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Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells.

Viruses of the Potyviridae family have VPg protein covalently attached to the 5’ end of their linear RNA genome. The protein interacts with the host translation initiation factor eIF4E that occurs in plant cells in two isoforms, one being the preferable target of a given VPg, the remaining one still acting in host protein synthesis. In animal cells only one form of eIF4E is directly involved in protein synthesis. The human eIF4E is known to be an oncogene; elevated expression of eIF4E leads to oncogenic transformation, cancers in animal models and poor prognosis in human cancers, while reduction of the eIF4E level can reverse the transformed phenotype. We show that VPg protein delivery to cells containing only one eIF4E isoform involved in protein synthesis resulted in immobilization of eIF4E in the cytoplasm. The region of VPg involved in the interaction with eIF4E has been partially identified. Peptides derived from this region interacted better with eIF4E than complete VPg protein. Here we characterized one of VPg peptides, VPg5 and we show that VPg5 delivered to colon carcinoma HCT116 cells is able to inhibit cell growth, which is accompanied by reduction in eIF4E level.List of abbreviationsAcMNPVAutographa californica multicapsid nucleopolyhedrovirusCBBCoomassie Brilliant BlueClYVVClover yellow vein viruseIF4Eeukaryotic initiation translation factor 4E4EBP, eIF4E binding proteinFCSfetal calf serumLMVLettuce Mosaic VirusMOImultiplicity of infectionMWmolecular weightONovernightTMB3,3,5,5-tetramethylbenzidineT. niTrichoplusia ni cellsPBSTPBS buffer containing 0.05% Tween-20pipost infectionPIpropidium iodideTEVtobacco etch virusTVMVtobacco vein mottling virusPVYpotato virus YVPggenome-linked viral protein

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth.

One of the major factors limiting the effectiveness of cancer chemotherapy is inefficient drug delivery. Systems enabling efficient delivery and enhanced intracellular uptake appear particularly promising in this respect. Virus-like particle, adenoviral dodecahedron (Dd), employs receptor-mediated endocytosis for cell penetration and is able to deliver intracellularly dozens of cargo molecules attached to one particle. We focused on studying Dd properties in the context of cancer treatment, showing that intratumoral injection of Dd, assessed in mouse xenograft model, results in vector accumulation in tumor without spreading in off-target organs. Moreover, we demonstrated that Dd is a promising vector targeting leukocytes and drug-resistant cancer cells. Dd uptake by human blood cells analyzed in vitro indicated the preference for leukocytes in comparison to red blood cells and platelets. Furthermore, internalization of Dd-doxorubicin conjugate by drug-resistant cells leads to increased nuclear accumulation of doxorubicin and significant enhancement of cytotoxicity against target cancer cells.

Virus-like particles (VLPs) are multimeric, sometimes multiprotein nanostructures assembled from viral structural proteins. They carry no replicative genetic information and can be produced in heterologous expression systems on a large scale. VLPs present repetitive high-density displays of viral surface proteins. Importantly, they contain functional viral proteins responsible for cell penetration of the virus, ensuring efficient cell entry and, thus, tissue-specific targeting characteristic of the virus of origin. The foremost application of VLPs is in vaccinology, where they provide delivery systems that combine good safety profiles with strong immunogenicity and constitute a safe alternative to inactivated infectious viruses. These stable and versatile nanoparticles display excellent adjuvant properties capable of inducing innate and cognate immune responses, resulting in the control of pathogenic microorganisms. Two VLP vaccines are already in use and several others will soon follow. More audacious utilization of VLPs properties is for drug delivery, whereby their vectoring properties are harnessed. In this approach advantage is taken of the remarkably efficient cell entry of VLPs, their repetitive surfaces providing polyvalency, and sometimes their native affinity conceivably ensuring specific targeting. However, such utilization of VLPs is in their infancy and has yet to be implemented in clinics.

