Biomedical subjects

Pierre Boulanger

Publications and source records attributed to Pierre Boulanger.

12 recordsLinked to original sources

PubMed2005

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses.

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.

Adenoviridae↗

PubMed2004

Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells.

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.

Adenoviruses, Human↗

PubMed2003

The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain.

We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-stranded region F of the knob (residues Leu-485-Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiDeltaHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in vivo.

Adenoviruses, Human↗

PubMed2003

Spatial navigation in virtual reality environments: an EEG analysis.

Past research has linked theta oscillations (electroencephalographic activity in the 4-8-Hz range) to spatial navigation in rodents and humans, and to the encoding and retrieval of spatial information in rodents. In the present study, electroencephalographic activity was measured while humans navigated through virtual mazes. Results confirmed previous findings that the frequency of theta episodes is directly related to the difficulty of maze navigation. We were also able to show that theta episodes occur most likely at points in a maze where new hallways come into view, or after navigational mistakes have been realized and are being corrected. This indicates that, just as in rodents, theta episodes in humans are related to the encoding and retrieval of spatial information.

Adult↗

PubMed2003

The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1.

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

Amino Acid Sequence↗

PubMed2003

Integrin alpha3beta1 is an alternative cellular receptor for adenovirus serotype 5.

Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of alpha3beta1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin alpha3beta1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-alpha3 and anti-beta1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with alpha3beta1, which involved multiple additional contact sites.

Adenoviruses, Human↗

PubMed2002

A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5.

Here we describe the development of a two-step chromatography process based on the use of ion-exchange resins for the purification of recombinant adeno-associated virus (rAAV) serotypes-2 and-5. In vitro and in vivo results demonstrate that this method, which does not require any prepurification step of the cell lysate, can be applied to obtain highly pure rAAV2 and rAAV5 stocks. As such,this procedure can be easily transferred in vector cores and also scaled up, allowing the direct comparison of these two, and potentially other, AAV serotypes in large animal models.

Animals↗

PubMed2002

The HIV-1 Nef protein enhances the affinity of reverse transcriptase for RNA in vitro.

Several viral proteins, including nucleocapsid protein, integrase, Vif, Tat, and Nef have been proposed to act as cofactors of HIV-1 reverse transcription. Using two viral RNA probes, one overlapping the primer-binding site (PBS) and the other representing the ribosomal frameshifting signal (FS) of HIV-1 RNA, we found that recombinant full-length Nef protein (NefLAI) increased the affinity of reverse transcriptase (RT) for RNA in vitro, and interacted directly with RT in protein co-precipitation assays. The effect on RT-RNA binding and the capacity of Nef to interact with RT was also observed with N-terminal deletion mutant NefDelta57 and NefSF2, although to a lesser level. NefDelta57 corresponded to the processed Nef protein present in the internal core of mature virions, and lacked the N-myristoylated N-terminus and N-terminal region implicated in virus infectivity and pathogenicity in vivo. NefSF2, a Nef allele from a highly pathogenic strain of HIV-1, differed from NefLAI by the amino acid sequence and immunoreactivity of its N-terminal domain. The effect observed with NefSF2 and NefDelta57, and data from phage biopanning experiments suggested that the RT-binding region in Nef involved the C-terminal flexible loop of its C-terminal domain, but the function in RT-RNA binding was also influenced by its N-terminal domain.

Amino Acid Sequence↗

PubMed2002

Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response.

The capacity of recombinant adenoviruses (rAd) to induce immunization against their transgene products has been well documented. In the present study, we evaluated the vaccinal adjuvant role of rAd independently of its vector function. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along with AdE1 degrees (10(9) particles), a first-generation rAd empty vector. Although coinjected with a suboptimal dose of lipopeptides, AdE1 degrees significantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected with AdE1 degrees plus LP6 developed both a polyspecific T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epitopes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted virions. When mice were immunized with LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the results obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen. Our results thus have some important implications not only in vaccinology but also for gene therapy using rAd vectors.

Adenoviridae↗

PubMed2002

Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

Adenoviridae↗

PubMed2001

Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag-Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing.

Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag-Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Deltap6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag-Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag-Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA-CA and CA-NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag-Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag-Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag-Pol precursor.

Animals↗

PubMedDate not supplied

Genetic retargeting of adenovirus vectors: functionality of targeting ligands and their influence on virus viability.

BACKGROUND: We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions. METHODS: Polypeptide ligands of cell surface molecules, including single-chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity. RESULTS: A major limiting factor for recovering viable recombinant Ad5 carrying fiber-fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber-ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number. CONCLUSIONS: Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber-ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors.

Adenoviridae↗