A novel CAV derived cell-penetrating peptide efficiently delivers exogenous molecules through caveolae-mediated endocytosis.

Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ß-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.

Identification of the interaction and interaction domains of chicken anemia virus VP2 and VP3 proteins.

Chicken anemia virus (CAV) is a small, single-stranded DNA virus of Anelloviridae family. Its genome segments encode three proteins, VP1, VP2, and VP3. This study identified an interaction between VP2 and VP3 and mapped the interaction domains. Through the yeast two-hybrid (Y2H) system, VP2 was found to interact with VP3. The presence of the VP2-VP3 complex in CAV-infected chicken cells was confirmed by co-immunoprecipitation. Confocal microscopy showed that VP2 and VP3 were expressed in the cytoplasm in cotransfected Vero cells. In the Y2H system, the interaction domains were identified as being within the N-terminal aa 1-30 and C-terminal aa 17-60 for VP2 and the N-terminal aa 46-60 and C-terminal aa 1-7 for VP3. This study showed the interaction between VP2 and VP3 of CAV and identified multiple independent interactive domains within the two proteins. This provides novel information for investigating the biological functions of these proteins.

VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin.

Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.

C-terminal region of apoptin affects chicken anemia virus replication and virulence.

BACKGROUND: Chicken anemia virus (CAV) causes anemia and immune suppression, which are important diseases in the poultry industry. CAV VP3, also referred as 'apoptin', has been shown to selectively kill tumor cells, raising great hopes for its utilization as an anticancer therapy. The ability of apoptin to induce apoptosis is closely related to its nuclear localization. The C-terminal region of apoptin contains a bipartite nuclear localization signals (NLS), and a nuclear export signal (NES) is located between the arms of the NLS. Most previous studies have expressed apoptin of different lengths in vitro to understand the relationship between its localization and its induction of apoptosis. METHODS: In this study, we investigated the replication of CAV and its induction of apoptosis in vitro and in vivo with VP3-truncated infectious virus. Quantitative PCR was used to detect viral replication in MDCC-MSB1 cells, and the viral localization was observed by confocal microscopy. Flow cytometry was uesed to analyze virus-induced apoptosis in MDCC-MSB1 cells. Additionally, chickens infected with the rescued viruses compared with the parental virus rM9905 to evaluate the viral replication in vivo and virulence. RESULTS: Based on the infectious clone, we rescued two viruses in which were deleted NES-NLS2 (rCAV-VP3N88) or NLS1-NES-NLS2 (rCAV-VP3N80) in the C-terminal region of apoptin. The viral load of rCAV-VP3N88 decreased significantly between 60 and 108 hpi, and was always 10-100-fold lower than that of the parental virus rM9905. The levels of rCAV-VP3N80 were also 10-100-fold lower than that of rM9905 and declined significantly at three time points. There was almost no difference in the viral loads of rCAV-VP3N88 and rCAV-VP3N80. Additionally, rM9905 induced 85.39 ± 2.18% apoptosis at 96 hpi, whereas rCAV-VP3N88 and rCAV-VP3N80 induced 63.08 ± 4.78% and 62.56 ± 7.35% apoptosis, respectively, which were significantly (about 20%) lower than that induced by the parental virus. The rescued viruses altered the nuclear localization in MDCC-MSB1 cells. Moreover, deletion of C-terminal region of apoptin impaired viral replication in vivo and reduced the virulence of CAV in chickens. CONCLUSIONS: In summary, we have demonstrated that the C-terminal deletion of apoptin in infectious CAV affected the replication of the virus. The deletion of the C-terminal region of apoptin not only significantly reduced viral replication in vitro but also reduced its induction of apoptosis, which correlated with the loss of its nuclear localization. The deletion of the C-terminal region of apoptin also impaired the replication of CAV and attenuated its virulence in chickens.

[Effect of Low Dose of Chicken Infectious Anemia Virus in Attenuated Vaccine on SPF Chicken Body Weight and Vaccine Immune Antibody].

