Co-chaperone BAG3 and adenovirus penton base protein partnership.

The BAG family of Hsp70/Hsc70 co-chaperones is characterised by the presence of a conserved BAG domain at the carboxyl-terminus. BAG3 protein is the only member of this family containing also the N-terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N-terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co-migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co-chaperone activity, suggesting that this interaction is not related to the classical BAG3 co-chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad-infected cells, suggesting that the interaction between virus penton base protein and cellular co-chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host-pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co-chaperone.

Structure of the dodecahedral penton particle from human adenovirus type 3.

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.

Adenovirus dodecahedron, a new vector for human gene transfer.

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.

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E.

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 serotype 3 fibre protein is expressed as a trimer in Escherichia coli.

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.

The avian adenovirus penton: two fibres and one base.

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 could be 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 region homologous to the human virus E3 region was not found at the position where it normally occurs in the human virus genome.

Novel partner proteins of adenovirus penton.

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.

Lipoic acid-derived amphiphiles for redox-controlled DNA delivery.

BACKGROUND: Intracellular release of free DNA from the vector complex is one of the critical steps limiting the efficiency of non-viral gene delivery. The complex should be stable enough to prevent DNA degradation but it should be destabilized inside the cell to allow DNA release and transcription. Destabilization and degradation of synthetic vectors is also required to reduce their cytotoxicity and augment the life-time of transfected cells. RESULTS: Here we describe new cationic amphiphiles made from the natural pro-vitamin, lipoic acid, that reversibly binds and releases DNA, depending on the redox state of the lipoate moieties. In the oxidized state these amphiphiles condense DNA into homogeneous spherical particles, which, upon reduction, swell into DNA toroids with subsequent release of free DNA. Complex reduction and DNA release can be induced by various thiols as well as enzymatically, by thioredoxin reductase. Transfection with amphiphile-DNA complexes in vitro shows a several fold increase of transgene expression compared with DOTAP, and can be further augmented by attachment of the nucleus-targeting peptide to the amphiphile. The increase of transfection efficiency results from GSH- and NAD(P)H-dependent complex reduction and release of free DNA inside the cells. CONCLUSIONS: The present work demonstrates the principle of a redox-controlled gene delivery system that uses the reversibility of thiol-disulfide exchange reaction. Our data suggest that the efficiency of synthetic vectors can be augmented by their controlled destabilization inside the cells. Being formed from the natural non-toxic compound lipoic acid, these cationic amphiphiles provide a new promising class of synthetic vectors for gene delivery.

Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation.

Adenoviral dodecahedron (Dd) is a virus-like particle composed of twelve pentameric penton base (Pb) proteins, responsible for adenovirus cell penetration. It is generated spontaneously in the baculovirus system upon expression of the Pb gene of adenovirus serotype 3. This particle shows remarkable cell penetration ability with 2,00,000-3,00,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. We have used it in the past for delivery of small drugs as well as a vaccination platform, in which Dd serves as a particulate vaccine delivery system. Since development of new biomedicals depends strongly on the cost of their expression and purification, we attempted, albeit unsuccessfully, to obtain Dd expression in bacteria. We therefore retained its expression in the baculovirus/insect cells system but introduced significant improvements in the protocols for Dd expression and purification, leading to considerable savings in time and improved yield.

Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells.

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 degrees 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 fibers 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.

The penton base of human adenovirus type 3 has the RGD motif.

The gene encoding the penton base of human adenovirus (Ad) type 3 has been sequenced. The resulting amino-acid sequence has an Arg-Gly-Asp (RGD) motif located near its middle in a hydrophilic region. The same motif is found in serotypes 2, 5 and 12. This sequence was found [Wickham et al., Cell 73 (1993) 309-319] to be involved in the internalisation of Ad2 through an interaction with some specific integrins.

Determination of the nucleotide sequence for the penton-base gene of human adenovirus type 5.

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.

Human adenovirus 2 temperature-sensitive mutant 112 contains three mutations in the protein IIIa gene.

The temperature-sensitive (ts) mutant 112 of human adenovirus 2 is defective in the late stage of virus maturation. The region of functional mutation has been localised by marker rescue. It was observed that the ts mutation can be rescued by the left-hand part of the wild-type gene (nucleotides 12,301-12,891). By nucleotide sequencing, two mutations, both C to T (at position 12,386 and 12,741), were found in this region. The first one, in the glycine 20 codon, is silent, whereas the second changes alanine 145 to valine. A third mutation, which changed C to A (nucleotide 13,613), was identified in the right-hand part of the gene, resulting in the replacement of alanine-436 by threonine.