In the context of value added valorization of hemicelluloses (HCs), their soft extraction by autohydrolysis (AH) of softwood (SW) chips has been optimized via the temperature/time parameters (170°C/2 h, 170°C/1 h and 150°C/1 h). Two enzyme mixtures containing mainly a glucanase and a mannanase were used to decrease the degree of polymerization (DP) of the extracted HCs. Hydrolysates containing HCs were analyzed in terms of monomers and oligomers, molecular weight distribution (MWD) and chemical composition. The MW was strongly dependent on AH conditions: most of the water-soluble HCs with 1800 Da MW were obtained at 150°C/1 h. The parameters 170°C/2 h gave rise to MWs

Virus-like particles (VLPs) assemble spontaneously during the viral cycle or in heterologous systems during expression of viral structural protein. Depending on the complexity of the VLPs, they can be obtained by expression in prokaryotic or eukaryotic expression system from the suitable recombinant vectors, or formed in cell-free conditions. Moreover, they can be built from proteins of a single virus, or can present the proteins or peptides derived from a virus or cell on a platform derived from any other single virus, thus forming chimeric VLPs. VLPs are best known for their immunogenic properties, but the versatility of VLPs allows a wide variety of applications. They are lately in the centre of investigations in vaccinology, drug delivery and gene therapy. This review focuses on utilization of VLPs for drug delivery.

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102-1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed.

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.

The sub-viral dodecahedral particle of human adenovirus type 3, composed of the viral penton base and fiber proteins, shares an important characteristic of the entire virus: it can attach to cells and penetrate them. Structure determination of the fiberless dodecahedron by cryo-electron microscopy to 9 Angstroms resolution reveals tightly bound pentamer subunits, with only minimal interfaces between penton bases stabilizing the fragile dodecahedron. The internal cavity of the dodecahedron is approximately 80 Angstroms in diameter, and the interior surface is accessible to solvent through perforations of approximately 20 Angstroms diameter between the pentamer towers. We observe weak density beneath pentamers that we attribute to a penton base peptide including residues 38-48. The intact amino-terminal domain appears to interfere with pentamer-pentamer interactions and its absence by mutation or proteolysis is essential for dodecamer assembly. Differences between the 9 Angstroms dodecahedron structure and the adenovirus serotype 2 (Ad2) crystallographic model correlate closely with differences in sequence. The 3D structure of the dodecahedron including fibers at 16 Angstroms resolution reveals extra density on the top of the penton base that can be attributed to the fiber N terminus. The fiber itself exhibits striations that correlate with features of the atomic structure of the partial Ad2 fiber and that represent a repeat motif present in the amino acid sequence. These new observations offer important insights into particle assembly and stability, as well as the practicality of using the dodecahedron in targeted drug delivery. The structural work provides a sound basis for manipulating the properties of this particle and thereby enhancing its value for such therapeutic use.

The use of cytotoxic agents to eliminate cancer cells is limited because of their nonselective toxicity and unwanted side effects. One of the strategies to overcome these limitations is to use latent prodrugs that become toxic in situ after being enzymatically activated in target cells. In this work we describe a method for producing tumor-specific toxins by using a ubiquitin fusion technique. The method is illustrated by the production of recombinant toxins by in-frame fusion of ubiquitin to saporin, a toxin from the plant Saponaria officinalis. Ubiquitin-fused toxins were rapidly degraded via the ubiquitin-proteasome system, significantly reducing their nonspecific toxicity. The insertion of the protease-cleavage sequence between ubiquitin and saporin led to the removal of ubiquitin by the protease and resulted in protease-dependent stabilization of the toxin. We engineered toxins that can be stabilized by specific proteases such as deubiquitinating enzymes and prostate-specific antigen (PSA). Both constructs were activated in vitro and in cultured cells by the appropriate enzyme. Processing by the protease resulted in a greater than 10-fold increase in the toxicity of these constructs. Importantly, the PSA-cleavable toxin was able to kill specifically the PSA-producing prostate cancer cells. The ubiquitin fusion technique is thus a versatile and reliable method for obtaining selective cytotoxic agents and can easily be adapted for different kinds of toxins and activating proteases.

Ubiquitylation, the modification of cellular proteins by the covalent attachment of ubiquitin, is critical for diverse biological processes including cell cycle progression, signal transduction and stress response. This process can be reversed and regulated by a group of proteases called deubiquitylating enzymes (DUBs). Otubains are a recently identified family of DUBs that belong to the ovarian tumour (OTU) superfamily of proteins. Here, we report the first crystal structure of an OTU superfamily protein, otubain 2, at 2.1 A resolution and propose a model for otubain-ubiquitin binding on the basis of other DUB structures. Although otubain 2 is a member of the cysteine protease superfamily of folds, its crystal structure shows a novel fold for DUBs. Moreover, the active-site cleft is sterically occluded by a novel loop conformation resulting in an oxyanion hole, which consists uniquely of backbone amides, rather than the composite backbone/side-chain substructures seen in other DUBs and cysteine proteases. Furthermore, the residues that orient and stabilize the active-site histidine of otubain 2 are different from other cysteine proteases. This reorganization of the active-site topology provides a possible explanation for the low turnover and substrate specificity of the otubains.

