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ABSTRACT: CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun). The two resulting chimeric proteins, DIII-DENV1-RIgE and DIII-WNVKun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55GagHIV in insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens.
Virology Journal 04/2013; 10(1):129. · 2.34 Impact Factor
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03/2012; , ISBN: 978-953-51-0287-8
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ABSTRACT: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.
A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 μM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway.
The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.
Retrovirology 02/2012; 9:17. · 6.47 Impact Factor
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ABSTRACT: Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.
PLoS ONE 01/2012; 7(12):e52326. · 4.09 Impact Factor
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ABSTRACT: Tumor cells can escape the immune system by overexpressing molecules of the B7 family, e.g. B7-H1 (PD-L1 or CD86), which suppresses the anti-tumor T-cell responses through binding to the PD-1 receptor, and similarly for B7.1 (CD80), through binding to CTLA-4. Moreover, direct interactions between B7-H1 and B7.1 molecules are also likely to participate in the immunoevasion mechanism. In this study, we used a mouse model of tumor dormancy, DA1-3b leukemia cells. We previously showed that a minor population of DA1-3b cells persists in equilibrium with the immune system for long periods of time, and that the levels of surface expression of B7-H1 and B7.1 molecules correlates with the dormancy time. We found that leukemia cells DA1-3b/d365 cells, which derived from long-term dormant tumors and overexpressed B7-H1 and B7.1 molecules, were highly permissive to Ad5FB4, a human adenovirus serotype 5 (Ad5) vector pseudotyped with chimeric human-bovine fibers. Both B7-H1 and B7.1 were required for Ad5FB4-cell binding and entry, since (i) siRNA silencing of one or the other B7 gene transcript resulted in a net decrease in the cell binding and Ad5FB4-mediated transduction of DA1-3b/d365; and (ii) plasmid-directed expression of B7.1 and B7-H1 proteins conferred to Ad5FB4-refractory human cells a full permissiveness to this vector. Binding data and flow cytometry analysis suggested that B7.1 and B7-H1 molecules played different roles in Ad5FB4-mediated transduction of DA1-3b/d365, with B7.1 involved in cell attachment of Ad5FB4, and B7-H1 in Ad5FB4 internalization. BRET analysis showed that B7.1 and B7-H1 formed heterodimeric complexes at the cell surface, and that Ad5FB4 penton, the viral capsomere carrying the fiber projection, could negatively interfere with the formation of B7.1/B7-H1 heterodimers, or modify their conformation. As interactors of B7-H1/B7.1 molecules, Ad5FB4 particles and/or their penton capsomeres represent potential therapeutic agents targeting cancer cells that had developed immunoevasion mechanisms.
Molecular Cancer 08/2011; 10:105. · 3.99 Impact Factor
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ABSTRACT: The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.
PLoS ONE 01/2011; 6(11):e27234. · 4.09 Impact Factor
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ABSTRACT: Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.
PLoS ONE 01/2011; 6(5):e18205. · 4.09 Impact Factor
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ABSTRACT: Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.
Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.
Expression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.
BMC Biotechnology 01/2010; 10:80. · 2.35 Impact Factor
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Taco G Uil,
Jeroen de Vrij,
Jort Vellinga,
Martijn J W E Rabelink,
Steve J Cramer,
On Ying A Chan,
Margherita Pugnali,
Maria Magnusson,
Leif Lindholm, Pierre Boulanger,
Rob C Hoeben
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ABSTRACT: Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context.
Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers.
In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent.
LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands.
The Journal of Gene Medicine 09/2009; 11(11):990-1004. · 2.48 Impact Factor
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Ophélia Granio,
Katherine J D Ashbourne Excoffon,
Petra Henning,
Patricia Melin,
Caroline Norez,
Gaëlle Gonzalez,
Philip H Karp,
Maria K Magnusson,
Nagy Habib,
Leif Lindholm,
Frédéric Becq, Pierre Boulanger,
Joseph Zabner,
Saw-See Hong
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ABSTRACT: In vivo gene transfer to the human respiratory tract by adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response, and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on CFTR function. This negative interference did not result from a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype that uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared with Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from patients with CF (CF-HAE) via the apical surface, restored chloride channel function at relatively low vector doses, and showed relatively stable expression of GFP-CFTR for several weeks.
Human gene therapy 09/2009; 21(3):251-69. · 4.20 Impact Factor
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Ophélia Granio,
Marine Porcherot,
Stéphanie Corjon,
Kuntida Kitidee,
Petra Henning,
Assia Eljaafari,
Andrea Cimarelli,
Leif Lindholm,
Pierre Miossec, Pierre Boulanger,
Saw-See Hong
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ABSTRACT: Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.
