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VP3 (Bullenkamp et al., 2012). At this time, it is not clear if VP3 nucleus and reduced the degree of apoptosis. These results show
of other anelloviruses also have apoptotic activity. that NLS1 and 2 are needed for optimal accumulation in the
nucleus and that nuclear accumulation correlates with apoptosis.
Apoptin: use as an anti-cancer therapy Similar results were reported by Poon et al. (2005). Surprisingly,
The mechanism of apoptosis induction in chicken cells in vivo or AA 1–69 and AA 80–121 are both involved in cell killing. AA
chicken cell lines in vitro has not been fully elucidated. However, 1–69 fused to a heterologous NLS and transfected into Saos-2
the discovery that transfection of certain human tumour cell lines cells, which lack p53, accumulated in the nucleus of the cells
with VP3 caused apoptosis has led to a large number of papers resulting in increased induction of apoptosis but still below the
examining the interactions between Apoptin and human tumour level caused by Apoptin (Danen-Van Oorschot et al., 2003). Using
cell lines (see below). Some of the information has relevance for Apoptin protein it was shown that the AA1–69 and AA80–121
the understanding of the development of apoptosis after infec- fragments could bind to naked DNA (Leliveld et al., 2004). Phos-
tion with CAV. However, the interaction between VP2 and VP3 is phorylation of AA T108 mediated by tumour-specific kinases is
important in the chicken system but this interaction is absent in important for the induction of apoptosis perhaps by facilitating
the transfection experiments with Apoptin. the transfer into the nucleus and/or activation of Apoptin (Rohn
Shortly after the discovery that transfection of MSB1 cells et al., 2002) or preventing export from the nucleus in tumour cells
with Apoptin caused apoptosis, Zhuang et al. (1995a,b) showed (Poon et al., 2005). Rohn et al. (2002) quote unpublished data
that transfection of human osteosarcoma cell lines and four that VP3 undergoes phosphorylation after infection of MSB1
human lymphoblastoid cell lines with ORF3 caused apoptosis cells with CAV.
in these cell lines. The induction of apoptosis was independent Danen-Van Oorschot et al. (2003) were unable to determine if
of the presence or absence of p53, a known inducer of apoptosis, the NES at AA 33–46 was functional or a cytoplasmic retention
and high expression of the proto-oncogene bcl-2. Transfection signal. Wang et al. (2004) suggested that the sequence was a cyto-
with VP3tr (see previous section) resulted in a delayed onset of plasmic retention signal rather than a NES. However, a true NES
apoptosis and retention of VP3 in the cytoplasm. Transfection was located at AA 97–105 (Poon et al., 2005) which is functional
of several types of normal, non-transformed human cell lines did in normal but not in tumour cells. Phosphorylation of T108 inhib-
not result in apoptosis and in these cells the Apoptin remained its the export of Apoptin from the nucleus into the cytoplasm in
in the cytoplasm while it localized in the nucleus of transformed tumour cells. Fig. 9.8 provides a schematic model summarizing
cells (Danen-Van Oorschot et al., 1997). Additional studies by how Apoptin may remain in the nuclei of tumour cells. Recently,
Noteborn and collaborators confirmed that transformed cells, but several groups have identified different kinases involved in the
not normal cells are susceptible to Apoptin-induced apoptosis phosphorylation of Apoptin. Depending on the tumour cell
(reviewed in Rohn and Noteborn, 2004; de Smit and Noteborn, line examined, abnormal phosphatidylinositol-3kinase/Akt
2009; Noteborn, 2009). Several nude mouse models with activation resulting in the activation of cyclin-dependent kinase
xenografts of human tumours have shown that Apoptin reduces 2 (Maddika et al., 2009), protein kinase Cβ (Jiang et al., 2010;
tumour growth in vivo (e.g. Pietersen and Noteborn, 2000; Back- Bullenkamp et al., 2015) or checkpoint kinase (Chk)1 and Chk2
endorf et al., 2008; Backendorf and Noteborn, 2014; Liu et al., (Kucharski et al., 2016) are phosphorylating Apoptin. Interest-
2016). Healthy rats remained healthy after repeated injections ingly and relevant for understanding the induction of apoptosis
with replication-deficient adenovirus vectors expressing Apoptin in MSB1 cells, inhibition of Chk1/2 activity significantly reduced
(AdMLP-VP3) and transgenic mice expressing Apoptin in many the percentage of MSB1 cells undergoing apoptosis and the virus
tissues did not show negative effects (reviewed in Pietersen and titre.
Noteborn, 2000) suggesting that treatment with AdMLP-VP3 The entrée and retention of Apoptin into the nucleus of
may be safe. Currently, several different methods to deliver Apop- transformed cells has been elucidated but the actual mechanism
tin including virus, plasmid, and Salmonella vectors expressing of the induction of apoptosis is not fully resolved (Noteborn,
VP3 and as recombinant protein are proposed for clinical trials 2004). Phosphorylated Apoptin can interact with different
(Backendorf and Noteborn, 2014). Tavassoli et al. (2005), how- proteins all leading to apoptosis in dividing and non-dividing
ever, questioned the specificity of Apoptin for transformed cells. tumour cells. The interested reader is referred to a recent review
Wadia et al. (2004) suggested that a concentration-dependent article by Bullenkamp and Tavassoli (2004) for more detailed
NLS rather than a tumorigenic NLS was responsible for the high information.
levels of expression of Apoptin needed to induce apoptosis in
tumour as well as normal cells, which was disputed in a response Stages of viral replication
by Rohn et al. (2005). The replication cycle of CAV has not been studied in great detail
VP3 consists of 121 AA with a putative nuclear export signal in cell culture nor in chickens and remains poorly understood.
(NES) at AA 33–46 and a bipartite NLS at AA 82–88 (NLS1)
and at AA 111–121 (NLS2) (Danen-Van Oorschot et al., 2003). Attachment, penetration and uncoating
Fusion of VP3 AA 80–121 to green fluorescent protein (GFP) The mechanism of attachment of CAV to target cells has not
redirected GFP to the nucleus, while fusion of AA 100–121, been elucidated. Infection of lymphoblastoid cell lines has
lacking NLS1, to GFP failed to do so. Mutation of AA 86–90 been reported for several MDV-transformed T-cell lines, but
(KKRSC) to alanines impaired the localization of GFP to the as mentioned before the susceptibility may vary with passage