Effect of dinucleotides on wheat germ translation system.

The effect of ribodinucleoside monophosphates on total protein synthesis was studied in a wheat germ cell-free system, using brome mosaic virus (BMV) RNA as a messenger. Dinucleotides inhibit total protein synthesis to different extents. Of those tested the most inhibitory is CpA. The inhibitory effect of dinucleotides is due to their adverse effect on initiation and not on elongation of polypeptide synthesis. It seems that the dinucleotides complementary to the initiation codon are able to compete with the initiator tRNA during initiation of protein synthesis. The comparison of the effect exerted by different dinucleotides suggests that under conditions of the in vitro protein synthesis RNA 4 is an mRNA molecule with the initiation codon and its immediate neighbourhood being exposed.

Rapid sequence-independent cellular response to oligodeoxynucleotides.

The presence of receptors for oligodeoxynucleotides (OdN) on the surface of L929 cells has previously been described. To study the possible coupling of the receptor to cellular signal transducing systems, the effect of phosphodiester OdN of different sequences on cellular phospholipase C and protein kinase C (PKC) activities in L929 fibroblasts was studied. Treatment of cells with OdN induced an increase in 32P labeling of phosphatidic acid which was accompanied by a gradual decrease in diacylglycerol. These effects seem to be independent of the OdN sequence. PKC activity in membranes isolated from OdN-treated cells was found to be lower than that in membranes of control cells. SDS-PAGE of the 32P-labeled cellular proteins revealed that OdN treatment caused a decrease in phosphorylation of the 26 and 73 kDa cellular proteins in the cells.

The interaction of wheat germ tyrosyl-tRNA synthetase and the tRNA-like end of brome mosaic virus RNA has no effect on in vitro viral protein synthesis and on in vitro encapsidation.

The effect of aminoacylation of the tRNA-like end of brome mosaic virus RNA during in vitro protein synthesis and in vitro viral encapsidation was investigated. The components of the homologous system were: BMV RNA, wheat germ cell-free protein synthesizing system and pure tyrosyl-tRNA synthetase from wheat germ. During in vitro protein synthesis directed with tyrosylated as well as non-tyrosylated BMV RNA, no differences were observed in the amount and in the class of polypeptides formed neither in the velocity of the translation reaction. Excess active TyrRS was added during in vitro translation, without modifying the translation efficiency. BMV RNA and active TyrRS were preincubated prior to translation in order to interact without the translation system components and then subjected to translation in vitro. Similar results were obtained when BMV RNA was preincubated with inactive TyrRS or BSA. These results indicate that the aminoacylation of BMV RNA has no pronounced effect on viral protein synthesis in vitro. During BMV RNA encapsidation either tyrosylated or non-tyrosylated BMV RNA 4 could be encapsidated in a similar way.

Standardization of procedure for efficient ex vivo gene transfer into porcine pancreatic islets with cationic liposomes.

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.

Human adenovirus serotype 3 (Ad3) and the Ad3 fiber protein bind to a 130-kDa membrane protein on HeLa cells.

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 host cell fraction that increases the infectivity of bacteriophage f2.

A fraction that increases infectivity of bacteriophage f2 was isolated from uninfected E. coli cells. The greatest effect was obtained when the fraction was added to the phage reconstituted in vitro. The fraction isolated from the ribosome-free supernatant consisted of proteins, lipids, carbohydrates, and unidentified material. Cleavage of protein by the treatment with trypsin did not significantly affect the infectivity-restoring activity. It is suggest that lipids may play an essential role in the activity of the fraction isolated from the host cell.

Structural proteins of adenovirus. Expression in Escherichia coli.

The fiber proteins of adenovirus serotype 2 Ad2 and serotype 3 Ad3 and structural protein IIIa of wild type Ad2 and Ad2 ts 112 mutant were cloned and expressed in E. coli. For the expression of both fiber proteins a gene expression system based on bacteriophage T7 RNA polymerase was used. The expressed proteins constituted 1-3% of total host cell protein. Both proteins were insoluble and inclusion bodies were observed. The proteins could be purified from cellular debris by extraction with 6 M urea followed by chromatography in the presence of diminishing concentration of urea. The folding of recombinant fiber proteins was assessed by sensitivity to proteases and gel filtration. Both proteins were synthetized as trimers. Ad2 recombinant fiber has a much less compact structure than native Ad2 fiber, since on gel filtration it is excluded before the native fiber. It is also much more sensitive to chymotrypsin digestion than the native protein. Contrary to that, Ad3 recombinant fiber is much less sensitive to proteolytic cleavage and on gel filtration has the same exclusion volume as the trimeric native fiber of Ad3.