Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

Abstract

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1-and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. Human papillomaviruses (HPVs) are small double-stranded DNA viruses that replicate and cause hyperproliferative lesions within stratified epithelia (reviewed in references 26 and 34). Among the 100 described HPV genotypes, more than 30 infect the anogenital mucosa (3, 15). These mucosal types are classified as low-or high-risk viruses according to their capacity to cause benign or cancerous lesions, respectively. High-risk HPVs are the etiological agents of cervical cancer, the second leading cause of cancer in women worldwide (47). These viral types have also been linked with other malignancies of the genital tract and with the majority of anal cancers in both men and women (2,13,24,48). Specific high-risk types, and in particular HPV type 16 (HPV16), are also increasingly being associated with a subset of head and neck cancers (32).HPVs infect keratinocytes of the basal cell layer. Upon infection, the viral genome is established as a multicopy episome in the nuclei of these cells through several rounds of viral DNA replication. Maintenance of the viral episome at 50 to 100 copies in these undifferentiated cells is then achieved through low levels of DNA replication and is essential for the persistence of the infection, itself a risk factor for virus-induced carcinogenesis. As the infected cells differentiate and reach the upper layers of the ep...

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

“…The plasmids encoding GFP-E1 and yellow fluorescent protein (YFP)-E1 from HPV31 were previously described (7,24). Those expressing codon-optimized 3F-E1 (p31E1), 3F-E2 (p31E2), and red fluorescent protein (RFP)-E2 were also described earlier (8,25). The plasmid used to express PHLPP1 fused to GFP was constructed by inserting the PHLPP1 ORF (obtained from Life Technologies; Mammalian Gene Collection clone 3916044) between the SacI and BamHI sites of plasmid pGFP2-N1 (BioSignal Packard-PerkinElmer).…”

“…The plasmids used to express UAF1 and its truncated derivatives tagged at their N terminus with a triple-Flag epitope (3F) were previously described (8). The vector expressing UAF1 fused to green fluorescent protein (GFP) was constructed by inserting the UAF1 open reading frame (ORF) between the EcoRV and KpnI sites of plasmid pGFP2-C2 (BioSignal Packard-Perkin-Elmer).…”

“…Little is known about the molecular determinants of E1 that mediate its interaction with UAF1, other than our previous observations that a peptide spanning the E1 UAF1-binding site (UBS; amino acids [aa] 1 to 40) is intrinsically disordered in solution and that mutations of conserved residues within this region abrogate binding (7,8). Our understanding of the E1-binding surface on UAF1 is also limited.…”

“…However, because SLD1 shows only weak sequence similarity to other ubiquitin-like domains, additional evidence is needed to ascertain its function. We previously reported that a fragment of UAF1 encompassing both the WD repeat region and SLD1, but lacking SLD2, is sufficient for interaction with HPV E1, thus raising the possibility that the two SLDs may function independently (8). Clearly, additional studies are needed to elucidate how E1 interacts with UAF1-USP complexes and, more generally, how UAF1-containing deubiquitinating enzymes recognize their substrates.…”

“…Several regulatory motifs have been identified in this region, including a bipartite nuclear localization signal (NLS) and, for most HPV types, a nuclear export signal (NES) and a cyclin-binding motif (CBM) that promote the nucleocytoplas-mic shuttling of E1 in a Cdk2 phosphorylation-dependent manner (4). We previously reported that the first 40 amino acids of E1 from anogenital HPV types contain a binding site for the USP1-associated factor 1 (UAF1) cellular protein that is required for efficient viral DNA replication and for episomal maintenance of the viral genome in immortalized keratinocytes (7,8). In noninfected cells, UAF1 forms separate complexes with the three deubiquitinating enzymes USP1, USP12, and USP46 and stimulates their activity (9)(10)(11)(12).…”