Borna disease virus (BDV) replicates and transcribes its negative-sense RNA genome in the nucleus. The BDV phosphoprotein (P) is localized in the nucleus of infected cells and cells transfected with P expression constructs. To identify the nuclear localization signal (NLS) of P, COS-7 cells were transfected with wild-type or mutant forms of P fused with green fluorescent protein (GFP). Whereas GFP alone was exclusively cytoplasmic, P or P-GFP were nuclear. Analysis of carboxy- and amino-terminal truncation mutants of P indicated that amino acids (aa) 20-37 are sufficient to promote efficient nuclear accumulation of the fusion protein. Residual nuclear import of GFP was observed with portions of P including aa 33-134 or aa 134-201, suggesting the presence of additional NLS motifs. The major NLS of P appears to be bipartite. It consists of two basic aa domains, R22RER25 and R30PRKIPR36, separated by four non-basic aa, S26GSP29.


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