Hepatitis: Rubinstein E

Charrin S, le Naour F, Silvie O, Milhiet PE, Boucheix C, Rubinstein E. · Inserm, U, Villejuif, France. · Biochem J. · Pubmed #19426143 No free full text.

Abstract: Despite high expression levels at the plasma membrane or in intracellular vesicles, tetraspanins remain among the most mysterious transmembrane molecules 20 years after their discovery. Several genetic studies in mammals and invertebrates have demonstrated key physiological roles for some of these tetraspanins, in particular in the immune response, sperm-egg fusion, photoreceptor function and the normal function of certain epithelia. Other studies have highlighted their ability to modulate cell migration and metastasis formation. Their role in the propagation of infectious agents has drawn recent attention, with evidence for HIV budding in tetraspanin-enriched plasma membrane domains. Infection of hepatocytic cells by two major pathogens, the hepatitis C virus and the malaria parasite, also requires the tetraspanin CD81. The function of tetraspanins is thought to be linked to their ability to associate with one another and a wealth of other integral proteins, thereby building up an interacting network or 'tetraspanin web'. On the basis of the biochemical dissection of the tetraspanin web and recent analysis of the dynamics of some of its constituents, we propose that tetraspanins tightly regulate transient interactions between a variety of molecules and as such favour the efficient assembly of specialized structures upon proper stimulation.

Rocha-Perugini V, Lavie M, Delgrange D, Canton J, Pillez A, Potel J, Lecoeur C, Rubinstein E, Dubuisson J, Wychowski C, Cocquerel L. · Institut de Biologie de Lille, CNRS-UMR8161, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France. · BMC Microbiol. · Pubmed #19476617 links to  free full text

Abstract: BACKGROUND: Three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for Plasmodium infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM). RESULTS: In our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels. CONCLUSION: In contrast to Plasmodium infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.

Yalaoui S, Zougbédé S, Charrin S, Silvie O, Arduise C, Farhati K, Boucheix C, Mazier D, Rubinstein E, Froissard P. · Université Pierre et Marie Curie-Paris6, UMR S511, Paris, France. · PLoS Pathog. · Pubmed #18389082 links to  free full text

Abstract: Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.

Rocha-Perugini V, Montpellier C, Delgrange D, Wychowski C, Helle F, Pillez A, Drobecq H, Le Naour F, Charrin S, Levy S, Rubinstein E, Dubuisson J, Cocquerel L. · Institut de Biologie de Lille (UMR8161), CNRS, Universités de Lille I et Lille II, Institut Pasteur de Lille, Lille, France. · PLoS One. · Pubmed #18382656 links to  free full text

Abstract: Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.

Rubinstein E. · Department of Infectious Diseases, Sheba Medical Center, Tel Hashomer, Israel. · Isr Med Assoc J. · Pubmed #15274538 links to  free full text

This publication has no abstract.

Charrin S, Le Naour F, Labas V, Billard M, Le Caer JP, Emile JF, Petit MA, Boucheix C, Rubinstein E. · INSERM U268, Institut André Lwoff, Hôpital Paul Brousse, 14 Av Paul Vaillant Couturier, Villejuif 94807 Cedex, France. · Biochem J. · Pubmed #12708969 links to  free full text

Abstract: Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.

Silvie O, Rubinstein E, Franetich JF, Prenant M, Belnoue E, Rénia L, Hannoun L, Eling W, Levy S, Boucheix C, Mazier D. · INSERM U511 Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, Centre Hospitalo-Universitaire Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France. · Nat Med. · Pubmed #12483205 No free full text.

Abstract: Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.

Delpuech O, Buffello-Le Guillou DB, Rubinstein E, Féray C, Petit MA. · INSERM E. 99-41, Centre Hépato-Biliaire, Hôpital Paul-Brousse, 12, avenue Paul-Vaillant Couturier, 94800 Villejuif, France. · Eur Cytokine Netw. · Pubmed #11282549 No free full text.

Abstract: Patients with chronic hepatitis C present an imbalance of Th1/Th2 cytokine production. Therefore, we investigated whether the exposure of the CD4+ T cell line H9 to HCV could induce activation of cells through synthesis of IL-10. Three infection protocols were performed to enhance HCV propagation. Viral particles were prepared by ultracentrifugation of serum from patients. From 3 to 81 days post-infection (p.i.), HCV-RNA was monitored both in supernatants and cells by nested RT-PCR, IL-10 protein in medium by ELISA, and IL-10 mRNA in cells by semi-quantitative RT-PCR. The expression of tetraspanins was analyzed by flow cytometry. The PKC signal pathway was studied using specific inhibitors. The H9 cells express CD81. HCV-RNA (+) was detected in cells until 21 days p.i, and in culture media over 39 days p.i. Up to day 81 p.i., HCV exposure induced a specific, 2-fold increase of IL-10 production by H9 cells. IL-10 production was inhibited by a PKC inhibitor (Calphostin C). This study shows that even if the infection of H9 T cells did not result in any viral progeny, HCV induced the activation of IL-10 secretion, which supports the role of IL-10 in HCV pathogenesis.