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

Abstract

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. IMPORTANCE 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.

Werner syndrome protein (WRN) regulates cell proliferation and the human papillomavirus 16 life cycle during epithelial differentiation

“…We demonstrate that in CIN1, 2 and 3 lesions there is an increasing tendency for elevated SIRT1 expression and reduced WRN expression. This inverse correlation of expression of these proteins reflects what we observe in our C33a studies where SIRT1 activity deacetylates and destabilizes WRN, although the deacetylated WRN is active in interacting with the E1-E2 replicating DNA and promoting high fidelity DNA replication (25). Others have also demonstrated regulation of WRN function by SIRT1 deacetylation (44-46) SIRT1 is essential for the HPV31 life cycle in cervical keratinocytes, demonstrated by using shRNA to eliminate SIRT1 expression (30).…”

“…Removal of SIRT1 resulted in hyper-acetylation and stabilization of WRN. In addition, removal of WRN expression from C33a cells by CRISPR/Cas9 resulted in a similar phenotype to that of SIRT1 deletion; increased DNA replication that has a reduced fidelity (25). Therefore, a SIRT1 controls the recruitment of WRN to E1-E2 replicating DNA, and depletion of SIRT1 or WRN results in mutagenic HPV16 E1-E2 DNA replication.…”

“…Our recent work demonstrated that deacetylation of WRN by SIRT1 promotes binding of WRN to E1-E2 replicating DNA (25). Removal of SIRT1 resulted in hyper-acetylation and stabilization of WRN.…”

“…In the absence of SIRT1, E1-E2 replication has a reduced fidelity and one reason for this could be the lack of WRN recruitment to the replicating DNA (26). CRISPR/Cas9 editing of WRN from C33a cells resulted in a similar phenotype to that of SIRT1 deletion: increased DNA replication that has a reduced fidelity (25). Therefore, a SIRT1-WRN axis controls recruitment of WRN to E1-E2 replicating DNA, and a lack of SIRT1 or WRN results in mutagenic HPV16 E1-E2 DNA replication.…”

“…HPV16 is responsible for around 50% of HPV positive cervical cancers and 80-90% of HPV positive oropharyngeal cancers. We have identified several proteins as being involved in E1-E2 DNA replication including TopBP1, SIRT1 and WRN, all proteins that have key roles in the DNA damage response, HR and DNA replication (25-30). Our recent work on WRN demonstrated that deacetylation of this protein by SIRT1 (a protein that also deacetylates TopBP1) promotes binding of WRN to HPV16 origin containing plasmid DNA being replicated by E1-E2 (25).…”