DNA replication in the DNA viruses usually occurs in the nuclei of infected cells. Thus, viral DNA needs to cross the nuclear envelope through protein-mediated nuclear transportation to establish a productive infection after infecting the cells . CAV is a non-enveloped, small DNA virus containing a circular ssDNA genome . VP1, which is a major capsid protein of CAV, interacts with cells during virus infection, and the viral genome should theoretically be carried by VP1 to enter cells. The main question is what protein is conferred a function to direct a viral genome into the nucleus for sequential DNA replication? VP1 is thought to have a functional role to bind and direct the viral genome into the nucleus. However, there is still a lack of direct evidence to support this speculation. In our results from computational prediction, four putative DNA binding motifs were combined with the amino acid sequence of the CAV VP1 protein. Although the exact DNA binding motif was not determined in this study, it did not affect the characterization of VP1 DNA binding activity. After performing a DNA-binding assay to examine DNA migration, the VP1 protein of CAV was confirmed to have DNA-binding activity. Additional experiments are still needed for identifying major DNA binding motifs within VP1 protein. The confirmed DNA-binding activity in VP1 might be useful to further verify the underlying mechanisms of viral DNA replication. In addition, GFP-VP1 has demonstrated nucleo-cytoplasm shuttling activity (Fig. 3). The results imply VP1 is a nuclear protein to binds DNA molecules, such as those in the viral genome and travels into the nucleus during its early life cycle. In fact, we have not only predicted the presence of nuclear localization signals (NLSs) with PSORT II but also predicted nuclear exporting signals (NESs) with NetNES server (http://www.cbs.dtu.dk/services/NetNES/). These putative NLS motifs were within the amino acid sequence of VP1, which spanned from amino acid residues 3 to 19 (NLS1) and 24 to 47 (NLS2). For NESs, the putative motifs within the VP1 protein spanned from amino acid residues 76 to 84 (NES1), 109 to 119 (NES2) and 375 to 387 (NES3) (Fig. 1a). In terms of the observation of fluorescent of GFP-VP1, this result was confirmed according to computational predictions. In the early life cycle of the virus, viral DNA replication is an important stage for the establishment of productive infection . Previous studies have reported circular, negative ssDNA of the CAV genome might be replicated through rolling-circle amplification . During this stage, previous researchers isolated the viral replicative form (RF) DNA, which is an open circular dsDNA obtained from MDCC-MSB1 cells after being infected with the virus for 30 h . In addition to the presence of closed and open circular dsDNA, circular ssDNA with genome-sized and small linear dsDNA of 800 bp were observed in the later stages of DNA replication . In this study, all similar DNA molecules with different conformations, including linear ssDNA, which is derived from circular ssDNA, were used to examine the possible functional roles of the VP1 protein in DNA-binding activities. Additionally, other putative DNA binding motifs, especially for the initiation of rolling-circle amplification (RCR) within the VP1 protein, had also been reported and predicted in a previous study . Three putative motifs were proposed that spanned from amino acid 313 to 320 (FATLTALG), 350 to 358 (GQRWHTLVP), and 399 to 408 (TATYALKEPV). These motifs might be interaction sites, such as the origin (Ori) site of the CAV genome, for interacting with VP1 for regulating DNA replication .