Caractérisation de la fonction de la protéine cellulaire p80/UAF1 dans la réplication du génome du virus du papillome humain

Human papillomaviruses (HPVs) are the etiological agents of cervical cancers, as well as multiple other anogenital and oropharyngeal neoplesias. The viral proteins E1 and E2, in concert with the host DNA replication machinery, mediate the replication of the double-stranded DNA genome of HPV. E1 exploits its helicase activity to unwind DNA ahead of the replication fork and orchestrates synthesis of the viral genome. Our previous work demonstrated that E1 contains in its N-terminus a binding motif for the host protein p80/UAF1, a domain that is highly conserved amongst anogenital HPVs. The integrity of this region was essential for the maintenance of the viral episome.  The research presented here first demonstrated that the UAF1-binding motif is not required for the assembly of the E1-E2-Origin pre-replisome, but important for the following viral DNA replication. We have determined that UAF1 is relocalized, in presence of E1 and E2, in nuclear foci reminiscent of viral DNA synthesis sites. UAF1 also physically interacted, through E1-binding, with the viral origin of replication. Moreover, we have shown that inhibition of E1-UAF1 interaction through the overexpression of an E1-derived and UAF1-binding peptide, N40, interferes with HPV DNA replication. This is in agreement with the model according to which E1 recruits UAF1 to the replisome to promote viral DNA replication.  UAF1 is a WD40-containing protein with no enzymatic activity and presumed to function through interactions with other cellular factors. We have investigated the UAF1 interaction network in cervical cells and discovered that UAF1 associates with the deubiquitinating enzymes USP1, USP12 and USP46, as well as with the phosphatase PHLPP1. E1 was found to assemble as a ternary complex with UAF1 and any of the associated USPs: USP1, USP12 or USP46. These USPs were also relocalized by E1 to the nucleus and they associated with the viral origin in presence of E2. Moreover, their enzymatic function was essential for optimal viral genome replication. In contrast, PHLPP1 did not associate with E1, and the interactions of the latter proteins with UAF1 were shown to be mutually exclusive. PHLPP1 contains a UAF1-binding motif homologous to the one encoded within E1. This PHLPP1-derived peptide, P1, interacts with the UAF1-USP complex and, similarly to N40, competes with E1-UAF1 interaction. Accordingly, P1 overexpression leads to inhibition of HPV DNA replication. The fusion of the peptide P1 to an E1 protein (E1Δ) defective for UAF1-binding restored its capacity to interact with UAF1 and USP46, as well as to relocalize UAF1 into E1-E2-containing nuclear foci. This artificial recruitment of UAF1 and of the associated USPs increased viral DNA replication, a process that involved the enzymatic activity of the USPs.  Collectively, our work suggests that HPV E1 interacts with UAF1 in order to recruit to the replisome a deubiquitinating complex whose activity is required for optimal viral DNA replication.

Keyphrases: Papillomavirus, UAF1, p80, USP1, USP12, USP46, WDR48, HPV, E1 helicase, DNA replication, PHLPP1