JC virus (JCV) infects 70-90% of the human population and causes progressive multifocal leukoencephalopathy (PML) in severely immunocompromised individuals. This virus is difficult to culture, and this has interferred with studying the three viral capsid proteins: VP1, VP2, andVP3. Thus, it is unknown how these proteins are synthesized and assembled into the capsids. The ultrastructure of the JCV capsid has not been determined. To study the synthesis and structure of the JCV capsid proteins, first a complete sequence of JCV Tokyo-1 strain (5128 bp) was determined. Open-reading frames (ORFs) of four late proteins were deduced: agnoprotein, nt 275-290; VP2, nt 425-1558; VP3, nt 881 - 1558; and VP1, not 1467 - 2531. Second, mRNAs for the capsid proteins were analyzed. One unspliced mRNA and three alternatively spliced mRNAs were detected, and splice sites for each spliced mRNA were determined. The unspliced mRNA could translate VP2 and VP3. One of the three spliced mRNAs could translate VP1. The other two spliced mRNAs could encode new proteins; this has to be experimentally determined. Finally, a highly efficient eukaryotic expression vector for capsid protein which I developed with Drs. Nagashima and Yasui (in Japan) was analyzed. A viral gene fragment (nt 409 - 2531) of JCV Tokyo-1 strain, including introns, was subcloned into an eukaryotic expression vector, pcDLSRI-296, to construct VP231-SRI vector. Six different mRNAs were detected from the VP231-SRI transfected cells. Four of the six mRNAs were identical to those detected from the wild type JCV. Of the six mRNAs, VP1 mRNA was dominantly expressed. VP231-SRI expressed a very large amount of VP1, which was efficiently transported to the nucleus and assembled into recombinant capsids. In future studies, (1) the function of the leader sequence encoding the agnoprotein will be analyzed since analysis of the expression vector suggested that the leader sequence has an important role in regulating transcription and/or translation levels and ratio of the three capsid proteins; (2) regulation of the alternative splicing(s) of the capsid proteins will be further investigated; (3) nuclear transport of VP1, VP2 and VP3 will be analyzed since our data have suggested that the nuclear transport of VP1 is highly enhanced by expression of VP2 and VP3; and (4) the fine structure of recombinant JCV capsids will be determined.
