Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle. HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

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.

16Human papillomaviruses (HPV) are double stranded DNA viruses causative in a host of human diseases 17 including several cancers. Following infection two viral proteins, E1 and E2, activate viral replication in 18 association with cellular factors, and stimulate the DNA damage response (DDR) during the replication 19 process. E1-E2 uses homologous replication (HR) to facilitate DNA replication, but an understanding of 20 host factors involved in this process remains incomplete. Previously we demonstrated that the class III 21 deacetylase SIRT1, which can regulate HR, is recruited to E1-E2 replicating DNA and regulates the level 22 of replication. Here we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the 23 absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-24 E2 replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and 25 fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role 26 for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of 27 WRN, but a reduced ability to interact with E1-E2 replicating DNA. We present a model in which E1-E2 28 replication turns on the DDR stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN 29 interaction with E1-E2 replicating DNA to control the quantity and fidelity of replication. As well as 30 offering a crucial insight into HPV replication control, this system offers a unique model for investigating 31 the link between SIRT1 and WRN in controlling replication in mammalian cells. 32 Importance 33HPV16 is the major viral human carcinogen, responsible for between 3 and 4 % of all cancers worldwide. 34Following infection this virus activates the DNA damage response (DDR) to promote its life cycle, and 35 recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during 36replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 37 replication interacts with the DDR remains incomplete. Here we demonstrate that the cellular deacetylase 38 SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein 39 WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 40 3 replication. Overall the results suggest a mechanism where SIRT1 deacetylation of WRN promotes its 41 interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication. 42 43 44 HPV16 E1-E2 DNA replication uses translesion synthesis to by-pass replication polymerases on UV 126 damaged DNA (66), and that replication in the presence of DNA damaging agents is mutagenic (48). We 127 now demonstrate that deletion of SIRT1 from C33a cells resulted in an elevation in mutation frequency of 128 3 to 4 fold (Fig 1c). Restoration of SIRT1 expression during E1-E2 DNA replication in the SIRT1 CRISPR 129 knock out cells ...

ObjectivesWe evaluated the performance and time to result for pathogen identification (ID) and antimicrobial susceptibility testing (AST) of the Accelerate Pheno™ system (AXDX) compared with standard of care (SOC) methods. We also assessed the hypothetical improvement in antibiotic utilization if AXDX had been implemented.MethodsClinical samples from patients with monomicrobial Gram-negative bacteraemia were tested and compared between AXDX and the SOC methods of the VERIGENE® and Bruker MALDI Biotyper® systems for ID and the VITEK® 2 system for AST. Additionally, charts were reviewed to calculate theoretical times to antibiotic de-escalation, escalation and active and optimal therapyResultsID mean time was 21 h for MALDI-TOF MS, 4.4 h for VERIGENE® and 3.7 h for AXDX. AST mean time was 35 h for VITEK® 2 and 9.0 h for AXDX. For ID, positive percentage agreement was 95.9% and negative percentage agreement was 99.9%. For AST, essential agreement was 94.5% and categorical agreement was 93.5%. If AXDX results had been available to inform patient care, 25% of patients could have been put on active therapy sooner, while 78% of patients who had therapy optimized during hospitalization could have had therapy optimized sooner. Additionally, AXDX could have reduced time to de-escalation (16 versus 31 h) and escalation (19 versus 31 h) compared with SOC. ConclusionsBy providing fast and reliable ID and AST results, AXDX has the potential to improve antimicrobial utilization and enhance antimicrobial stewardship.