Hepatitis: Budkowska A

Burlone ME, Budkowska A. · Pasteur Institute, Hepacivirus and Innate Immunity, 75724 Paris Cedex 15, France. · J Gen Virol. · Pubmed #19264629 No free full text.

Abstract: Hepatitis C virus (HCV), a major cause of chronic liver disease, is a single-stranded positive sense virus of the family Flaviviridae. HCV cell entry is a multi-step process, involving several viral and cellular factors that trigger virus uptake into the hepatocyte. Tetraspanin CD81, human scavenger receptor SR-BI, and tight junction molecules Claudin-1 and occludin are the main receptors that mediate HCV entry. In addition, the virus may use glycosaminoglycans and/or low density receptors on host cells as initial attachment factors. A unique feature of HCV is the dependence of virus replication and assembly on host cell lipid metabolism. Most notably, during HCV assembly and release from the infected cells, virus particles associate with lipids and very-low-density lipoproteins. Thus, infectious virus circulates in patient sera in the form of triglyceride-rich particles. Consequently, lipoproteins and lipoprotein receptors play an essential role in virus uptake and the initiation of infection. This review summarizes the current knowledge about HCV receptors, mechanisms of HCV cell entry and the role of lipoproteins in this process.

André P, Perlemuter G, Budkowska A, Bréchot C, Lotteau V. · INSERM U503, IFR 128 Biosciences Lyon Gerland, 21 avenue Tony Garnier, 69365 Lyon cedex 07, France. · Semin Liver Dis. · Pubmed #15732001 No free full text.

Abstract: The majority of infectious hepatitis C particles are present in the low-density fractions from plasma of infected patients, suggesting an association of the virus with lipoproteins and the use of lipoprotein receptors for cell entry. Although classical hepatitis C virus (HCV) virions have been reported by some investigators, their role in the HCV life cycle has not been clearly identified. Moreover, two other forms of particles have been characterized: low-density lipo-viro-particles (LVPs) and high-density particles. The latter are nonenveloped nucleocapsids that have immunoglobulin G Fcgamma binding capacity. LVPs are spherical particles enriched in triglycerides. At a minimum, they contain apolipoprotein B, HCV RNA, and core protein. The main source of LVPs is likely to be the enterocytes rather than the hepatocytes, suggesting an interaction between chylomicron and LVP assembly. In experimental systems, HCV core protein inhibits the microsomal triglyceride transfer protein, binds to apolipoprotein AII, and induces accumulation of cytoplasmic lipid droplets. A model of LVP and HCV core-lipid droplet generation is proposed.

Roohvand F, Maillard P, Lavergne JP, Boulant S, Walic M, Andréo U, Goueslain L, Helle F, Mallet A, McLauchlan J, Budkowska A. · Unité des Hépacivirus et Immunité Innée, Institut Pasteur, 25/28 Rue du Dr. Roux, Paris 75724, France. · J Biol Chem. · Pubmed #19269968 No free full text.

Abstract: Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified beta- and alpha-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.

Boulant S, Douglas MW, Moody L, Budkowska A, Targett-Adams P, McLauchlan J. · MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK. · Traffic. · Pubmed #18489704 No free full text.

Abstract: Attachment of hepatitis C virus (HCV) core protein to lipid droplets (LDs) is linked to release of infectious progeny from infected cells. Core progressively coats the entire LD surface from a unique site on the organelle, and this process coincides with LD aggregation around the nucleus. We demonstrate that LD redistribution requires only core protein and is accompanied by reduced abundance of adipocyte differentiation-related protein (ADRP) on LD surfaces. Using small hairpin RNA technology, we show that knock down of ADRP has a similar phenotypic effect on LD redistribution. Hence, ADRP is crucial to maintain a disperse intracellular distribution of LDs. From additional experimental evidence, LDs are associated with microtubules and aggregate principally around the microtubule-organizing centre in HCV-infected cells. Disrupting the microtubule network or microinjecting anti-dynein antibody prevented core-mediated LD redistribution. Moreover, microtubule disruption reduced virus titres, implicating transport networks in virus assembly and release. We propose that the presence of core on LDs favours their movement towards the nucleus, possibly to increase the probability of interaction between sites of HCV RNA replication and virion assembly.

