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P Roingeard

Publications and source records attributed to P Roingeard.

At least 19 recordsLinked to original sources
PubMed2007

The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model.

BACKGROUND AND AIMS: The prevalence and severity of liver steatosis are higher in patients infected with genotype 3 hepatitis C virus (HCV) than in patients infected with other genotypes. HCV core protein is known to affect lipid metabolism, inducing lipid droplet accumulation both in vitro and in vivo. An in vitro cellular model was used to investigate whether an HCV core protein with residues specific to genotype 3 increased this phenomenon. METHODS: Sequence comparisons for HCV core protein domain II, which is known to interact with lipid droplets, identified the phenylalanine (F) residue at position 164 as the only residue specific to genotype 3. The area covered by lipid droplets in sections of cells producing a wild-type genotype 1a HCV core protein was compared with that in cells producing a Y164F mutant protein. RESULTS: Cumulative lipid droplet area was significantly greater in sections of cells producing the Y164F mutant HCV core protein than in cells producing the wild-type protein (p<0.001). The frequency of cell sections containing more than 3 mum(2) of lipid droplets, in particular, was higher for the mutant than for the wild-type protein. CONCLUSION: The data provide a molecular explanation for HCV genotype 3-specific lipid accumulation. This difference between genotypes may be due to phenylalanine having a higher affinity for lipids than tyrosine (Y). These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis.

Animals↗
PubMed2001

The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

Cell Membrane↗
PubMed2000

Identification of the glycoprotein 41(TM) cytoplasmic tail domains of human immunodeficiency virus type 1 that interact with Pr55Gag particles.

We investigated the protein/protein interactions that occur during human immunodeficiency virus (HIV-1) budding. We evaluated the binding to Pr55Gag particles of peptides mapping to the cytoplasmic tail of gp41TM and of host-cell proteins, in a cell-free, in vitro assay. Host-cell proteins and irrelevant viral envelope peptides did not bind. Peptides corresponding to a large central domain of the gp41TM cytoplasmic tail (93 residues) bound to Pr55Gag particles. This demonstrates that a Gag/Env interaction is responsible for the specific incorporation of the Env glycoprotein into nascent HIV-1 virions, and defines more accurately the gp41TM domain involved in this interaction.

Amino Acid Sequence↗
PubMed2000

Antigenic properties of recombinant envelope glycoproteins derived from T-cell-line-adapted isolates or primary human immunodeficiency virus isolates and their relationship to immunogenicity.

The native envelope glycoproteins of primary HIV-1 virions have weaker antigenicity than do T-cell laboratory-adapted (TCLA) viruses. These antigenic properties require further evaluation if recombinant envelope glycoproteins are produced as part of a vaccine strategy. In this study, we compared the antigenicity of recombinant envelope glycoproteins derived from three primary isolates (PI) (HIV-1(BX08), HIV-1(CHA), and HIV-1(133)) and two TCLA viruses (HIV-1(HXB2) and HIV-1(MN)) produced using the Semliki Forest virus (SFV) system. This analysis was performed by radioimmunoprecipitation assays and flow cytometry. The results suggest that the SFV produces envelope glycoproteins with features in common with the envelopes found in naturally occurring virions. In particular, the PI envelopes had weak heterogeneous antigenic properties. However, the cytometric analysis also showed that there was less envelope glycoprotein on the cell surface for the PI envelopes than for those of TCLA viruses, suggesting differences in their intracellular trafficking. The immunogenic properties of the various envelope glycoproteins were evaluated in mice using recombinant SFV particles as vaccine vectors. The PI envelopes were less immunogenic than the TCLA envelopes, probably due to both their low antigenicity and cell surface expression level. Thus, it may be difficult to design an effective vaccine based on native recombinant PI envelopes.

Adaptation, Physiological↗
PubMed2000

DNA-containing and empty hepatitis B virus core particles bind similarly to envelope protein domains.

DNA synthesis within the hepatitis B virus (HBV) nucleocapsid appears to be coupled to nucleocapsid envelopment. The nature of the envelopment signal is unknown, but is thought to involve a conformational change at the surface of the capsid that facilitates interaction with HBV envelope proteins. In binding assays in vitro, it was found that empty HBV core particles bound synthetic peptides corresponding to HBV envelope protein domains with the same affinity as did HBV DNA-containing core particles. This suggests that the selection of replication-competent nucleocapsids for envelopment is not related to the capacity of DNA-containing core particles to bind specifically to HBV envelope proteins, and that there must be an alternative mechanism.

Capsid↗
PubMed1998

Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis.

