The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.