An investigation of post-translational modification of human papillomavirus E4 and the role of modified E4 proteins during the virus life cycle

[摘要] Infection with human papillomaviruses (HPV) is the etiological basis for cervical cancer. The HPV E1^E4 protein is proposed to be a major regulator of the HPV life cycle and the multiple and diverse activities associated with E1^E4 suggest that it is a multi-functional protein. This thesis has sought to address the hypothesis that phosphorylation and proteolysis contribute towards the pleiotrophic functions of the E1^E4 protein, and has investigated the functional significance of these E1^E4 post-translational modifications during the HPV life cycle. This study has uncovered the novel finding that the E1^E4 protein of HPV type 18 (HPV18) exists as a phospho-protein within cells and is a substrate for multiple cellular kinases in vitro. The phospho-acceptor residue for cyclin-dependent kinases (CDK) 1 and 2 has been identified as threonine 23, whilst serine 58 is phosphorylated by protein kinase A (PKA). Furthermore, a cyclin binding motif (\(^{43}\)RRL\(^{45}\)) within the HPV18 E1^E4 protein is required for association with active CDK complexes and this association may influence CDK activity since the activity of CDK2-cyclin A was shown to be reduced in the presence of HPV18 E1^E4. This thesis has revealed that HPV18 E1^E4 is a target for N-terminal proteolysis, and this post-translational modification occurs during the HPV18 replication cycle. Key elements necessary for proteolysis have been mapped to a conserved leucine-rich sequence (LLXLL) present at the N-terminus of the HPV18 E1^E4 protein. Since E1^E4 expression is required for HPV18 genome amplification, N-terminally truncated E4 species may contribute towards its role in the replication cycle. To examine this hypothesis, mutations that attenuate E1^E4 proteolysis were introduced into HPV18 genomes and transfected into human foreskin keratinocytes (HFK). Mutation of the leucine-rich motif prevented efficient proteolysis of the E1^E4 protein during the HPV life cycle and resulted in reduced viral genome amplification within differentiating HFKs suggesting that efficient E1^E4 proteolysis may be required for this E4 function.

[发布机构] University:University of Birmingham;Department:Institute of Cancer Studies

[关键词] R Medicine;R Medicine (General)