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Human herpesvirus 7 (HHV-7) DNA sequences colinear with the HHV-6 lytic-phase origin of DNA replication (oriLyt) were amplified by PCR. Plasmid constructs containing these sequences were replicated in HHV-7-infected cord blood mononuclear cells but not in HHV-6-infected cells. In contrast, plasmids bearing HHV-6 oriLyt were replicated in both HHV-6- and HHV-7-infected cells. Finally, the minimal HHV-7 DNA element necessary for replicator activity was mapped to a 600-bp region which contains two sites with high homology to the consensus binding site for the HHV-6 origin binding protein. At least one of these binding sites was shown to be essential for replicator function of HHV-7 oriLyt.