Andréo U, Maillard P, Kalinina O, Walic M, Meurs E, Martinot M, Marcellin P, Budkowska A. · Unité Hépacivirus Institut Pasteur, 25/28 Rue du Dr Roux, 75724 Paris Cedex 15, France. · Cell Microbiol. · Pubmed #17517063 No free full text.

Abstract: The host-virus interactions leading to cell infection with hepatitis C virus (HCV) are not fully understood. The tetraspanin CD-81 and human scavenger receptor SR-BI/Cla1 are major receptors mediating virus cell entry. However, HCV in patients' sera is associated with lipoproteins and infectious potential of the virus depends on lipoproteins associated to virus particles. We show here that lipoprotein lipase (LPL), targeting triglyceride-rich lipoproteins (TRL) to the liver, mediates binding and internalization of HCV to different types of cells, acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate proteoglycans (HSPG). The dimeric structure and catalytic activity of LPL are required for LPL-mediated HCV uptake to cells. Unexpectedly, exogenous LPL significantly inhibits HCVcc infection in vitro. This effect is prevented by anti-LPL antibodies and by tetrahydrolipstatin (THL) a specific inhibitor of LPL enzymatic activity. In addition, we show that antibodies directed to apolipoprotein B (ApoB)-containing lipoproteins efficiently inhibits HCVcc infection. Our findings suggest that LPL mediates HCV cell entry by a mechanism similar to hepatic clearance of TRL from the circulation, promoting a non-productive virus uptake. These data provide new insight into mechanisms of HCV cell entry and suggest that LPL could modulate HCV infectivity in vivo.

Namboodiri AM, Budkowska A, Nietert PJ, Pandey JP. · Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA. · Mol Immunol. · Pubmed #17485114 links to  free full text

Abstract: Immunoglobulin GM allotypes are associated with the outcome of several infections, including hepatitis C virus (HCV) infection, but the underlying mechanisms are not known. HCV employs sophisticated strategies to evade host immunosurveillance. One such strategy might involve the scavenging of the Fc gamma domain of the anti-HCV IgG antibodies by its Fc gamma receptor-like site formed by HCV core protein, potentially interfering with the Fc gamma-mediated host defense mechanisms. We tested the hypothesis that GM allotypes modulate this viral strategy through differential binding to the core protein. Here we show that the absorbance values for binding to the HCV core protein were significantly higher for IgG1 with GM 3 allotype than that for the allelic GM 1,2,17 determinants (p=0.0003). These results provide a mechanistic explanation for the involvement of GM allotypes in the outcome of HCV infection. These findings also shed light on the possible evolutionary selective mechanism that maintains GM polymorphism.

Roohvand F, Aghasadeghi MR, Sadat SM, Budkowska A, Khabiri AR. · Hepatitis and AIDS Department, Pasteur Institute of Iran, Pasteur Ave., Tehran 13164, Iran. · Biochem Biophys Res Commun. · Pubmed #17250802 No free full text.

Abstract: An efficient vaccine against Hepatitis C virus (HCV) infection requires induction of strong humoral and cellular responses against viral proteins. We evaluated the immunogenicity of HCV core protein (HCVcp), a prime vaccine candidate, formulated in various human compatible adjuvants. An Escherichia coli-expressed HCVcp, purified in native conditions was used for murine immunization in separate groups of: free HCVcp (Ag), Ag+C/IFA (Freunds), Ag+CpG, Ag+M720 (Montanide ISA 720), Ag+F127 (Pluronic acid) and cocktails of Ag+F127+CpG and Ag+M720+CpG. Mice immunized with M720(+CpG) developed the highest HCVcp-specific titers of total IgG, IgG1, 2a, 2b, and that of IFN-gamma and IL-4 cytokines compared to all other groups. HCVcp-specific-CTLs against relevant MHC class I peptides were detected only for Ag+M720+CpG, Ag+M720, and Ag+CpG groups and could be blocked by antimouse-CD8 antibodies. While CTLs were stable, only F127 formulated groups demonstrated detectable IgG antibodies one year post-immunization. Hence, HCVcp formulated in M720 (with a synergistic effect by inclusion of CpG) could induce balanced and strong Th1/Th2 responses with long-lived CD4(-)CD8(+) CTLs.

