The gene of the minor capsid protein VP2 of human polyomaivurs JCV was cloned into a prokaryotic expression
vector, PGEX4T-1, and expressed in E.coli . The 1035 base-pair DNA of VP2 was sequenced. The amino acid sequence of VP2 was translated and the restriction sites were also analyzed by a computer program. It was found that there are 8 amino acids differences among Mad-1, GS/B and Taiwan-3 JCV VP2. VP2 protein was expressed in E. coli and purified by elentroelution. VP2 antiserum was generated in a rabbit by using the purified recombinant VP2 protein. After synthesized in the cytoplasm, VP2 is transported into the nucleus for virion assembly. In order to identify the nuclear localization signal (NLS) of VP2, full-length VP2 and C-terminal truncated VP2 were cloned into a eukaryotic expression vector, pEGFP-C1, to express a fluorescent VP2 fusion protein in Cos-7 cells. After transfection of Cos-7 cells, intracellular localization of the fusion protein was visualized under UV microscope. The results showed that wild-
type VP2 was able to translocate the fusion protein into the nucleus. The last 13 amino acids truncated VP2 did not loss its
function for nuclear transport. However, the last 29 amino acids truncated VP2 was unable to transport the fusion protein into the nucleus and retained in the cytoplasm. Therefore, the NLS of VP2 was demonstrated to located at the C-terminal last 29 amino acids of VP2. Agter transported into the nucleus, VP2 should interact with viral genome for virion assembly. Therefore, DNA binding activity of VP2 was further investigated. Southwestern blot was employed for DNA binding assay in this study . Our results showed that VP2 was able to bind to DNA but the last 13 amino acids truncated VP2 was not. Furthermore, the last 13 amino acids were mutated by site-directed mutagenesis to determine the crucial amino acid(s) for DNA binding. The results showed that Lys-332 and Lys-336 mutated VP2 reduced DNA binding activity drastically. The results in this study demonstrated that the NLS and the DNA binding domain of VP2 were located at C-terminus but at different regions. These findings may provide some important information in the mechanisms of virion assembly
and infection pathway of JC virus.
