M. H. M. Noteborn

Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope

Chicken anaemia virus (CAV) expresses three proteins, VP1, VP2 and VP3, but its capsid contains only the VP1 protein. In this paper, we report that for production of the neutralizing epitope, co-synthesis of (recombinant) VP1 and VP2 has to take place. We show via immunofluorescence that recombinant-baculovirus-infected Sf9 cells synthesizing VP1 (or VP2) alone react very poorly with CAV-specific neutralizing antibodies. In contrast, Sf9 cells co-infected with VP1- and VP2-recombinant baculoviruses, or infected with a single recombinant baculovirus co-expressing both VP1 and VP2, react strongly with the neutralizing antibodies. Furthermore, immunoprecipitation assays show that VP1 and VP2 interact directly with each other, which indicates that the non-structural protein VP2 might act as a scaffold protein in virion assembly. Recombinant baculovirus expressing VP1 and VP2 is, therefore, a potential production system for a subunit vaccine against CAV infection.

The viral death effector Apoptin reveals tumor-specific processes.

Several natural proteins, including the cellular protein TRAIL and the viral proteins E4orf4 and Apoptin, have been found to exert a tumor-preferential apoptotic activity. These molecules are potential anti-cancer agents with direct clinical applications. Also very intriguing is their possible utility as sensors of the tumorigenic phenotype. Here, we focus on Apoptin, discussing recent research that has greatly increased our understanding of its tumor-specific processes. Apoptin, which kills tumor cells in a p53- and Bcl-2-independent, caspase-dependent manner, is biologically active as a highly stable, multimeric complex consisting of 30 to 40 monomers that form distinct superstructures upon binding cooperatively to DNA. In tumor cells, Apoptin is imported into the nucleus prior to the induction of apoptosis; this contrasts with the situation in primary or low-passage normal cell cultures where nuclear translocation of Apoptin is rare and inefficient. Apoptin contains two autonomous death-inducing domains, both of which exhibit a strong correlation between nuclear localization and killing activity. Nevertheless, forced nuclear localization of Apoptin in normal cells is insufficient to allow induction of apoptosis, indicating that another activation step particular to the tumor or transformed state is required. Indeed, a kinase activity present in cancer cells but negligible in normal cells was recently found to regulate the activity of Apoptin by phosphorylation. However, in normal cells, Apoptin can be activated by transient transforming signals conferred by ectopically expressed SV40 LT antigen, which rapidly induces Apoptin’s phosphorylation, nuclear accumulation and the ability to induce apoptosis. The region on LT responsible for conferring this effect has been mapped to the N-terminal J domain. In normal cells that do not receive such signals, Apoptin becomes aggregated, epitope-shielded and is eventually degraded in the cytoplasm. Finally, Apoptin interacts with various partners of the human proteome including DEDAF, Nmi and Hippi, which may help to regulate either Apoptin’s activation or execution processes. Taken together, these recent advances illustrate that elucidating the mechanism of Apoptin-induced apoptosis can lead to the discovery of novel tumor-specific pathways that may be exploitable as anti-cancer drug targets.

