Mathieu H. M. Noteborn

Chicken anaemia virus infection: molecular basis of pathogenicity.

Chicken anaemia virus (CAV) is a small virus of a unique type with a particle diameter of 23 to 25 nm and a genome consisting of a circular single-stranded (minus-strand) DNA. This DNA multiplies in infected cells via a circular double-stranded replicative intermediate, which was recently cloned. DNA analysis of CAV strains isolated in different continents revealed only minor differences among the various isolates. Apparently, all CAV isolates belong to a single serotype. CAV is not related to other known animal single-stranded circular-DNA viruses, such as porcine circovirus and psittacine beak-and-feather-disease virus. The major transcript from the CAV genome is an unspliced polycistronic mRNA of about 2100 nucleotides encoding three proteins of 51.6 kDa (VP1), 24.0 kDa (VP2) and 13.6 kDa (VP3 or apoptin). All three predicted CAV proteins are synthesized in CAV-infected cells. Immunization with (recombinant) VP1 and VP2 synchronously synthesized in the same cells elicits a protective response and can be used as subunit vaccine against chicken infectious anaemia. CAV causes clinical and subclinical disease in chickens, and is recognized as an important avian pathogen worldwide. In young chickens, CAV causes a transient severe anaemia due to destruction of erythroblastoid cells in the bone marrow and immunodeficiency due to depletion of cortical thymocytes. The depletion of the cortical thymocytes is considered to cause a (transient) immunodeficiency resulting in enhanced concurrent infections and to vaccination failures. The depletion of thymocytes and most likely also of erythroblastoid cells occurs via CAV-induced apoptosis. The CAV-encoded protein apoptin is the main inducer of this phenomenon.

The Chicken Anemia Virus-Derived Protein Apoptin Requires Activation of Caspases for Induction of Apoptosis in Human Tumor Cells

ABSTRACT The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome c is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843–5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vec- tors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.

Immunogenic and protective properties of chicken anaemia virus proteins expressed by baculovirus

The coding information for three putative chicken anaemia virus proteins (VP1, VP2, VP3) was inserted into a baculovirus vector and expressed in insect cells. The immunogenic properties of the chicken anaemia virus (CAV) proteins produced separately or together in insect-cell cultures were analysed by inoculating them into chickens. Only lysates of insect cells which have synthesised equivalent amounts of all three recombinant CAV proteins or cells which synthesised mainly VP1 plus VP2 induced neutralising antibodies directed against CAV in inoculated chickens. Progeny of those chickens were protected against clinical disease after CAV challenge. Inoculation of a mixture of lysates of cells that were separately infected with VP1-, VP2- and VP3-recombinant baculovirus did not induce significant levels of neutralising antibody directed against CAV and their progeny were not protected against CAV challenge. Our results indicate that expression in the same cell of at least two CAV proteins, VP1 plus VP2, is required to obtain sufficient protection in chickens. Therefore, recombinant CAV proteins produced by baculovirus vectors can be used as a sub-unit vaccine against CAV infections.

Antigenic parvovims B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system

Abstract Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VPlL) and the 1.8 kb coding region for VP2 (AcB19VP 2L), placed under the control of the polyhedrin promoter. The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. AcB19VPlL-in-fected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence. The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique. VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa β-galactosidase B19 fusion protein expressed in E. coli . Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots. VP2 did not react with the fusion protein-specific mAbs. The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing

AbstractApoptin, a protein from chicken anemia virus without an apparent cellular homologue, can induce apoptosis in mammalian cells. Its cytotoxicity is limited to transformed or tumor cells, making Apoptin a highly interesting candidate for cancer therapy. To elucidate Apoptins mechanism of action, we have searched for binding partners in the human proteome. Here, we report that Apoptin interacts with DEDAF, a protein previously found to associate with death effector domain (DED)-containing pro-apoptotic proteins, and to be involved in regulation of transcription. Like Apoptin, after transient overexpression, DEDAF induced apoptosis in various human tumor cell lines, but not in primary fibroblasts or mesenchymal cells. DEDAF-induced cell death was inhibited by the caspase inhibitor p35. Together with the reported association of DEDAF with a DED-containing DNA-binding protein in the nucleus and the transcription regulatory activity, our findings may provide a clue for the mechanism of Apoptins actions in mammalian cells.

