The E1 protein of bovine papillomavirus (BPV) plays several key roles in viral DNA replication. E1 binds the viral origin, unwinds template DNA at the replication fork and recruits cellular replication machinery to the viral DNA. E1 is phosphorylated at multiple sites, and phosphorylation of E1 regulates E1 function and viral DNA replication. A consensus motif for the cellular kinase CK2 was identified at serine 584 near the carboxyl terminus of BPV E1, and found to be highly conserved among papillomavirus E1 proteins. Serine 584 was identified as a substrate of CK1 and CK2 in vitro by mutational and biochemical analysis, and was phosphorylated by a cellular kinase in cultured cells.