The intracellular accumulation of empty hepatitis B virus (HBV) particles of filamentous shape leads to a direct cytopathic effect in so-called ground-glass hepatocytes. The aim of this study was to investigate how these filaments can be structurally formed at the cellular level. By electron microscopy, we reexamined the HBV-producer HepG2T-14 cells, which have been described as producing a substantial amount of empty HBV filaments compared with the other forms of HBV particles. Examination of ultrathin sections of HepG2T14 cells revealed the presence of HBV virions and filaments at the periphery of extremely large intracellular cisternae, probably related to a pre-Golgi compartment. Very long filaments appeared to be formed by a tubular budding of a long portion of the cisterna membrane. This phenomenon may be identical to that observed in the hepatocytes of HBV chronic carriers, in which the inability of the infected cell to export long HBV filamentous particles through the cellular secretion pathway seems to be at the origin of a direct cytopathic effect.

Actin Cytoskeleton↗
PubMed1998

Ultrastructural and physicochemical characterization of the hepatitis C virus recovered from the serum of an agammaglobulinemic patient.

Hepatitis C virus (HCV) morphology and physicochemical properties remain unclear because HCV usually circulates in a complexed form in association with immunoglobulins. In the present work, we were interested in the characterization of HCV particles derived from the serum of an anti-HCV negative/HCV RNA positive agammaglobulinemic patient suffering from chronic type C hepatitis. Physicochemical properties of the virus particles were determined by serum centrifugation on a 10-60% isopycnic sucrose density gradient. HCV RNA quantified by bDNA was found in a major peak at density 1.13 g/ml and in a minor peak at densities 1.05-1.07 g/ml. By electron microscopy, 45 nm large core-like particles were found at the 1.13 g/ml density while 60 nm large virus-like particles similar to other members of the Flaviviridae family were visualized at the 1.06-1.07 g/ml densities. This confirms some studies reporting the low density of HCV as compared to other members of the Flaviviridae family.

Agammaglobulinemia↗
PubMed1998

Interaction between hepatitis delta virus-encoded proteins and hepatitis B virus envelope protein domains.

Hepatitis delta virus (HDV) packaging requires prenylation of the HDV large protein (p27), as well as a direct protein-protein interaction between HDV proteins and hepatitis B virus (HBV) envelope protein domains. To investigate this interaction, we have analysed the binding capacity of baculovirus-expressed delta p24 and p27 proteins to synthetic peptides specific for the HBV envelope. Although a higher degree of binding was observed with p27, both p24 and p27 could bind HBV envelope peptides. One such peptide corresponded to residues 56-80 located in the cytosolic loop of the small HBV envelope protein, and another corresponded to 23 carboxy-terminal residues of the pre-S1 specific to the large HBV envelope protein. This indicates that in addition to p27, p24 may contribute to packaging of HDV through a protein-protein interaction with HBV envelope domains, and that an interaction between the pre-S1 polypeptide and delta proteins may play a role in infectivity.

Amino Acid Sequence↗
PubMed1997

Both pre-S1 and S domains of hepatitis B virus envelope proteins interact with the core particle.

The three envelope proteins of the hepatitis B virus (HBV) are encoded by a single open reading frame in the genome containing three separate in-phase AUG codons. This organization defines three protein domains (pre-S1, pre-S2, S) which form the small (S), middle (M, pre-S2/S), and large (L, pre-S1 /pre-S2/S) proteins. Mature virions are generated by the budding of preformed nucleocapsids through endoplasmic reticulum (ER) membranes containing S and L proteins, whereas the M protein is not necessary. This suggests an important function for the pre-S1 domain. To investigate the protein-protein interactions involved during the maturation process of the HBV virion, we studied in vitro the binding affinity to purified HBV core particles of various synthetic peptides identical to regions of the envelope proteins. Data previously obtained with deletion mutants were confirmed and refined. The 13 C-terminal amino acids of pre-S1 bound efficiently to core particles, whereas other pre-S domains did not. Moreover, the amino acid sequence 56-80 in the cytosolic loop of S bound efficiently to the HBV core. This double interaction between the HBV capside and both S and pre-S1 domains may be required for virion morphogenesis.

Amino Acid Sequence↗
PubMed1997

Comparison of antibody responses to different forms of HIV-1 core antigens by epitope mapping.

The specificity of antibodies to HIV-1 capsid (p24CA) and matrix (p17MA) proteins, produced in mice against unprocessed immature assembled polyprotein (wild-type p55 virus-like particles or chimeric p55 virus-like particles) or against the monomeric mature form (rp24CA/rp17MA), was analyzed by a microplate epitope mapping assay using a panel of synthetic peptides covering the entire p24CA plus p17MA sequences of HIV-1LAI. All immunized mice developed anti-p24CA and anti-p17MA antibodies, although the spectrum of specificity of these antibodies was different. Four p24 CA epitopes (residues 176-192, 201-218, 233-253, 285-304) were recognized by anti-rp24CA/rp17MA antibodies, whereas one p17MA epitope (residues 11-25) and one p24CA epitope (residues 176-192) were constantly recognized by anti-p55 virus-like particle antibodies. These results suggest a different specificity pattern of anti-p24CA and anti-p17MA antibodies depending on whether they are produced against the soluble mature form or the immature assembled form of the gag proteins.