Hepatocyte survival depends on beta1-integrin-mediated attachment of hepatocytes to hepatic extracellular matrix.

Abstract: Background: A major drawback of allogeneic hepatocyte transplantation is the lack of sustained survival of the transplanted cells in the recipient liver parenchyma. The purpose of this study was to determine the effect of the presence or absence of hepatic extracellular matrix (ECM) molecules on hepatocyte survival and function following hepatocyte isolation for transplantation purposes, and the role of β1‐integrin molecules therein.

Recombinant apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion

Apoptin, a protein derived from chicken anemia virus, induces apoptosis in human transformed or tumor cells but not in normal cells. When produced in bacteria as a recombinant fusion with maltose-binding protein (MBP-Apoptin), Apoptin forms a distinct, stable multimeric complex that is remarkably homogeneous and uniform. Here, using cytoplasmic microinjection, we showed that recombinant MBP-Apoptin multimers retained the characteristics of the ectopically expressed wild-type Apoptin; namely, the complexes translocated to the nucleus of tumor cells and induced apoptosis, whereas they remained in the cytoplasm of normal, primary cells and exerted no apparent toxic effect. In normal cells, MBP-Apoptin formed increasingly large, organelle-sized globular bodies with time postinjection and eventually lost the ability to be detected by immunofluorescence analysis. Costaining with an acidotrophic marker indicated that these globular structures did not correspond to lysosomes. Immunoprecipitation studies showed that MBP-Apoptin remained fully antibody-accessible regardless of buffer stringency when microinjected into tumor cells. In contrast, MBP-Apoptin in normal cells was only recoverable under stringent lysis conditions, whereas under milder conditions they became fully shielded with time on two epitopes spanning the entire protein. Further biochemical analysis showed that the long-term fate of Apoptin protein aggregates in normal cells was their eventual elimination. Our results provide the first example of a tumor-specific apoptosis-inducing aggregate that is essentially sequestered by factors or conditions present in the cytoplasm of healthy, nontransformed cells. This characteristic should reveal more about the cellular interactions of this viral protein as well as further enhance its safety as a potential tumor-specific therapeutic agent.

Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor.

Specificity is a prerequisite for systemic gene therapy of hepatocarcinoma. In vitro, the tumor-specific viral death effector Apoptin selectively induces apoptosis in malignant hepatic cells. Intratumoral treatment of xenografted subcutaneous hepatomas with Apoptin results in tumor regression. Here, we report a systemic delivery vehicle containing the Apoptin gene linked to asialoglycoprotein (Asor), which targets asialoglycoprotein receptor (ASGPR) present only on the surface of hepatocytes. In vitro, the protein–DNA complex Asor–Apoptin induced apoptosis in HepG2 hepatocarcinoma cells but not in normal L-02 hepatocytes. Non-hepatocyte-derived tumorigenic human A549 cells lacking the membrane ASGPR were not affected by Asor–Apoptin. In vivo systemic delivery of Asor–Apoptin via the tail vein into mice bearing in situ hepatocarcinoma resulted in specific and efficient distribution of Apoptin in both hepatocarcinoma cells and normal hepatocytes. Five days after injection of Asor–Apoptin, the in situ hepatocarcinomas showed significant signs of regression, whereas the surrounding normal hepatocytes did not. Systemically delivered Asor–LacZ expressing non-apoptotic LacZ gene did not inhibit tumor growth. Our data reveal that systemic delivery of Asor–Apoptin specifically induces apoptosis in malignant hepatocytes and thus constitutes a powerful and safe therapeutics against hepatocarcinomas.

Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin.

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell–specific, p53-independent, Bcl-2–insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)–transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1–induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector. Cancer Gene Therapy (2001) 8, 958–965