hypercalcemia, its clinical manifestations, and the principles of its management

Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening.

International audienceThe entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh-7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down-regulation of different factors implied in clathrin-mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4-kinase type III-alpha (PI4KIIIalpha) prevented infection by HCVpp or by cell-culture grown JFH-1-based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIIIalpha knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIbeta in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME-dependant entry route of HCV and identified 2 kinases, PI4KIIIalpha and beta, as relevant potential therapeutic targets