Human enteric adenovirus Ad41 is associated with children gastroenteritis. To infect gastrointestinal cells, the invading virus must be acid-stable and resistant to inactivation by bile salts and proteases. In addition, it has to cross the mucus barrier before it infects mucosa cells. We show that Ad41 infectivity is not diminished by acid exposure, a condition limiting the infectivity of the respiratory Ad. This feature can be attributed to a large extent to the global basic charge of enteric Ad virions and to the stability of Ad41 fiber, a viral protein mediating virus attachment. Upon exposure to pH shock, the respiratory Ad2 loses its ability to interact with lipids while enteric Ad41 still binds to the major phospholipids of gastric and intestine mucus. In addition, contrary to respiratory Ad, enteric Ad41 interacts with several sphingolipid components of plasma membranes. These results show that the molecular bases of the Ad41 enteric tropism stem from its particular physicochemical properties.

Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.

This review presents data on commercial and experimental virus-like particle (VLP) vaccines, including description of VLP vaccines against influenza. Virus-like particles are multimeric, sometimes multiprotein nanostructures assembled from viral structural proteins and are devoid of any genetic material. VLPs present repetitive high-density displays of viral surface proteins. Importantly, they contain functional viral proteins responsible for cell penetration by the virus, ensuring efficient cell entry and thus tissue-specific targeting, determined by the origin of the virus. The foremost application of VLPs is in vaccinology, where they provide delivery systems that combine good safety profiles with strong immunogenicity and constitute a safe alternative to inactivated infectious viruses. These stable and versatile nanoparticles display excellent adjuvant properties capable of inducing innate and cognate immune responses. They present both, high-density B-cell epitopes, for antibody production and intracellular T-cell epitopes, thus inducing, respectively, potent humoral and cellular immune responses. Uptake of VLPs by antigen-presenting cells leads to efficient immune responses resulting in control of pathogenic microorganisms.

Adenovirus fibres are trimeric proteins that protrude from the 12 fivefold vertices of the virion and are the cell attachment organelle of the virus. They consist of three segments: an N-terminal tail, which is noncovalently attached to the penton base, a thin shaft carrying 15 amino acid pseudo repeats, and a C-terminal globular head (or knob) which recognizes the primary cell receptor. Due to their exceptional stability, which allows easy distinction of native trimers from unfolded forms and folding intermediates, adenovirus fibres are a very good model system for studying folding in vivo and in vitro. To understand the folding and stability of the trimeric fibres, the unfolding pathway of adenovirus 2 fibres induced by SDS and temperature has been investigated. Unfolding starts from the N-terminus and a stable intermediate accumulates that has the C-terminal head and part of the shaft structure (shown by electron microscopy). The unfolded part can be digested away using limited proteolysis, and the precise digestion sites have been determined. The remaining structured fragment is recognized by monoclonal antibodies that are specific for the trimeric globular head and therefore retains a native trimeric structure. Taken together, our results indicate that adenovirus fibres carry a stable C-terminal domain, consisting of the knob with five shaft-repeats.

BACKGROUND New strategies to improve the outcome of encapsulated porcine islet transplantation may involve the transfer of gene sequences affecting islet viability. While adenoviral vectors appear as the most efficient gene transfer system so far established for islets, non-viral-based vectors are most likely to fulfill microbiological safety criteria and be retained in the clinical setting. Our aim was to standardize the procedures of gene transfer into adult porcine islets using cationic liposome DOTAP. METHODS Porcine islets obtained by collagenase digestion and density gradient purification were lipofected with plasmids coding for luciferase or beta-galactosidase under the control of simian virus 40 or cytomegalovirus promoter. The following parameters were explored: exposure time to vector (1-48 hr), DNA amount (1-15 microg/500 islets), and DOTAP to DNA ratio (2-16). Reporter gene expression was determined 48-72 hr after lipofection. RESULTS Efficiency and reproducibility of transfection were maximal with the following procedure: 3-hr exposure time followed by islet washing, 12 microg of DNA per 500 islets (150 microm equivalent), and DOTAP to DNA ratio of 12 microl/microg. Freshly isolated islets in large aliquots (n=4000 in 50-ml tubes) were efficiently transduced with this procedure, and distribution of gene expression was homogenous when islets were subsequently plated in 500-islet aliquots. Luciferase gene expression was detected for a minimum of 7 days after lipofection. Gene expression was also evident up to 4 weeks after islet transplantation beneath the kidney capsule of athymic mice. Transfection of islets using the beta-galactosidase vector yielded 25% positive islets. Islet viability was not adversely affected. CONCLUSIONS This islet lipofection procedure may help achieve the local release of a bioactive peptide in the graft environment and have therapeutic applications in islet transplantation.