Relevance of Apoptin's Integrity for Its Functional Behavior

Wadia et al. (7) recently claimed that apoptin, a virally encoded protein with tumor-selective apoptosis activity, contains a concentration-dependent nuclear localization signal (NLS) that is not tumor selective as previously reported (1, 2, 4). Their Fig. ​Fig.1B1B shows, however, that under all the conditions tested, nuclear import of green fluorescent protein (GFP)-apoptin70-121 remains 200 to 300% higher in tumor cells than in primary cells. The apparent concentration dependence of apoptins NLS is intriguing. We agree with their interpretation that presentation of multiple apoptin NLS domains by a molecular aggregate could generate more efficient nuclear trafficking than NLS exposure at a single site. This result is, however, somewhat academic, as full-length apoptin can only be harvested as a multimer from live cells, with a dissociation rate constant (koff) that is so slow that it can hardly be measured, whereas the C-terminal fragment forms only monomers (5, 9). This could explain why Wadia et al. could measure a cooperative, concentration-dependent effect of apoptins NLS, as they used a GFP fusion of a C-terminal fragment of apoptin lacking the multimerization domain (5). Had they used full-length apoptin, all the NLS sequences would likely have been clustered, resulting in effective nuclear import over all concentration ranges. Nevertheless, we suggest caution in using GFP-apoptin fusions in functional studies. For example, fusion of GFP to full-length apoptin results in increased levels of nuclear GFP-apoptin versus wild-type apoptin in primary cells (Fig. ​(Fig.1A1A). FIG. 1. (A) Apoptin but not GFP-apoptin is mainly localized in the cytoplasm of human primary cells. Human mesenchymal stem cells were microinjected with plasmids encoding GFP-apoptin (left) or wild-type apoptin (right). GFP-apoptin was analyzed directly by fluorescence ... The current hypothesis is that nuclear trafficking and tumor-specific phosphorylation of apoptin at Thr108 are essential for induction of apoptosis (3, 6, 8). Wadia et al. reported a failure to detect phosphorylation with radioactive labeling of GFP-apoptin70-121; we do not know why this is so, as phosphorylation of full-length apoptin has been thoroughly documented by the use of mass spectrometry and a phospho-specific antibody (6). Our preliminary experiments suggest that abolishing the phosphorylation site of apoptin does not significantly disrupt its nuclear import in tumor cells. In attempting to address this observation with a GFP-apoptin70-121 T108A mutant, Wadia et al. used a construct that is not phospho-null in vivo; in our hands, as Thr108 is the last in a run of three threonine residues, the adjacent Thr107 becomes opportunistically phosphorylated instead, which yields the same phenotype. Mutation at both positions 107 and 108 is required to eliminate phosphorylation and function. As Fig. ​Fig.1B1B indicates, phosphorylation of apoptin is required for apoptosis induced by its C-terminal death domain (3); a control experiment confirms that the apoptosis-competent fragment is phosphorylated on Thr108 in vivo (Fig. ​(Fig.1C1C). Apoptins tumor-specific activity does not result from a single characteristic. Its multimerization behavior, its potential to be phosphorylated, its cooperativity in nuclear trafficking, and many of its other physical, chemical, and functional activities all shape the mechanism by which it induces tumor-specific apoptosis. Extrapolations of results obtained from studies that disrupt apoptins integrity should therefore be approached with caution.

BAG-1 inhibits p53-induced but not apoptin-induced apoptosis.

BAG-1 has been identified as a Bcl-2-binding protein that inhibits apoptosis, either alone or in co-operation with Bcl-2. Here we show that BAG-1 inhibits p53- induced apoptosis in the human tumour cell line Saos-2. In contrast, BAG-1 was unable to inhibit the p53-independent pathway induced by apoptin, an apoptosis-inducing protein derived from chicken anaemia virus. Whereas BAG-1 seemed to co-operate with Bcl-2 to repress p53-induced apoptosis, co-expression of these proteins had no inhibitory effect on apoptin-induced apoptosis. Moreover, Bcl-2, and to some extent also BAG-1, paradoxically enhanced the apoptotic activity of apoptin. These results demonstrate that p53 and apoptin induce apoptosis through independent pathways, which are differentially regulated by BAG-1 and Bcl-2.

Detection of chicken anaemia virus by DNA hybridization and polymerase chain reaction.

A clone containing the complete genome of chicken anaemia virus (CAV) was used in hybridizations with DNA from various field isolates of CAV. CAV DNA from all field isolates was detected in a polymerase chain reaction with oligonucleotides derived from the sequence of the cloned CAV DNA as primers. By way of Southern blot analysis with (32)P-labelled DNA probes derived from cloned CAV DNA, all field isolates were shown to contain DNA molecules of about 2.3 kb, i.e. the size of cloned CAV DNA. In a dot-blot assay it was demonstrated that non-radioactively-labelled cloned CAV DNA hybridized specifically to DNA from field isolates. The cloned CAV DNA is highly similar to the DNA of field isolates, as borne out by restriction-enzyme mapping. We conclude that our cloned CAV genome is representative for CAV in the field. The described PCR and hybridization techniques, may, therefore, be used for research and diagnosis of CAV infections.

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing.

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens.

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.