Amino Acid Sequence↗
PubMed1996

Assembly and immunogenicity of chimeric Gag-Env proteins derived from the human immunodeficiency virus type 1.

We evaluated the potential of the precursor Gag protein (Pr55) of the human immunodeficiency virus type 1 (HIV-1) as a carrier for the presentation of envelope epitopes. Recombinant chimeric core-envelope protein-expressing constructs were derived by deletion of regions within the gag gene, especially of regions encoding p24 capsid epitopes. Sequences encoding either the principal neutralization determinant (PND) and/or the CD4-binding domains (CD4BS) were then inserted. Deletion of residues 196-226 within the p24 capsid protein did not prevent self-assembly into virus-like particles (VLPs) whereas deletion of residues 299-328 completely abolished VLP formation. Thus the major homology region (MHR) and proximal sequences are required for capsid assembly. An immunization study in mice showed that assembled chimeric proteins elicited strong anti-Gag, weak anti-envelope, and no neutralizing humoral responses. Nonassembled chimeric proteins were poor immunogens. Mapping of Pr55 antigenic sites using sera from immunized mice and peptides overlapping the entire Gag precursor showed that p24 capsid and p17 matrix epitopes presented to the immune system differed from the mature form (p24 or p17) and the multimeric immature form (Pr55).

Amino Acid Sequence↗
PubMed1995

Quantitative analysis of immunogold labellings of collagen types I, III, IV and VI in healthy and pathological human corneas.

BACKGROUND: We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. METHODS: Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. RESULTS: Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. CONCLUSION: The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.

Adult↗
PubMed1995

A simple procedure to generate chimeric Pr55gag virus-like particles expressing the principal neutralization domain of human immunodeficiency virus type 1.

The Pr55gag human immunodeficiency virus type 1 (HIV-1) precursor protein that is capable of auto-assembling was used as a carrier for a consensus sequence of the principal neutralization domain (PND) of the HIV-1 envelope. For this purpose, a modified HIV-1 gag gene with deletion of the sequence encoding a previously described p24 epitope (amino acids 196-228 of Pr55gag) was first obtained using PCR with degenerate primers, and then cloned. This deleted gag gene allowed in a second time the insertion of a synthetic oligonucleotide cassette encoding the North American/European consensus PND precisely in place of the p24 epitope. The chimeric gene was then inserted into a baculovirus transfer vector and expressed in insect cells. The construct formed 100-140 nm virus-like particles that were released into the extracellular medium. The use of a serum-free medium that supports growth of insect cells facilitated the downstream purification of the extracellular particles. The chimeric particles were recognized by monoclonal antibodies directed to V3 by Western blot but not by immune electron microscopy, suggesting that, although the inserted sequence was still antigenic it was not exposed at the surface of the particles. The results show the ability of Pr55gag to serve as a carrier for easy insertion, in a precisely defined region, of selected epitopes of gp120 surface envelope protein, and to still auto-assemble in virus-like particles. However, the data indicate that exposed epitopes of the mature p24 protein are not presented similarly in the Pr55 precursor, and therefore that different constructs with various insertions in different places must be generated. Such constructs offer an attractive approach for HIV vaccine development and will need evaluation for both antigenicity and immunogenicity.

Amino Acid Sequence↗
PubMed1995

Direct investigation of protein RNA-binding domains using digoxigenin-labelled RNAs and synthetic peptides: application to the hepatitis delta antigen.

A new method is described for the characterization of RNA binding domains of a protein and applied to the study of the interaction between proteins and nucleic acid of the human hepatitis delta virus (HDV). The method uses synthetic peptides coated onto an ELISA plate and tested for their ability to bind digoxigenin-labelled RNAs. RNA binding is quantified with peroxidase-conjugated anti-digoxigenin. The hepatitis delta antigen (HDAg) is an RNA-binding protein that specifically binds HDV RNAs. In a previous study, it was shown that HDAg sequences corresponding to residues 2-27 and 79-107 bound to both genomic and antigenomic strands. Further investigations are reported on HDAg/HDV RNA binding, using additional HDAg peptides and the full-length HDV genomic and antigenomic strands. In order to validate the method, the efficiency of peptide coating onto the ELISA plate was assessed with human antibodies against HDAg. The two arginine-rich motifs potentially involved in the RNA-binding activity (97-107 and 136-146) were explored and the residues 2-27 and 79-211 were mapped using synthetic peptides. Only peptides corresponding to residues 2-17, 2-27, 79-107 and 84-126 of the HDAg bound to the genomic and antigenomic strands. The second arginine-rich motif represented by peptides 130-144 and 128-152 did not bind to HDV RNAs in this assay. This second arginine-rich domain may be involved in this interaction without a direct ability to bind HDV RNAs.

Amino Acid Sequence↗