CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 interaction with mitotic chromatin and the viral life cycle

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle

Why Human Papillomaviruses Activate the DNA Damage Response (DDR) and How Cellular and Viral Replication Persists in the Presence of DDR Signaling

Human papillomaviruses (HPV) require the activation of the DNA damage response (DDR) in order to undergo a successful life cycle. This activation presents a challenge for the virus and the infected cell: how does viral and host replication proceed in the presence of a DDR that ordinarily arrests replication; and how do HPV16 infected cells retain the ability to proliferate in the presence of a DDR that ordinarily arrests the cell cycle? This raises a further question: why do HPV activate the DDR? The answers to these questions are only partially understood; a full understanding could identify novel therapeutic strategies to target HPV cancers. Here, we propose that the rapid replication of an 8 kb double stranded circular genome during infection creates aberrant DNA structures that attract and activate DDR proteins. Therefore, HPV replication in the presence of an active DDR is a necessity for a successful viral life cycle in order to resolve these DNA structures on viral genomes; without an active DDR, successful replication of the viral genome would not proceed. We discuss the essential role of TopBP1 in this process and also how viral and cellular replication proceeds in HPV infected cells in the presence of DDR signals

Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20–50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein–protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1–E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair

Activating the DNA Damage Response and Suppressing Innate Immunity: Human Papillomaviruses Walk the Line

Activation of the DNA damage response (DDR) by external agents can result in DNA fragments entering the cytoplasm and activating innate immune signaling pathways, including the stimulator of interferon genes (STING) pathway. The consequences of this activation can result in alterations in the cell cycle including the induction of cellular senescence, as well as boost the adaptive immune response following interferon production. Human papillomaviruses (HPV) are the causative agents in a host of human cancers including cervical and oropharyngeal; HPV are responsible for around 5% of all cancers. During infection, HPV replication activates the DDR in order to promote the viral life cycle. A striking feature of HPV-infected cells is their ability to continue to proliferate in the presence of an active DDR. Simultaneously, HPV suppress the innate immune response using a number of different mechanisms. The activation of the DDR and suppression of the innate immune response are essential for the progression of the viral life cycle. Here, we describe the mechanisms HPV use to turn on the DDR, while simultaneously suppressing the innate immune response. Pushing HPV from this fine line and tipping the balance towards activation of the innate immune response would be therapeutically beneficial

Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation

HPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.Human papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells