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Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVβ3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells., Graphical Abstract, Vectors targeting myeloid cells are scarce and yet crucial for the improvement of therapy efficacy and tolerability against myeloid neoplasia. This study highlights promising features of a non-infectious dodecahedral adenovirus-like nanoparticle to target preferentially acute myeloid leukemia blasts and hematopoietic stem and progenitor cells.

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.

We have analyzed the prebiotic effect of softwood hemicelluloses on specific bacterial species in vitro and on microbiota diversity and metabolism in healthy mice in vivo. The ethanol-precipitated glycans from the softwood hemicellulose autohydrolysate were able to stimulate in vitro the growth of Bifidobacterium adolescentis, but to a much lesser extent than that of adherent-invasive E. coli. B. adolescentis was the best producer of SCFA. When mice were fed with the ethanol precipitate fraction, the relative abundance of Bacteroidetes increased while that of Proteobacteria diminished, suggesting change towards a less obesogenic microbiome. Following treatment, lipid analysis showed a decrease in cholesterol, bile acids and free fatty acids, indicating a potential cardioprotection role. Furthermore, analysis of fermentation metabolites revealed an increase in metabolites important for immune health and well-being. Our results highlight the value of hemicelluloses as a potential source of prebiotics supporting both a healthy digestive tract and immune system.

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.

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.

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.

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.

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.

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.

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.

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; 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.

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.

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.

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

Adenovirus binds to its receptor via the head domain of its fiber protein. We have crystallized the adenovirus serotype 2 (subgroup C) receptor binding domain and solved the structure at 1.5 Å resolution by the molecular replacement technique using the known adenovirus type 5 head structure. Included in the high-resolution model are 306 water molecules, five alternative side chain conformations, and individual anisotropic temperature factors for each atom. The overall structure of the serotype 2 head is very similar to its serotype 5 homologue, apart from differences in some of the flexible loops. All but subgroup B adenoviruses are believed to use the recently identified protein CAR (Coxsackievirus and adenovirus receptor) as receptor. By comparison of the two structures and sequence alignment of CAR binding and non-CAR binding serotype fiber heads, we discuss possible receptor binding sites and propose a receptor binding site in a crevice between two monomers on the side of the trimer. The structural basis of the extraordinary stability of the fiber head trimer is also discussed.

Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

The adenovirus fiber appears as a long, thin projection terminated by a knob (head). The fiber consists of a trimeric protein whose head domain is thought to interact with cell receptors. The head part (amino acids 388 to 582) of adenovirus type 2 fiber was produced in a baculovirus expression system. The purified protein was shown to cross-link into trimers. It was very resistant to proteolytic attack and seemed to attain a high degree of compactness. The head domain efficiently inhibited attachment of adenovirus to receptors on the surface of HeLa cells, thereby confirming the hypothesis that the head domain interacts with viral receptors.

The gene coding for the enzyme malate dehydrogenase (MDH) of the extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced. The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da. The deduced amino acid sequence of the enzyme was found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs. The structural gene was cloned in the Escherichia coli expression vector pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl. The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria. These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme. Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln. This modification produced an enzyme that has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH). The mutation also caused a modification in the relative activities of the enzyme at different salt concentrations. The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.

RNA bacteriophage f2, treated in vitro with rifampicin, loses infectivity dramatically. Rifampicin interacts with phage RNA, binding to a few specific sites. Inhibition of phage RNA infectivity occurs at 10-100 times lower molar excess of rifampicin than inhibition of infectivity of intact phage particles. Thus the phage capsid acts as a barrier, diminishing interaction of the drug with phage RNA.

The major structural proteins of adenovirus (Ad), which form the external capsid, are hexon, penton base and fiber. The primary structure of the Ad5 penton base has been deduced from the nucleotide sequence of the corresponding gene. It has 98.6% homology with the sequence of the analogous protein from Ad2. This result is in contrast with the significantly lower homology found for the two other major structural proteins, the hexon and the fiber.

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 °C. The synthesized protein constituted about 1 % of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fiber runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.

RNA-protein cross-links were introduced into brome mosaic virus in situ by using the heterobifunctional agent p-azidophenylglyoxal. An improved RNA isolation method, without phenol extraction, was used to isolate RNA cross-linked with protein. RNA of the covalently linked complex was acid-digested and the oligonucleotides still attached to protein were 5'-end-labelled with 32P. The complexes were digested with trypsin and the tryptic peptides were purified by reversed-phase high-performance liquid chromatography. Amino acid analyses of cross-linked tryptic peptides revealed that out of the total 188 amino acids of brome mosaic virus coat protein only the 80 N-terminal amino acids are involved in the interaction with viral RNA. These results are discussed in connection with a predicted secondary structure of the coat protein. Both alpha helix (for amino acids 11-19) and other structures (between amino acids 20 and 80) are implicated in the coat protein-viral RNA interactions.

The amino acid sequence of the fiber from adenovirus type 5 has been deduced from the nucleotide sequence of the fiber gene. This sequence is compared with that of adenovirus type 2, a closely related serotype. We find 69% homology for the fiber protein whereas the known nonstructural proteins of these two serotypes have 99% sequence homology. A detailed sequence analysis was performed in the context of the model proposed by Green et al., [(1983), EMBO J., 8, 1357–1365] for the folding of the polypeptide chain of the adenovirus type 2 fiber. The N-terminal region, which in the virion is associated with the capsid, is identical for these two serotypes. In the shaft of the fiber the features, such as periodicity of the prolines and of the hydrophobic residues in the amino acid sequence, on which the model for the adenovirus type 2 is based are very well preserved in adenovirus type 5. On the other hand, there are large differences all along the sequence of the shaft of the fiber showing that there is a very limited homology between the amino acids in the two serotypes when they do not have a key role in establishing the structure. In the knob the homology between serotypes is 64%. These results are consistent with the differences between serotypes being confined to the exposed proteins.

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.

We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.