Thermodynamic, phylogenetic and structural studies of the mechanism of DNA sequence specific binding by papillomavirus *E2 proteins

Abstract

'High risk' human papillomaviruses (HPVs) are double-stranded DNA viruses that infect epithelial tissue causing cervical and other cancers. Of the over 20 HPV types associated with cancer, HPV-16 is the most prevalent and represents ∼50% of infections. Cancer progression has been linked to PV genome host integration and E2 gene disruption, leading to the upregulation of E6/E7 oncogene expression and breakdown of cell cycle control. E2 proteins from papillomaviruses bind with high affinity to palindromic DNA sequences consisting of two highly conserved four base pair sequences flanking a variable 'spacer' of identical length (ACCG NNNN CGGT). Discrimination of DNA sequences by their conformational and/or dynamic properties in the absence of direct contact is termed 'indirect readout'. This analysis utilized three different cognate sequences with unique conformational and dynamic properties to reveal a novel mechanism of 'indirect readout' as a key contribution to E2 protein sequence affinity and specificity. Furthermore, we explored the E2 DNA binding domain from 122 viral types and 24 HPV16 variants to assess the contributions of the structural and energetic characteristics that may contribute to a 'high risk' viral phenotype. Overall, the results show the retention of the overall fold within a 2 A RMSD and confirm the importance of the E2 DNA binding domain for viral viability. Analysis of the HPV 16 variants reveals conserved variations in the E2 protein DNA binding domain localized to the surface that interacts with the viral replication protein E1. Together this work presents a novel mechanism for E2 sequence recognition, highlights the importance of the E2 gene both structurally and energetically, suggests a mechanism for the increased cancer risk in HPV16 Non-European variants, and provides a hypothesis that will directly affect human biology and cancer treatment.