Distribution and function of JCV agnoprotein

JC virus (JCV), the causative agent of progressive multifocal leukoencephal opathy (PML), encodes six major proteins including agnoprotein, the functio n of which is unknown. To explore its function, we initially studied the ex pression and localization of agnoprotein in both cultured cells and PML bra in using immunohistochemical methods. Employing a specific polyclonal antib ody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendr oglial cells in PML brain. The immunohistochemistry indicated that the cyto plasm of oligodendroglial cells was relatively well-preserved in the demyel inated 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 susceptibl e 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 l arge T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and wh en coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibiti on of protein kinase A by its inhibitor H-89, however, reversed the cytopla smic localization of agnoprotein to the nuclear compartment. Our results su ggest that JCV agnoprotein may "shuttle" between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.