CAV is a small non-enveloped, single-stranded, circular DNA virus and was first isolated in Japan . This virus belongs to the genus Gyrovirus of the Circoviridae family and causes a severe immunosuppressive syndrome and anemia in chickens . It is a ubiquitous pathogen of chickens and has a worldwide distribution. According to epidemiological studies, it has been shown that almost all newly hatched chicks are susceptible to CAV as a clinical syndrome, but not mature chickens . Normally, young chickens, generally fewer than 2 weeks of age, are very susceptible to this virus through vertical transmission via hatching eggs . The virus typically induces aplasia of the bone marrow and damage to lymphoid tissue, which causes anemia and acute immunodeficiency syndrome [5, 6]. Up to the present, a large number of isolates, including strains from Australia, Bangladesh, Brazil, China, Germany, Malaysia, Nigeria, Slovenia, Taiwan and USA, have been reported and have had full or partial sequences published [7–9].
The DNA genome of CAV is about 2.3 kb in size [10–12] and there are three ORFs present on the negative sense genome. At least three proteins are produced from a single polycistronic 2.1 kb mRNA that is reliable produced as a single molecule and contains a promoter, TATA-box, and poly (A) signal [11, 13, 14]. The three translated proteins are called VP1, VP2 and VP3. VP1 is a 51 kDa protein that is the structure protein involved in assembly of the viral caspid . VP2 is a 24 kDa protein that contains a dual-specificity phosphatase (DSP) activity and some apoptotic activity [2, 16, 17]. However, VP2's apoptotic activity is much weaker than that of VP3. VP3 is a 13 kDa protein, also named apoptin, which induces apoptosis in infected chicken cells and human tumor cell lines [2, 18, 19]. During virus infection, VP2 and VP3 are detected very early, namely 12 h post infection, while VP1 is detected only after 30 h post infection . Some additional proteins have been reported to be translated after further splicing of the mRNA, but their biological functions have not been elucidated .
As mention above, VP2 is a DSP enzyme . The key catalytic residues of active site have been identified to be serine, threonine, and tyrosine. The cysteine residues, respectively, are located at positions 95 and 97 in the catalytic motif of VP2. Furthermore, mutation of these residues to serine results in reduced virus replication efficiency in the cell . This effect indicates that the phosphatase activity of VP2 is required for virus replication. It has been reported that co-expression of VP1 and VP2 allows neutralizing antibodies to be raised [21, 22] and it has been suggested that VP2 is a scaffold protein  that corrects the conformation of VP1. Therefore, it is expected that VP2 is a multifunctional protein with roles in virus infection, assembly and replication. VP2 possess a putative NLS and the protein is known to accumulate to a large extent in the nucleus of infected chicken cells [16, 24]. A recent study has shown that VP2, when fused to GFP, shows nuclear localization and this result indicates that the NLS of VP2 is also functional in plants .
Until now, the specific mechanism for the cellular localization of VP2 is not well understood. In this study, we first use bioinformatics to analysis the amino acid sequence of various different isolates of VP2, and were able to predict and examine for the presence of putative NLS and NES motifs, which have never been characterized previously. We generated GFP fused to various versions of VP2 created by truncation, site directed mutagenesis, and multiple site directed mutagenesis in order to confirm the locations of these putative NLS and NES sequences. Leptomycin B (LMB) can be used to identify the presence of a NES motif in a protein because it inhibits the CRM1 pathway and such a result has been found for VP3 of CAV. Using LMB, our results suggest that VP2 does not contain a functional NES and also that VP2 is CRM1 independent. Additionally, using a co-immunoprecipitation assay, we also found that VP2 associates with MCM3 and that this interaction does not require DSP activity.