Journal of Virology 05/2009; 83(12):6048-66. · 5.40 Impact Factor
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ABSTRACT: DSB, the 3-O-(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.
Wild-type Vif (Vifwt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.
The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.
Virology Journal 01/2009; 5:162. · 2.34 Impact Factor
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ABSTRACT: Adenovirus serotype 5 protein IX (pIX) has two domains connected by a flexible linker. Three N-terminal domains form triskelions on the capsid facets that cement hexons together, and the C-terminal domains of four monomers form complexes toward the facet periphery. Here we present a cryoelectron microscopy structure of recombinant adenovirus with a peptide tag added to the C terminus of pIX. The structure, made up by several C termini of pIX, is longer at both ends than the wild-type protein, and Fabs directed against the tag bind to both ends of the oligomer, demonstrating that the pIX C termini associate in an antiparallel manner.
Journal of Virology 12/2008; 83(2):1135-9. · 5.40 Impact Factor
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Laure Franqueville,
Petra Henning,
Maria Magnusson,
Emmanuelle Vigne,
Guy Schoehn,
Maria E Blair-Zajdel,
Nagy Habib,
Leif Lindholm,
G Eric Blair,
Saw See Hong, Pierre Boulanger
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ABSTRACT: Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.
PLoS ONE 02/2008; 3(8):e2894. · 4.09 Impact Factor
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ABSTRACT: The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies.
Our data suggested that the C-terminal domain of EED was folded as a seven-bladed beta-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique alpha-helix in the fourth beta-blade. More importantly, the position of flexible loops and accessible beta-strands on the beta-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the beta-propeller.
Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed beta-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the beta-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible.
Virology Journal 02/2008; 5:32. · 2.34 Impact Factor
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ABSTRACT: Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.
American Journal of Respiratory Cell and Molecular Biology 01/2008; 37(6):631-9. · 5.13 Impact Factor
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ABSTRACT: Abstract
Background
DSB, the 3- O -(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.
Results
Wild-type Vif (Vif<sup>wt</sup>) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H<sup>108</sup>C<sup>114</sup>C<sup>133</sup>H<sup>139 </sup>coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vif<sup>wt </sup>showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.
Conclusion
The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vif<sup>wt</sup>, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.
Virology Journal. 01/2008;
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ABSTRACT: Abstract
Background
The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies.
Results
Our data suggested that the C-terminal domain of EED was folded as a seven-bladed β-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique α-helix in the fourth β-blade. More importantly, the position of flexible loops and accessible β-strands on the β-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the β-propeller.
Conclusion
Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed β-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the β-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible.
Virology Journal. 01/2008;
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ABSTRACT: Transient overexpression of genes involved in lung regulation might prevent alveolar developmental disorders (ADDs) in premature neonates. However, adenovirus 5 (Ad5) vectors per se, and not isolated capsid proteins, induce ADDs after tracheal administration to newborn rats. To test the hypothesis that Ad5 capsid components are mainly responsible for ADDs, we evaluated newborn rats' lung development by morphometry after tracheal administration of a panel of Ad5 vectors with mutations in the fiber or penton base. Three distinct patterns of lung response were observed on postnatal day (PD) 21: (i) emphysematous-like lesions, common to Ad5 overexposing RGD motifs; (ii) altered septation, representative of the wild-type capsid Ad5 lesion; (iii) absence of lung toxicity, shown by Ad5 vectors with fibers shortened to seven repeats. None of these patterns correlated with the degree of lung inflammation or gene transduction. In contrast, a more impaired elastogenesis associated with emphysema was preceded by a significantly increased level of activated caspase 3 on PD11. Moreover, the altered septation was associated with a persistent and significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive alveolar septal cells on PD21. Our results underline the deleterious effects of Ad-induced apoptosis, which is not only responsible for limited transgene expression but also involved in lung development disorders.
Molecular Therapy 12/2007; 15(11):2008-16. · 6.87 Impact Factor
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ABSTRACT: The human EED protein, a member of the superfamily of Polycomb group (PcG) proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA), the integrase enzyme (IN) and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells.
During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefDelta57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefDelta57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm.
Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic RNA packaging and virus assembly, resulting possibly from a mistrafficking of viral genomic RNA (gRNA) or gRNA/Gag complex. Nef reversed the EED negative effect on virus production, a function which required the integrity of the Nef N-terminal domain, but not its N-myristoyl group. The antagonistic effect of Nef correlated with a cellular redistribution of both EED and Nef.
Retrovirology 02/2007; 4:37. · 6.47 Impact Factor
