Enveloped particles in the serum of chronic hepatitis C patients

Petit, Marie-Anne; Lièvre, Marjory; Peyrol, S; Sequeira, Sylvie De; Berthillon, Pascale; Ruigrok, Rob W.H.; Trépo, Christian

doi:10.1016/j.virol.2005.03.023

Cited by 32 publications

(50 citation statements)

References 33 publications

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“…Immunization with peptides corresponding to HVR-1 can lead to the generation of sera capable of protection against homologous virus (25), and therefore induction of AP33-type antibodies, at titers capable of inducing neutralization, should be possible. That said, studies with the broadly neutralizing human immunodeficiency virus type 1 antibodies 2G12, b12, and 2F5 show that protection from in vivo challenge might require higher concentrations of antibody than their in vitro IC 50 (52). However, many factors affect in vivo challenge and in vitro neutralization results.…”

Section: Discussionmentioning

confidence: 99%

“…HCV particles in infected patients are associated with plasma lipoprotein (3), which may mask epitope availability, although antibodies to HVR-1 are clearly able to neutralize infection in chimpanzees (25). Furthermore, Petit et al (50) more recently demonstrated that native virions have at least some E1E2 epitopes surface exposed. Nevertheless, it will be important to define whether or not MAb AP33 is indeed capable of neutralizing infection by patient-derived virions and, if so, whether passive transfer of this antibody can protect in animal model challenge experiments.…”

Section: Discussionmentioning

confidence: 99%

“…The proportion of infected cells was determined by measurement of GFP by fluorescence-activated cell sorting as described above. The neutralizing activity was expressed as the IC 50 , defined as the concentration of antibody required to achieve 50% inhibition of infection.…”

Section: Methodsmentioning

confidence: 99%

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Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

Owsianka

Tarr

Juttla

et al. 2005

J Virol

254

305

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Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein.Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 g/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.Hepatitis C virus (HCV), a positive-strand RNA virus belonging to the Flaviviridae family, is the major cause of non-A, non-B viral hepatitis. HCV has infected approximately 200 million people worldwide and current estimates suggest that as many as 3 million individuals are newly infected each year (4). Approximately 80% of those infected fail to clear the virus; a chronic infection ensues, frequently leading to severe chronic liver disease, cirrhosis, and hepatocellular carcinoma (2, 41). Current treatments for chronic infection are ineffective for approximately 50% of patients, and there is a pressing need to develop preventative and therapeutic vaccines.Due to the error-prone nature of the RNA-dependent RNA polymerase and the high replicative rate in vivo (30, 46), HCV exhibits a high degree of genetic variability. Crucially, this propensity for genetic change allows the virus to respond to and overcome a variety of selective pressures, including host immunity and antiviral therapy (18,26,37,44,53). HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nucleotide level, respectively (59, 61). A significant challenge for the development of vaccines will lie in identifying protective epitopes that are conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope proteins, the natural targets for the neutralizing response, are two of the most variable proteins (10).The envelope proteins E1 and E2 are responsible for cell binding and entry (5,8,16,51,57). They are N-linked glycosylated (23,31,43,62) transmembrane proteins with a Nterminal ectodomain and a C-terminal hydrophobic membrane anchor (12,21,22). In vitro ex...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

Owsianka

Tarr

Juttla

et al. 2005

J Virol

254

305

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“…Core protein resolved from the liver or serum of HCV-infected patients was reported to have an apparent molecular mass comparable with that of p21 expressed in tissue culture cells, leading to the plausible suggestion that the HCV virion capsid is composed of the SPP-processed form of core protein (21,52). Isolation and characterization of HCV virions have proven to be difficult, however, and a higher molecular mass was reported for core protein recovered from the circulating blood of HCV-infected patients in other studies (48,53). Moreover, the efficiency of core protein processing by SPP has recently been suggested to differ among HCV genotypes (54).…”

Section: Discussionmentioning

confidence: 99%

Signal Peptide Peptidase-catalyzed Cleavage of Hepatitis C Virus Core Protein Is Dispensable for Virus Budding but Destabilizes the Viral Capsid

Vauloup‐Fellous

Pène

Garaud-Aunis

et al. 2006

Journal of Biological Chemistry

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The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell. Signal peptide peptidase (SPP)5 belongs to a novel family of polytopic membrane-associated aspartyl proteases that catalyze cleavage of peptide bonds within the hydrophobic environment of a lipid bilayer (for review see Ref. 1). SPP is highly conserved in higher eucaryotes from plants to humans, including insects (2, 3). This membrane protein with nine proposed transmembrane domains exerts its action in the endoplasmic reticulum (ER), where it resides by virtue of a classical ER retrieval signal (3-5). The SPP substrates identified so far have in common a type II (N terminus facing the cytosol and C terminus facing the lumen) orientation but do not unequivocally point to a common role for SPP activity. In humans, SPP carries out the intramembrane cleavage of signal peptides of polymorphic major histocompatibility complex class I molecules after they have been cleaved off by signal peptidase; the short N-terminal signal peptide remnants then serve as epitopes presented at the cell surface by nonpolymorphic human lymphocyte antigen-E molecules for immune surveillance (6). Thus, SPP activity may promote the liberation from the ER lipid bilayer of some bioactive signal peptide fragments. Another role of SPP activity may be to protect cells from the toxicity of some signal peptides, e.g. the signal peptide of eosinophil cationic protein (7). Finally, the substrate spectrum of SPP may not be restricted to signal peptides but e...

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“…HCV RNA-containing particles have also been recovered after treatment with deoxycholic acid and NP-40, to remove lipid, and characterized by rate-zonal centrifugation, to determine the sedimentation coefficient and particle size. A panel of antibodies to host lipoproteins and viral glycoproteins was used to identify host and viral proteins exposed on the surfaces of HCV particles from serum and thus to contribute knowledge about ultrastructural features of the HCV lipoviroparticle (40,57).…”

mentioning

confidence: 99%

Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients

Nielsen¹,

Bassendine²,

Burt

et al. 2006

J Virol

308

324

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Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, <1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.

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Enveloped particles in the serum of chronic hepatitis C patients

Cited by 32 publications

References 33 publications

Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein

Signal Peptide Peptidase-catalyzed Cleavage of Hepatitis C Virus Core Protein Is Dispensable for Virus Budding but Destabilizes the Viral Capsid

Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients

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