Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.Human polyomavirus JC (JCV) persists asymptomatically in healthy individuals of most of the human population. However, in immunocompromised individuals, JCV can cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disorder of the central nervous system. JCV has a genome of a double-stranded circular DNA composed of the early region and the late region. Genome organization of JCV is closely related to that of simian virus 40 (SV40), which shares 70% identity in nucleotide sequence. The JCV replication cycle is divided into the early stage and the late stage. During the late stage, the capsid proteins are synthesized in the cytoplasm and then transported to the nucleus to be assembled into progeny virions. By electron microscopy, JCV virions are identified as round particles or filamentous forms in the nuclei of infected oligodendrocytes of PML brains (41,44,69).JCV Tokyo-1 strain was isolated from the brain tissue of a Japanese PML patient (43, 44), and the viral genomic DNA was cloned (40). The virus particles of Tokyo-1 were purified, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue. The detected protein components indicated the presence of the major capsid protein VP1 (43 kDa), the minor capsid proteins VP2 (39 kDa) and VP3 (27 kDa), and histones (16, 15, and 14 kDa) incorporated into the viral capsids (2).Based on the crystal structures of other polyomaviruses (26...