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.