Distribution and function of JCV agnoprotein.

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), encodes six major proteins including agnoprotein, the function of which is unknown. To explore its function, we initially studied the expression and localization of agnoprotein in both cultured cells and PML brain using immunohistochemical methods. Employing a specific polyclonal antibody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendroglial cells in PML brain. The immunohistochemistry indicated that the cytoplasm of oligodendroglial cells was relatively well-preserved in the demyelinated foci. Agnoprotein coprecipitated with tubulin in immunoprecipitation assays and the colocalization of agnoprotein with cytoplasmic tubulin was verified by double immunostaining with confocal microscopy. Transfection of an agnogene deleted JCV Mad1 strain [Mad1(Delta agno)] into the susceptible cell line failed to produce not only agnoprotein but also VP1 and large T mRNAs, whereas the wild-type JCV Mad1 resulted in the expression of both large T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and when coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibition of protein kinase A by its inhibitor H-89, however, reversed the cytoplasmic localization of agnoprotein to the nuclear compartment. Our results suggest that JCV agnoprotein may "shuttle" between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.