After 1 h of absorption, the supernatants were removed and fresh medium was added. The cells were then mixed with cell-free BDV. Proteins purified from mock-transfected cells using an anti-FLAG M2 affinity gel served as a control. OL cells were pretreated with 4 μg/ml GP1-TAP for 30 min. To further validate the binding of GP1-TAP, we tested whether GP1-TAP inhibits BDV infection. A flow cytometric analysis indicated that GP1-TAP binds to OL cells (Fig. To verify that GP1-TAP binds to OL cells, the cells were incubated with 4 μg/ml GP1-TAP, and binding was detected by anti-FLAG M2 antibody (Sigma). GP1-TAP was purified using anti-FLAG M2 affinity gel (Sigma). To extend our understanding of the role of BDV G in the interaction with the cell plasma membrane, we transfected GP1 fused with hemagglutinin-tobacco etch virus protease cleavage site-FLAG tags (GP1-TAP) into human oligodendroglioma OL cells. In addition, a previous work using a hippocampal culture system suggested that BDV G is required for viral dissemination in neurons ( 2) however, cellular factors involved in BDV cell entry, especially cell surface association, remain to be elucidated. Recent studies revealed that GP1 is involved in virus interaction with as-yet-unidentified cell surface receptor(s) and that GP2 mediates a pH-dependent fusion event between viral and cell membranes ( 2, 7, 27). BDV G is translated as a precursor protein, GP, which is posttranslationally cleaved by the cellular protease furin to generate two functional subunits of the N (GP1) and C (GP2) termini ( 28). BDV cell entry is mediated by endocytosis, following the attachment of viral envelope glycoprotein (G) to the cellular receptor ( 2, 7, 8). BDV infects a wide variety of host species and causes central nervous system (CNS) diseases in animals, which are frequently associated with behavioral disorders ( 14, 19, 29, 31). Borna disease virus (BDV) belongs to the Bornaviridae family of nonsegmented, negative-strand RNA viruses and is characterized by highly neurotropic and noncytopathic infection ( 18, 33).

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