Vincent Lotteau

Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles

ABSTRACT The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

Hepatitis C virus infection protein network

A proteome‐wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein–protein interactions between HCV and human proteins was identified by yeast two‐hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFβ pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

Autophagy Induction by the Pathogen Receptor CD46

Autophagy is a highly regulated self-degradative mechanism required at a basal level for intracellular clearance and recycling of cytoplasmic contents. Upon intracellular pathogen invasion, autophagy can be induced as an innate immune mechanism to control infection. Nevertheless, pathogens have developed strategies to avoid or hijack autophagy for their own benefit. The molecular pathways inducing autophagy in response to infection remain poorly documented. We report here that the engagement of CD46, a ubiquitous human surface receptor able to bind several different pathogens, is sufficient to induce autophagy. CD46-Cyt-1, one of the two C-terminal splice variants of CD46, is linked to the autophagosome formation complex VPS34/Beclin1 via its interaction with the scaffold protein GOPC. Measles virus and group A Streptococcus, two CD46-binding pathogens, induce autophagy through a CD46-Cyt-1/GOPC pathway. Thus, upon microorganism recognition, a cell surface pathogen receptor can directly trigger autophagy, a critical step to control infection.

Oxidized Low-Density Lipoprotein Promotes Mature Dendritic Cell Transition from Differentiating Monocyte

Proinflammatory oxidized phospholipids are generated during oxidative modification of low-density lipoproteins (LDL). The production of these proinflammatory oxidized phospholipids is controlled by secreted enzymes that circulate as proteins complexed with LDL and high-density lipoprotein. During the acute phase response to tissue injury, profound changes occur in lipoprotein enzymatic composition that alter their anti-inflammatory function. Monocytes may encounter oxidized phospholipids in vivo during their differentiation to macrophages or dendritic cells (DC). In this study we show that the presence of oxidized LDL (oxLDL) at the first day of monocyte differentiation to DC in vitro yielded phenotypically atypical cells with some functional characteristics of mature DC. Addition of oxLDL during the late stage of monocyte differentiation gave rise directly to phenotypically mature DC with reduced uptake capacity, secreting IL-12 but not IL-10, and supporting both syngeneic and allogeneic T cell stimulation. In contrast to known mediators of DC activation, oxLDL did not trigger maturation of immature DC. An intriguing possibility is that a burst of oxidized phospholipids is an endogenous activation signal for the immune system, which is tightly controlled by lipoproteins during the acute phase response.

IRGM is a common target of RNA viruses that subvert the autophagy network.

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.

VirHostNet: a knowledge base for the management and the analysis of proteome-wide virus–host interaction networks

Infectious diseases caused by viral agents kill millions of people every year. The improvement of prevention and treatment of viral infections and their associated diseases remains one of the main public health challenges. Towards this goal, deciphering virus–host molecular interactions opens new perspectives to understand the biology of infection and for the design of new antiviral strategies. Indeed, modelling of an infection network between viral and cellular proteins will provide a conceptual and analytic framework to efficiently formulate new biological hypothesis at the proteome scale and to rationalize drug discovery. Therefore, we present the first release of VirHostNet (Virus–Host Network), a public knowledge base specialized in the management and analysis of integrated virus–virus, virus–host and host–host interaction networks coupled to their functional annotations. VirHostNet integrates an extensive and original literature-curated dataset of virus–virus and virus–host interactions (2671 non-redundant interactions) representing more than 180 distinct viral species and one of the largest human interactome (10 672 proteins and 68 252 non-redundant interactions) reconstructed from publicly available data. The VirHostNet Web interface provides appropriate tools that allow efficient query and visualization of this infected cellular network. Public access to the VirHostNet knowledge-based system is available at http://pbildb1.univ-lyon1.fr/virhostnet.

Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

Mature Dendritic Cell Generation Promoted by Lysophosphatidylcholine

During the acute phase response, the interplay between high density lipoproteins and low density lipoproteins (LDL) favors transient generation of oxidized LDL with proinflammatory activities. We hypothesized that oxidative modification of LDL is an endogenous signal for the immune system, and we have shown that oxidized LDL promotes mature dendritic cell transition from monocyte, therefore linking the nonspecific acute phase response to adaptive immunity. Lysophosphatidylcholine (LPC) is a major lipid component of oxidized LDL with reported proinflammatory activities. We now report that LPC acts through G protein-coupled receptors on differentiating monocytes to generate mature dendritic cells with the ability to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. LPC is most effective in lipoprotein-deprived serum and can be inhibited by an excess of native LDLs reflecting normal plasma conditions. Therefore, by controlling the balance between native and oxidized lipoproteins and the resulting production of LPC, the acute phase reactants may provide a context of Ag presentation that is transiently favorable to immune activation. Intralipid, a therapeutic lipid emulsion for parenteral nutrition with unexplained immunomodulatory properties, also blocked LPC activity. This opens perspectives for the understanding and treatment of acute and chronic inflammatory diseases.

CD4 T Cell Tolerance to Nuclear Proteins Induced by Medullary Thymic Epithelium

Thymic epithelium is involved in negative selection, but its precise role in selecting the CD4 T cell repertoire remains elusive. By using two transgenic mice, we have investigated how medullary thymic epithelium (mTE) and bone marrow (BM)-derived cells contribute to tolerance of CD4 T cells to nuclear beta-galactosidase (beta-gal). CD4 T cells were not tolerant when beta-gal was expressed in thymic BM-derived cells. In contrast, CD4 T cells of mice expressing beta-gal in mTE were tolerized. Tolerance resulted from presentation of endogenous beta-gal by mTE cells but not from cross-priming. mTE cells presented nuclear beta-gal to a Th clone in vitro, while thymic dendritic cells did not. The data indicate that mTE but not thymic BM-derived cells can use a MHC class II endogenous presentation pathway to induce tolerance to nuclear proteins.

Benchmarking a luciferase complementation assay for detecting protein complexes

1Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA. 2British Columbia Cancer Agency, Canada’s Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada. 3Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California, USA. 4Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, California, USA. 5Brain Tumor Research Center, Department of Neurosurgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, Santa Cruz, California, USA. 6Howard Hughes Medical Institute, Santa Cruz, California, USA. 7Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri, USA. e-mail: twang@genetics.wustl.edu or jcostello@cc.ucsf.edu