Role of DNA binding in Varicella Zoster IE62 transactivation
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The Varicella Zoster Virus (VZV) IE62 protein is the major viral transactivator and is the most extensively studied VZV immediate early (IE) protein due to its importance in viral gene regulation. Little however is known about the role of IE62 DNA binding during transactivation. The DNA-binding domain (DBD) encompasses amino acids 417-647 of the IE62 sequence and contains a helix-turn-helix motif. It is known that IE62 is capable of associating with DNA both non-specifically and in a sequence specific manner with a consensus site (ATCGT). However, the individual functions of these interactions are poorly understood. Sequence analysis of the VZV genome identified 245 IE62 consensus sites with an upper estimate of some 54 being present in approximately one-half of putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using a bacterially expressed IE62 fragment containing the DBD and duplex oligonucleotides showed that the DBD has a 4-fold increased affinity for its consensus site as compared to a non-consensus sequence. Luciferase reporter experiments indicate that the presence of the consensus sequence results in suppression of IE62-mediated transactivation in model and native VZV promoters. The small difference in affinity for consensus versus non-consensus DNA sequences suggests that binding to the consensus site may result in a conformation impaired for interaction with the cellular transcription apparatus. The role of specific protein-protein interactions with the IE62 DNA-binding domain and their effect on IE62 DNA-binding and transactivation was also examined. Affi-gel chromatography revealed that viral IE63 protein interacts with the DBD whereas the cellular general transcription factors TBP and TFIIB do not. EMSAs showed that the interaction of IE63 with the IE62 DBD caused a loss in DBD binding to non-specific DNA and an increase in site-specific DNA binding. In luciferase reporter experiments the loss of DNA binding activity correlated with a loss of transactivation. The presence of IE63 decreased the relative difference in affinity of the IE62 DBD for consensus vs. non-consensus DNA from 4-fold to 15-fold. Finally, a mutational analysis of the DBD indicated that mutation of lysine 548 to glutamate, previously suggested to be severely deficient in site-specific DNA binding and transactivation, in fact only had a minor defect in general and specific DNA binding. In gene reporter assays however, the K548E mutation caused a 70-97% decrease in IE62-mediated transactivation of all promoter tested. These data suggest that the K548E mutation is affecting IE62 function through a mechanism distinct from DNA binding.