International audience; : We exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering a multivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The delivery platform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remarkable endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the most abundant influenza internal protein with the conserved primary structure. Two different M1 immunodominant epitopes were separately inserted in Dd external positions without destroying the particles' dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system were properly presented by human dendritic cells triggering efficient activation of antigen-specific T cells responses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens. These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulate cellular immune responses.

Background The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. Results Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. Conclusions Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

The name of the first author was spelled incorrectly. The correct name is: Bastien Hermant. The correct citation is: Hermant B, Gudrun A, Potopalsky AI, Chroboczek J, Tcherniuk SO (2013) Amitozyn Impairs Chromosome Segregation and Induces Apoptosis via Mitotic Checkpoint Activation. PLoS ONE 8(3): e57461. doi:10.1371/journal.pone.0057461.

It was recently shown that co-expression of adenovirus type 3 (Ad3) penton base and fibre in the baculovirus system produces dodecahedral particles, as does the expression of the penton base alone. The structure of both of these dodecahedral particles, with and without fibre, has been determined by cryoelectron microscopy and 3-dimensional reconstruction techniques to a resolution of 25 and 20 A, respectively. The general form of the penton base resembles that of the base protein in the recent reconstruction of adenovirus type 2. There is a remarkable difference in the penton base structure with and without the fibre. The five small protuberances on the outer surface of each base move away from the 5-fold axis by approximately 15 A when the fibre is present. These protuberances are of relatively low density and most probably represent a flexible loop possibly containing the RGD site involved in integrin binding. The fibre is apparently bound to the outer surface of the penton base, rather than inserted into it. The fibre is flexible and the shaft contains two distinct globular regions 26 A in diameter. The volume of the inner cavity of the dodecahedron is 350 +/- 100 nm3. This small volume precludes the use of the inner cavity to house genetic information for gene therapy; however, the possibility remains of linking the gene to the dodecahedron surface in the hope that it will be internalized with the dodecahedron.

Human enteric adenoviruses of species F, HAdV-40 and HAdV-41 (Ad40 and Ad41), are associated with gastroenteritis in children. Ad attachment to the primary receptor on the cell surface is mediated by the distal head domain of the fiber protein, an antenna-like component of the adenovirus capsid. Differently from the majority of human Ads that possess one type of fiber on their capsid, the Ad40 and 41 have two distinct fibers. The long fiber recognizes the host membrane protein CAR, which permits virus attachment, but nothing is known about the role of the short fiber. Using the head domain of the Ad41 short fiber, we fished out a putative membrane protein that has never been previously described. This partner of the short fiber of enteric Ad41 (ParAd41) is a small, hydrophobic protein with three putative trans membrane domains, which interacts with the Ad41 short fiber but not with the Ad41 long fiber or with the fiber of respiratory Ad2 serotype. ParAd41 is localized in intracellular membranes including the nuclear membrane. Saturation of the short fiber with ParAd41 inhibits virus infectivity, which substantiates the putative role of ParAd41 in enteric Ads tropism. It is conceivable that the interaction of the short fiber with ParAd41 mediates virus postattachment endocytosis step as well as interaction with the nuclear membrane prior to the injection of viral DNA into the nucleus, thus enabling enteric adenovirus infection. This study is the first one to probe the molecular nature of enteric Ad41 tropism.

The trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus–cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain. To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus–cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed inEscherichia coliand (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus. The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes. At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein. The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration. Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D. Xia, L. J. Henry, R. D. Gerard, and J. Deisenhofer,Structure2, 1259–1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost β-sheets in this structure. Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides. This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber–cell interaction.

