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

Abstract

The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCEThe E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. H uman papillomaviruses (HPVs) are small DNA tumor viruses that infect squamous epithelia of the skin and mucosa. Infections by these viruses can lead to the development of benign and malignant tumors, depending on the viral types. Several HPV types are sexually transmitted and the causative agents of cervical cancer, the second most common cancer in women worldwide. Th...

Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

“…These results showed that the E1-UAF1 interaction is required for the replication of HPV11 episomes in U2OS cells, similarly to what has been observed previously for HPV31 in immortalized keratinocytes ( 11 – 14 ). Importantly, the results also revealed that the deubiquitinating activity of USP1 is essential for episomal replication of the HPV11 genome.…”

“…Also, the previously described transcription map of the HPV11 genome in U2OS cells is similar to the transcription map of the HPV11 genome in HPV-associated lesions ( 47 ), further validating the use of the U2OS cell line to study the replication of HPV episomes. The importance of the E1-UAF1-USP1 complex for efficient HPV replication has been previously reported ( 11 – 14 ); however, we demonstrated that the lower replication efficiency of mutant HPV11 genomes encoding a E1 protein defective for UAF1-binding is associated with decreased production of RIs generated by the unidirectional mechanism, while bidirectional theta replication was affected to a lesser degree ( Fig. 2 ).…”

“…UAF1 is associated with three deubiquitinating enzymes, USP1, USP12, and USP46, and HPV E1 forms a ternary complex with UAF1 and any one of these three USPs ( 12 ). The E1-UAF1-USP complex is recruited to the viral origin and is necessary for efficient replication of the HPV genome ( 11 – 14 ). While the UAF1-USP12 and UAF1-USP46 complexes regulate histone deubiquitination ( 15 ) and the cell survival- and growth-promoting Akt pathway ( 16 – 20 ), the UAF1-USP1 complex is involved in the Fanconi anemia (FA) DNA repair pathway ( 21 ).…”

Kinase Activity of Fibroblast Growth Factor Receptor 3 Regulates Activity of the Papillomavirus E2 Protein

“…The effect of FGFR3 kinase activity on E2-stimulated viral DNA replication was subsequently evaluated using C33A cells transfected with BPV-1 E1 and E2 expression vectors, along with a BPV-1 luciferase-linked reporter in the presence of FGFR3 WT, K650E, and K508R expression constructs. In this assay, luciferase expression is proportional to episomal copy number and is sensitive to DNA replication inhibitors (24)(25)(26). Firefly luciferase readout was normalized to Renilla luciferase levels as a transcription and transfection control (Fluc/Rluc).…”

High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

“…HPVs have been shown to recruit numerous cellular repair factors to their replication centers, mainly for HPV amplification [ 59 – 61 ]. Similarly, HPV E1 has been shown to interact with USP1-UAF1 complex to facilitate viral replication [ 62 ]. We speculate that increased Ub-FancD2 and foci formation may create an environment where FA repair proteins are readily available to support HPV replication.…”