It has been reported that phosphorylation of rabies virus N plays an important role in the process of viral transcription and replication. Rabies virus N is phosphorylated when expressed alone, indicating that cellular kinase phosphorylates rabies virus N. To identify what cellular kinase phosphorylates rabies virus N, the N was expressed in Escherichia coli and purified by metal affinity chromatography. The recombinant N was phosphorylated by BHK cellular extracts and by purified CK-II. In addition, the phosphorylation of the recombinant N in vitro can be blocked by a CK-II inhibitor, heparin. Furthermore, N phosphorylation in the virus-infected cells can be inhibited by a CK-II specific inhibitor, 5,6-dichloro-beta-D-ribofuranosyl benzimidazole. However, PKC did not phosphorylate the recombinant N in vitro; nor did staurosporine, a PKC and other kinase inhibitor, prevent rabies virus N from phosphorylation. Thus, our data demonstrate that cellular CK-II phosphorylates rabies virus N. (C) 2003 Elsevier Science (USA). All rights reserved.