The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.

The penton capsomer of mammalian adenoviruses consists of a trimeric, long and thin fibre inserted into a pentameric base. The avian adenoviruses possess a penton which presents another symmetry mismatch: each pentameric base is associated with two fibres. Here we have studied the morphology of the penton of CELO virus, an avian adenovirus, and we have determined the sequence of both fibres, one long and one short. The short fibre is probably associated with the base in the same way as the mammalian viral fibres and we will discuss how the long fibre coul dbe attached. The shafts of all known adenovirus fibres consist of a series of 15-residue repeats. The avian virus fibres show a more complicated and less regular shaft repeat structure with single, double and triple repeats. The sequences of the receptor binding (head) domains of both fibres are very different from all other known fibre head domains and very different from each other, suggesting that the two fibres might bind to different receptors. The genome organization of the sequenced region is rather different from that in human adenoviruses. In particular, a rgion homologous to the human virus E3 region was not found at the position where it normally occurs in the human virus genome.

The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.

International audience; During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

International audience; Amitozyn (Am) is a semi-synthetic drug produced by the alkylation of major celandine (Chelidonium majus L.) alkaloids with the organophosphorous compound N,N'N'-triethylenethiophosphoramide (ThioTEPA). We show here that the treatment of living cells with Am reversibly perturbs the microtubule cytoskeleton, provoking a dose-dependent cell arrest in the M phase. Am changed the dynamics of tubulin polymerization in vitro, promoted the appearance of aberrant mitotic phenotypes in HeLa cells and induced apoptosis by the activation of caspase-9, caspase-3 and PARP, without inducing DNA breaks. Am treatment of HeLa cells induced changes in the phosphorylation of the growth suppressor pRb that coincided with maximum mitotic index. The dose-dependent and reversible anti-proliferative effect of Am was observed in several transformed cell lines. Importantly, the drug was also efficient against multidrug-resistant, paclitaxel-resistant or p53-deficient cells. Our results thus open the way to further pre-clinical evaluation of Am.

International audience

International audience; The majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution.

Virus-coded VPg protein of Potato virus Y (PVY) does not have homologs apart from other VPgs. Since VPg is indispensable for the potyvirus life cycle, it appeared a good candidate for eliciting pathogen-derived resistance to PVY. Following agroinfection used to obtain PVY VPg-transgenic Arabidopsis thaliana plants, only few transgenic seeds were recovered giving rise to six transgenic plants that contained the VPg gene with the correct sequence. They generated VPg mRNA, but VPg protein was not detected. Some plants were immune to PVY infection suggesting post-transcriptional gene silencing. However, the likely PVY VPg toxicity exerted at an early stage of transformed seeds development precludes its use for engineering pathogen-derived resistance.

We report the sequence of 7558 nucleotides of the adenovirus type 5 genome. With this sequence and previously published data, the complete sequence of this genome is now available and can be compared with the already known sequence of the adenovirus type 2 genome. These two serotypes belong to the same subgroup and sequence comparison shows 94.7% homology between the two genomes. The differences are not at all randomly distributed. Transitions between C and T and between A and G account in total for 58.3% of the differences and even for 68.6% for the genome devoid of the fiber and the hexon genes (instead of 33% expected for an equal probability of changes). In the fiber gene the transitions account for 47% of the differences. The detailed analysis of the nucleotide substitution between the two genomes suggests that the Ad2 genome could derive from that of Ad5 one, with the exception of the fiber gene which is likely to be present in Ad2 genome as a result of genetic recombination. The homology between the amino acids sequences of the structural proteins varies from 100% (proteins pVII and IX) to only 69.2% for the fiber.

International audience; We propose a novel influenza vaccine composed of the adenovirus dodecahedron (Dd) as delivery platform carrying an internal influenza matrix protein M1. To attach the antigen to the vector we used WW domains interacting with Dd. Successful internalization of the Dd-M1WW complex was observed using biochemical and cell biology techniques. We show here that the complex of Dd with antigen is a potent activator of human myeloid dendritic cells (MDC), and that it is efficiently presented by MDC to M1-specific CD8+ T lymphocytes. These results show that proposed vaccine model is feasible and that adenovirus dodecahedron is a potent delivery platform for foreign antigens to human cells.