In order to observe the effect of the immune and weight of chickens after use the attenuated vaccine with low dose of chicken infectious anemia virus (CIAV). In this study, the effects of low dose of CIAV on the weight of SPF chickens and NDV antibody production were observed by simulated experiments. The results showed that 10 EID50 and 5 EID50 CIAV per plume attenuated NDV vaccines were used to cause the weight loss of SPF chickens. Compared with the use of the non contaminated vaccine group, it has significant difference. And NDV antibody levels compared with the use of the non contaminated groups also decreased after use the vaccine with two doses of CIAV contaminated. It has significant difference. A certain proportion of CIAV antibody positive was detected at the beginning of the second week after use the NDV vaccine with two doses of CIAV contaminated. The detection of a high proportion of CIAV nucleic acid was detected in the first week after the use of a contaminated vaccine. The results of the study demonstrate the effects of CIAV pollution on the production and immune function of SPF chickens, and it is suggested that increasing the detection of viral nucleic acid can help save time and improve the detection rate in the detection of exogenous virus contamination by SPF chicken test method.

Occurrence and Phylogenetic Studies of Chicken Anemia Virus from Polish Broiler Flocks.

Chicken anemia virus (CAV) is a widespread chicken pathogen of significant economic importance. In 2013, broiler chicken flocks in Poland were examined for the presence of CAV, and phylogenetic relatedness between the strains was established. Ten cloacal swabs from each of 106 broiler flocks (birds aged 3-6 wk) were collected in different regions of the country and tested with the use of real-time PCR (all samples) and conventional PCR (those samples positive in real-time PCR) assays. The presence of CAV was detected in 16 of the flocks tested. Phylogenetic analysis clearly confirmed the existence of genetic diversity within the group of circulating CAV strains and their distinctiveness from vaccine strains used in Poland.

Transcriptomic Profiling of Virus-Host Cell Interactions following Chicken Anaemia Virus (CAV) Infection in an In Vivo Model.

Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host's immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate <0.05) changes in expression in thymus and bone marrow on CAV infection were mainly associated with T-cell receptor signalling, immune response, transcriptional regulation, intracellular signalling and regulation of apoptosis. Expression levels of a number of adaptor proteins, such as src-like adaptor protein (SLA), a negative regulator of T-cell receptor signalling and the transcription factor Special AT-rich Binding Protein 1 (SATB1), were significantly down-regulated by CAV infection, suggesting potential roles for these genes as regulators of viral infection or cell defence. These results extend our understanding of CAV-induced immunosuppression and suggest a global immune dysregulation following CAV infection.

Comparative histopathological and immunological study of two field strains of chicken anemia virus.

Infection of poultry with chicken anemia virus (CAV) is implicated in several field problems in broiler flocks due to the immunosuppression generated and, consequently, the increased susceptibility to secondary infections. Recently, we have reported an increased occurrence of clinical cases caused by CAV strains distantly related to those commonly used for vaccination. In order to understand the behavior of two Argentinean CAV strains (CAV-10 and CAV-18) in two-week-old chickens, an immune and histopathological study was performed. Neither mortality nor clinical signs were observed in the infected or control groups. Thymus lobes from chickens infected with both CAV viruses were smaller compared to the negative control group. At 14 days post-infection (dpi), only chickens inoculated with CAV-10 show a severe depletion of lymphocytes in the thymus cortex and in follicles from the bursa of Fabricius. Also thymopoiesis disorders, such as reduction in the percentage of total DP (CD4 + CD8α+) thymocytes and alteration in the percentages of DP subpopulations, were more important in animals inoculated with the CAV-10 than the CAV-18 strain. In addition, only animals infected with CAV-10 show a decrease in CD8αß splenocytes. Altogether our results show that, although both Argentinean CAV strains produce subclinical infections in chickens causing immunosuppression at 14 dpi, they might differ in their in vivo pathogenicity.

Interactions of Plasmodium juxtanucleare and chicken anaemia virus: establishing a model.

The pathogens Plasmodium juxtanucleare and chicken anaemia virus (CAV) are easily transmitted and potentially harmful to chickens. In this study, we established an experimental model to investigate the effects of avian malaria caused by P. juxtanucleare in white leghorn specific-pathogen-free (SPF) chicks previously immunosuppressed with CAV. Parasitaemia, haematological variables and clinical and pathological parameters were determined in four different experimental groups: chicks coinfected by CAV and P. juxtanucleare strain (Coinfected group), chicks exclusively infected by CAV (CAV group) or P. juxtanucleare (Malaria group) and uninfected chicks (Control group). Our data demonstrated that P. juxtanucleare parasitaemia was significantly higher in the Coinfected group. Furthermore, haematological parameters, including the RBC, haematocrit and haemoglobin concentration were significantly reduced in coinfected chicks. In agreement with the changes observed in haematological features, the mortality among coinfected chicks was higher compared with animals with single infections. Clinical analysis indicated moderate changes related to different organs size (bursa of Fabricius, heart and liver) in coinfected birds. The experimental coinfection of SPF chickens with P. juxtanucleare and CAV may represent a research tool for the study of avian malaria after CAV immunosuppression, enabling measurement of the impacts caused by different pathogens during malarial infection.

Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift.

We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV+IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV+IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.

Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3.

BACKGROUND: VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. RESULTS: An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. CONCLUSIONS: VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Comparative susceptibility of some avian and mammalian lymphoblastoid cell lines to chicken anemia virus.

Although MSB1 is the most-commonly used lymphoblastoid cell line for isolation of chicken anemia virus (CAV), some researchers have reported a few biological drawbacks. None of them were supported by the results of the present study. Another four avian (HP1, HP2, BK3 and CU10) and two mammalian (BTL-26 and KO-1) cell lines were investigated for susceptibility to the TK-5803 and AH-9409 strains. Both strains caused CPE on BK3 like MSB1. The mean number of positive cells for each strain in MSB1 and BK3 were not significantly different. The majority of the HP2, CU10 and HP1 cells showed no CPE. The virus titers of both strains were higher in MSB1 and BK3 (10(6.5-7.5) TCID(50)/0.1 ml) than in HP2, CU10 and HP1 (10(3.5-4.5) TCID(50)/0.1 ml). BTL-26 and KO-1 were resistant to CAV. BK3 could be used for isolation of CAV.

Apoptin, a tumor-selective killer.

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.

Apoptosis-inducing proteins in chicken anemia virus and TT virus.

Torque teno viruses (TTVs) share several genomic similarities with the chicken anemia virus (CAV). CAV encodes the protein apoptin that specifically induces apoptosis in (human) tumor cells. Functional studies reveal that apoptin induces apoptosis in a very broad range of (human) tumor cells. A putative TTV open reading frame (ORF) in TTV genotype 1, named TTV apoptosis inducing protein (TAIP), it induces, like apoptin, p53-independent apoptosis in various human hepatocarcinoma cell lines to a similar level as apoptin. In comparison to apoptin, TAIP action is less pronounced in several analyzed human non-hepatocarcinoma-derived cell lines. Detailed sequence analysis has revealed that the TAIP ORF is conserved within a limited group of the heterogeneous TTV population. However, its N-terminal half, N-TAIP, is rather well conserved in a much broader set of TTV isolates. The similarities between apoptin and TAIP, and their relevance for the development and treatment of diseases is discussed.

Chicken anemia virus.

Chicken anemia virus (CAV), the only member of the genus Gyrovirus of the Circoviridae, is a ubiquitous pathogen of chickens and has a worldwide distribution. CAV shares some similarities with Torque teno virus (TTV) and Torque teno mini virus (TTMV) such as coding for a protein inducing apoptosis and a protein with a dual-specificity phosphatase. In contrast to TTV, the genome of CAV is highly conserved. Another important difference is that CAV can be isolated in cell culture. CAV produces a single polycistronic messenger RNA (mRNA), which is translated into three proteins. The promoter-enhancer region has four direct repeats resembling estrogen response elements. Transcription is enhanced by estrogen and repressed by at least two other transcription factors, one of which is COUP-TF1. A remarkable feature of CAV is that the virus can remain latent in gonadal tissues in the presence or absence of virus-neutralizing antibodies. In contrast to TTV, CAV can cause clinical disease and subclinical immunosuppression especially affecting CD8+ T lymphocytes. Clinical disease is associated with infection in newly hatched chicks lacking maternal antibodies or older chickens with a compromised humoral immune response.

Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV).

To test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(-), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(-). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(-) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT(108)I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T(108)I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10-36, 31-59, 59-88 and 80-112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(-). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV.

Inhibition of chicken anaemia virus replication using multiple short-hairpin RNAs.

