Functional studies of the Human Papillomavirus E7 protein.

Massimi, Paola (2000). Functional studies of the Human Papillomavirus E7 protein. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ff62

Abstract

El is the major Human Papillomavirus transforming protein. Mutations in regions of E7 result in a loss or reduction of transforming activity even through wild type levels of pRB binding are retained, indicating the existence of additional cellular targets.
I performed a series of experiments to investigate whether HPV E7, like AdEla, can bind to TBP. E7 from both benign- and malignancy-associated HPVs binds to TBP, both in vitro and in vivo. Alignment of E7 and Ela sequences shows that the core TBP binding domain on Ela is close to its CKII recognition site, and I found that the phosphorylation can increase the binding of both E7 and Ela to TBP. Mutational analysis confirmed that the highly conserved carboxy-terminal region of TBP is involved in the interaction with E7, and also in the binding with other proteins, such as E2, E6 and p53. In addition, two carboxy terminal mutants of E7 were found to be defective in the binding with TBP.
Two possible biological consequences have been shown for the E7-TBP interaction:
1 ) E7, like AdEla, inhibits p53’s transcriptional activity through the formation of a tripartite complex between E7, TBP and p53.
2) E7 mutants which are defective in TBP binding are less transforming, indicating a partial role for TBP in the transformation function of E7.
Considering the importance of E7 phosphorylation in transformation and in regulation of the association with TBP, I dedicated the second part of the study to analysing the E7 phosphorylation state in vivo. I identified a second E7 phosphorylation site which lies at Ser 71, and, more interestingly, I found changes in the phosphorylation level of E7 protein which could regulate the specificity of the E7-TBP interaction through well-defined phases of the cell cycle.

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