BACKGROUND:Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. PRINCIPAL FINDINGS:Dodecahedron (Dd) structure is preserved at up to about 50 degrees C at pH 7-8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37 degrees C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. CONCLUSIONS/SIGNIFICANCE:Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.

The adenovirus serotype 3 (Ad3) fibre has been expressed in Escherichia coli as an insoluble protein. The protein was solubilized by extraction with urea. Slow removal of urea during the purification procedure resulted in a soluble Ad3 fibre preparation. Polyacrylamide gel analysis of the purified fibre protein, as well as cross-linking experiments performed on cellular debris of expressing cells, suggest that the recombinant Ad3 fibre self-assembles as a trimer from identical polypeptide chains. Gel filtration gave the same exclusion volume for the purified recombinant fibre and for the native fibre in the protein mixture extracted from the Ad3-infected cells. The recombinant fibre was partially resistant to proteolytic degradation, suggesting a folded structure.

Background Direct protein transduction is a recent technique that involves use of peptide vectors. In this study, we demonstrate that adenovirus dodecahedron (Dd), a virus-like particle devoid of DNA and able to penetrate cells with high efficiency, can be used as a vector for protein delivery. Methods Taking advantage of Dd interaction with structural domains called WW, we have elaborated a universal adaptor to attach a protein of interest to this vector. Results A tandem of three WW structural domains derived from the Nedd4 protein enables the formation of stable complexes with Dd, without impairing its endocytosis efficiency. Our protein of interest fused to the triple WW linker is delivered by the dodecahedron in 100% of cells in culture with on average more than ten million molecules per cell. Conclusion These data demonstrate the great potential of adenovirus dodecahedron in combination with WW domains as a protein transduction vector. Copyright © 2006 John Wiley & Sons, Ltd.

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.

Adenovirus penton is a non-covalent complex composed of the penton base and fiber proteins, localized at the twelve vertices of the icosahedral virus capsid. In cells infected by adenovirus serotype 3 (Ad3), penton is found not only in the virus capsid but also self-assembled in dodecahedra formed through interactions between the twelve penton bases. In this study, the intracellular trafficking of penton proteins from the cytoplasm to the nucleus has been followed, and the nuclear re-arrangement induced by viral infection has been observed by electron microscopy of ultrathin sections. The amount of dodecahedra has been assessed in relation to the number of Ad3 infectious virions produced during the Ad3 replication cycle. It appears that dodecahedra are produced in a large excess over viral infectious particles and that they are located intranuclearly along the nuclear membrane of Ad3-infected cells at late times of infection.

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.

Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two alpha-helices connected by a beta-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

International audience; During adenovirus type 3 (Ad3) infection cycle, the penton (Pt) of the viral capsid, a noncovalent complex of fiber and penton base proteins, is produced in large excess and self-assembles to form a highly organized dodecahedral structure, termed dodecahedron (Dd). The physiological role of these particles is poorly understood, but we have recently reported that they can penetrate cells with high efficiency and thus may constitute an attractive tool for gene or protein delivery approaches. Surprisingly, Dd displayed the ability to enter cells non-permissive to Ad3, suggesting the existence of additional internalization modes. In this study, we show that Ad3 Dd binds to cell surface heparan sulfate (HS) through high affinity interaction with the penton base. Furthermore, binding to HS was found to be the prerequisite for a novel and Dd specific entry pathway that could not be used by Ad3. Overall, these data provide new insights in the possible role of Dd during viral infection and potential therapeutic applications.

Each of the 12 vertices of the adenovirus virion is made of penton, the complex of two oligomeric proteins: a pentameric penton base anchored in the capsid and an antenna-like trimeric fiber extending outwards. Adenovirus penton plays an essential role in the infection of host cells because it is indispensable for virus attachment and internalization. The initial interactions of penton with the primary and secondary receptors are well described. In contrast with that, the role of the penton components downstream of the initial cell contact is not known. This work shows for the first time that two adenovirus structural proteins, fiber and base, are able to interact intimately with different classes of cellular targets. In the case of penton base, a protein responsible for virus internalization, the partners include three ubiquitin-protein ligases that are involved in protein turnover, cell cycle control and endocytosis. Another base protein partner, BAG3, is involved in controlling Hsc70 chaperone activity. Virus attachment protein, fiber, interacts with many different partners, some of them involved in signal transduction and cell growth. Further work will illustrate the implications of these interactions for both the viral and cellular life cycles.