Deng Q, Zhai JW, Michel ML, Zhang J, Qin J, Kong YY, Zhang XX, Budkowska A, Tiollais P, Wang Y, Xie YH. · State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, and Ruijin Hospital, Department of Infectious Diseases, Shanghai, China. · J Virol. · Pubmed #17192308 links to  free full text

Abstract: A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.

Maillard P, Huby T, Andréo U, Moreau M, Chapman J, Budkowska A. · Hepacivirus Unit, Pasteur Institute, Paris, France. · FASEB J. · Pubmed #16476701 links to  free full text

Abstract: The possible role of candidate receptors in the cellular penetration of HCV from serum of infected patients remains unclear. SR-BI/Cla1 interacts with plasma HDL, native and modified LDL, and VLDL, and facilitates cellular cholesterol efflux to lipoprotein acceptors. SR-BI/Cla1 binds HCV E2 protein and interacts with HCV pseudotypes via the HVR1 of the E2 envelope glycoprotein. Our data reveal that functional SR-BI/Cla1 expressed on the surface of CHO cells mediates the binding and uptake of HCV from the sera of infected patients. Interaction between HCV and SR-BI/Cla1 is not sensitive to either anti-E2 or anti-HVR1 antibodies but is effectively inhibited by anti-betalipoprotein antibodies and competed out by apoB-containing lipoproteins and notably by VLDL. We interpret our data to indicate that VLDL associated with or incorporated into HCV plays a critical role in the primary interaction of HCV with SR-BI/Cla1, whereas the HCV E2 protein does not. In addition, our findings in hepatoma cell lines suggest that the interaction of HCV with human hepatocytes is equally mediated, at least in a part, by VLDL, and as such may represent an alternative pathway for infection. The association of HCV with ApoB-containing lipoproteins may promote cellular uptake of this virus in the presence of neutralizing antibodies.

Mavromara P, Sall A, Kalinina O, Horm V, Budkowska A, Anonymous00336. · EA 2689 Université de Lille 2 et Service de réanimation polyvalente, Hôpital Salengro CHRU Lille, 59037 Lille Cedex, France. · Med Mal Infect. · Pubmed #15978361 No free full text.

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Maillard P, Lavergne JP, Sibéril S, Faure G, Roohvand F, Petres S, Teillaud JL, Budkowska A. · Carcinogénèse Hépatique et Virologie Moléculaire, Unité des Venins, and Plateau Génomique Structurale, Institut Pasteur, 75724 Paris cedex 15, France. · J Biol Chem. · Pubmed #14610077 links to  free full text

Abstract: We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.

Pizarro JC, Vulliez-le Normand B, Riottot MM, Budkowska A, Bentley GA. · Unité d' Immunologie Structurale, Institut Pasteur, Paris, France. · FEBS Lett. · Pubmed #11749974 No free full text.

Abstract: The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37-43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ-line genes have been identified. The three-dimensional structure of the Fab fragment has been determined by X-ray crystallography to 2.6 A resolution.

Maillard P, Krawczynski K, Nitkiewicz J, Bronnert C, Sidorkiewicz M, Gounon P, Dubuisson J, Faure G, Crainic R, Budkowska A. · Epidémiolgie Moléculaire des Entérovirus, Institut Pasteur, 75724 Paris, France. · J Virol. · Pubmed #11483769 links to  free full text

Abstract: One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.