Human papillomavirus (HPV) E2 is essential for transcriptional regulation of viral oncoprotein expression and the replication and persistence of episomal HPV genomes. Episomal persistence is mediated by tethering of viral genomes to host cell chromosomes during mitosis. Previous work demonstrated that interaction of E2 with the cellular DNA helicase ChlR1 is necessary for viral genome tethering. Therefore, disruption of this interaction is a potential therapeutic target for persistent HPV infections. To investigate the use of fragment-based drug discovery in the development of novel inhibitors of the E2-ChlR1 interaction, a fragment library was screened to identify those that bind E2 and several hits were identified. Concurrently, the interaction between HPV16 E2 and ChlR1 was characterised and shown to be a direct protein-protein interaction. The binding sites within E2 and ChlR1 were mapped and this information was used to identify a mutant E2 protein unable to bind ChlR1 (E2-Y131A). E2-Y131A was functionally characterised. HPV16 genomes encoding E2 wild type and Y131A were transfected into primary human keratinocytes to study the differentiation-dependent virus life cycle. Mutant genomes failed to establish genome maintenance, providing strong evidence that the interaction between HPV16 E2 and ChlR1 is necessary for the persistence of HPV infection.