Role of the human mediator complex in varicella zoster virus IE62 mediated transcriptional activation
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The varicella zoster virus (VZV) major transactivator, IE62, contains a potent N-terminal acidic transcriptional activation domain (TAD). Our experiments revealed that the minimal IE62 TAD encompasses aa 19-67. We show that the minimal TAD interacts with the human Mediator complex. Site-specific mutation revealed residues throughout the minimal TAD are important for both activation and Mediator interaction. The TAD interacts directly with aa 402-590 of the MED25 subunit and site specific TAD mutations abolished this interaction. Two-dimensional NMR spectroscopy revealed the TAD is intrinsically unstructured. This suggests that transactivation may involve the TAD adopting a defined structure upon binding MED25. The effects of the TAD mutations were examined in recombinant viruses, and we found that TAD mutant viruses replicated to a similar extent as wild type virus in MeWo cells, and recruited Mediator to the nuclear viral replication compartments despite the significant reduction at the transcription step. The effects of the TAD mutations were also examined in the context of full-length IE62 in the absence of viral infection. The results showed that the TAD mutations significantly decreased, but did not completely abolish the transactivating activity of IE62 in MeWo cells. The observation that the TAD mutations differentially decreased transcriptional activity suggests that they also differentially affect the affinity of the TAD for its binding partner(s). The presence of additional transcription factors USF/Sp1 alleviated the effects of the mutations, suggesting that these factors may independently recruit Mediator. While effects of the mutations were similar in MeWo cells and Vero76 cells, they generally reduced IE62 activity more profoundly in SHSY5Y neuroblastoma cells, indicating the presence of cell-type specific factor(s) involved in the IE62 mediated transactivation. Furthermore, we showed the presence of an alternative mechanism independent of the N-terminal TAD, as TAD-deletion mutants continued to transactivate although to a significantly lesser extent. Finally, the transcriptional synergy between IE62 and NFκB was examined. The data showed, unlike observations with USF and Sp1, IE62 did not synergize with NFκB. This result, coupled with previous observations by other laboratories, suggests that VZV evolved to negate NFκB induced host antiviral responses by not requiring NFκB for viral gene expression, and sequestering NFκB into the cytoplasm to avoid expression of cellular antiviral genes. This thesis work advanced our understanding of VZV gene expression mediated by IE62, and showed IE62 utilizes human Mediator in conjunction with the cellular transcription apparatus. These interactions could be targets for intervention in VZV infection.