RNA interference is becoming a powerful tool in gene-specific silencing. New generation vaccines against many pathogens will attempt to incorporate these molecules. Here we report the efficient silencing of chicken anaemia virus (CAV) genes in vitro using short-hairpin RNAs (shRNAs) targeting the region of the CAV transcript encoding either viral protein (VP) 1, or overlapping sections of VP2/3 and VP1/2. The shRNAs were first validated against a EGFP-CAV fusion transcript reporter system and then against CAV grown in MDCC-MSB1 cells. The decrease in CAV replication was shown with a flow cytometry assay specific for VP3. Overall the results showed efficient silencing of CAV replication in tissue culture using shRNAs. It was also shown that the combination of three shRNAs being expressed from a single plasmid is less effective at silencing CAV replication than the most active shRNA alone.

Unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus.

Current clinical studies on human annelloviruses infections are directed towards finding an associated disease. In this review we have emphasized the many similarities between human anellovirus and avian circoviruses and the cell and tissue types infected by these pathogens. We have done this in order to explore whether knowledge acquired from natural and experimental avian infections could reflect and be extrapolated to the less well-characterized human annellovirus infections. The knowledge gained from the avian system may provide suggestions for decoding the enigmatic human anellovirus infections, and finding the specific disease or diseases caused by these human anellovirus infections. Each additional parallelism between chicken anemia virus (CAV) and Torque teno virus (TTV) further strengthens this premise. As we have seen information from human infections can also be used to better understand avian infections as well. Increased attention must be focused on the "hidden" or unrecognized, seemingly asymptomatic effects of circovirus and anellovirus infections. Understanding the facilitating effect of these infections on disease progression caused by other pathogens may help to explain differences in outcome of complicated poultry and human diseases. The final course of a pathogenic infection is determined by variations in the state of health of the host before, during and after contact with a pathogen, in addition to the phenotype of the pathogen and host. The health burden of circoviridae and anellovirus infections may be underestimated, due to lack of awareness of the need to search past the predominant clinical effect of identified pathogens and look for modulation of cellular-based immunity caused by co-infecting circoviruses, and by analogy, human anneloviruses.

Genome sequencing analysis of Brazilian chicken anemia virus isolates that lack MSB-1 cell culture tropism.

Specific amino acid (aa) substitutions in VP1, VP2 and VP3 genes were reported as a distinctive feature of the American CIA-1 strain, characterized as having a variable rate of growth and tropism for different MSB-1 cell sublines [Renshaw RW, Soiné C, Weinkle T, O'Connell PH, Ohashi K, Watson S, et al. A hypervariable region in VP1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 1996;70(12):8872-8]. DNA sequencing of 878 nucleotides from twelve Brazilian CAV, eight of which tested for in vitro isolation in three different sources of MDCC-MSB1 cell line and identified as lacking capacity to propagate in any of these cells, were compared to sequence data available for CAV strains propagated or not in cell culture. Alignment of the deduced aa resulted in a lack of singled out amino acid substitutions in the partial genomic sequences of Brazilian isolates that would entirely contrast them to viruses propagated in MSB-1 cells, indicating that the combined VP1, VP2 and VP3 substitutions observed may not entirely account as sole determinants of CAV isolation and propagation in MDCC-MSB-1 cells.

Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies.

The chicken anemia virus protein Apoptin selectively induces apoptosis in transformed cells while leaving normal cells intact. This selectivity is thought to be largely due to cell type-specific localization: Apoptin is cytoplasmic in primary cells and nuclear in transformed cells. The basis of Apoptin cell type-specific localization and activity remains to be determined. Here we show that Apoptin is a nucleocytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, since Apoptin fragments containing either the NES or the NLS fail to differentially localize in transformed and primary cells. Significantly, cell type-specific localization can be conferred in trans by coexpression of the two separate fragments, which interact through an Apoptin multimerization domain. We have previously shown that Apoptin interacts with the APC1 subunit of the anaphase-promoting complex/cyclosome (APC/C), resulting in G(2)/M cell cycle arrest and apoptosis in transformed cells. We found that the nucleocytoplasmic shuttling activity is critical for efficient APC1 association and induction of apoptosis in transformed cells. Interestingly, both Apoptin multimerization and APC1 interaction are mediated by domains that overlap with the NES and NLS sequences, respectively. Apoptin expression in transformed cells induces the formation of PML nuclear bodies and recruits APC/C to these subnuclear structures. Our results reveal a mechanism for the selective killing of transformed cells by Apoptin.