Investigation of origin dependent replication of VZV DNA
Khalil, Mohamed Ibrahim
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Varicella Zoster Virus (VZV) contains two origins of DNA replication (oriS) flanked by the ORF62 and ORF63 genes. The oriS contains a 46 bp A-T rich palindrome and three consensus binding sites for the VZV origin-binding protein (OBP). All three of the OBP-binding sites are upstream of the palindrome. A partial downstream OBP binding sequence was previously identified however there was no proof of binding of the VZV OBP to this site. The work presented in this thesis investigated the role that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes designated GC box 1 and GC box 2, in the downstream region. We found that two members of the Sp family of transcription factors (Sp1and Sp3) stably bind to GC box 1. A predicted binding site for the cellular factor YY1 was also identified. Supershift and mutational analysis confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding, an increase in origin-dependent replication efficiency and a decrease in expression from a bidirectional dual reporter plasmid containing the VZV oriS region flanked by luciferase reporters in the ORF62/63 positions. Mutation of the YY1 site had insignificant effect on DNA replication efficiency but increased transcription from both reporters. The role that the partial downstream OBP binding site, Box D, plays in oriS-dependent DNA replication was also investigated. We found that there are characteristic complexes formed with a Box D containing oligonucleotide using uninfected and infected nuclear extracts. Mutation of the core CGC motif resulted in loss of the infected cell complex. This mutation also resulted in an increase in origin-dependent DNA replication efficiency and a decrease in expression from the bidirectional reporter. Most importantly, in replication incompetent plasmids also containing a Box D mutation, no expression was observed from the luciferase reporter in the ORF63 position. These results indicate that the Box D sequence plays a key role in expression of the latency associated VZV ORF63 gene. These results suggest a model in which origin dependent DNA replication and viral transcription are coupled by the binding of Sp1, Sp3 and YY1 cellular transcription factors to the downstream region of the VZV replication origin and the formation of the infected cell complex with Box D sequence during lytic infection. They also have implications regarding the establishment or reactivation of viral latency.