DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

Bristol, Molly L.; Wang, Xu; Smith, Nathan W.; Son, Minkyeong P.; Evans, Michael R.; Morgan, Iain M.

doi:10.3390/v8060175

Cited by 20 publications

(38 citation statements)

References 49 publications

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“…1b; we have previously used this assay to investigate E1-E2 DNA replication (48, 66). It has demonstrated that HPV 16 E1-E2 DNA replication uses translesion synthesis to by-pass replication polymerases on UV damaged DNA (66), and that replication in the presence of DNA damaging agents is mutagenic (48). We now demonstrate that deletion of SIRT1 from C33a cells resulted in an elevation in mutation frequency of 3 to 4 fold (Fig 1c).…”

Section: Resultsmentioning

confidence: 99%

“…E1-E2 DNA replication activates the DNA damage response (DDR) (43-48) and recruits a variety of cellular factors required for homologous recombination (HR) to the viral genome (11, 12, 47, 49, 50); it has been proposed that E1-E2 DNA replication proceeds via HR in the presence of an active DDR (51). The reason the virus activates the DDR is related to the mode of E1-E2 replication where initiation is not restricted to only once per cell cycle; re-initiation of genomes already undergoing replication would result in torsional stress and potentially clashes of DNA replication forks that would activate the DDR (52).…”

Section: Introductionmentioning

confidence: 99%

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Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

Morgan

Das

Bristol

et al. 2018

Preprint

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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 ...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

Morgan

Das

Bristol

et al. 2018

Preprint

Self Cite

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“…Our initial hypothesis aligned with previous indications that the estrogen and the ERα increase the risk of cervical cancer, and we further predicted that high doses of estrogen would initiate the DNA damage response (DDR) (14–16, 18, 19, 55–58). Based on our previously published data, we further predicated that this increase in the DDR via estrogen would enhance HPV tumorigenicity and ultimately result in worse outcomes and disease progression(59). However, it soon became clear that our initial hypothesis was incorrect when HPV+ cells were specifically sensitized via estrogen treatment, while HPV-cells showed little to no response.…”

Section: Discussionmentioning

confidence: 99%

“…UMSCC104 (Millipore), and UMSCC152 (ATCC) cells were grown in Eagle’s Minimum Essential Medium (EMEM, Invitrogen) supplemented with non-essential amino acids (NEAA, Gibco) and 10% charcoal stripped fetal bovine serum. N/Tert-1 cells and all derived cell lines, as well as HTK+HPV16 cells (a generous gift from Dr. Craig Meyers, UPenn, Hershey) have been describe previously(24, 25, 52, 59) and were maintained in keratinocyte-serum free medium (K-SFM, Invitrogen), supplemented with a 1% (vol/vol) penicillin-streptomycin mixture (ThermoFisher Scientific). All N/Tert-1 cells were also supplemented with 4 μg/ml hygromycin B (Millipore Sigma).…”

Section: Methodsmentioning

confidence: 99%

Estrogen attenuates the growth of human papillomavirus positive epithelial cells

Bristol

James

Wang

et al. 2020

Preprint

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12Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are 13 significant risk factors in the development of cancer, and HPV accounts for 14 approximately 5% of all worldwide cancers. Recent studies using data from The Cancer 15 Genome Atlas (TCGA) have demonstrated that elevated levels of estrogen receptor 16 alpha (ERα) are associated with improved survival in oropharyngeal cancers, and these 17 elevated receptor levels were linked with human papillomavirus positive cancers 18 (HPV+cancers). There has been a dramatic increase in HPV-related head and neck 19 squamous cell carcinomas (HPV+HNSCCs) over the last two decades and therapeutic 20 options for this ongoing health crisis are a priority; currently there are no anti-viral 21 therapeutics available for combating HPV+cancers. During our own TGCA studies on 22 head and neck cancer we had also discovered the overexpression of ERα in 23 HPV+cancers. Here we demonstrate that 17β-estradiol (estrogen) attenuates the 24 growth/cell viability of HPV+cancers in vitro, but not HPV negative cancer cells. In 25 addition, N/Tert-1 cells (foreskin keratinocytes immortalized with hTERT) containing 26 HPV16 have elevated levels of ERα and growth sensitivity following estrogen treatment 27 when compared with parental N/Tert-1. Finally, we demonstrate that there are 28 potentially two mechanisms contributing to the attenuation of HPV+ cell growth following 29 estrogen treatment. First, estrogen represses the viral transcriptional long control region 30 (LCR) downregulating early gene expression, including E6/E7. Second, expression of 31 E6 and E7 by themselves sensitizes cells to estrogen. Overall our results support the 32 recent proposal that estrogen could be exploited therapeutically for the treatment of 33 HPV positive oral cancers.34 3 Importance 35Human papillomaviruses cause around 5% of all human cancers, yet there are no 36 specific anti-viral therapeutic approaches available for combating these cancers. These 37 cancers are currently treated with standard chemo-radiation therapy (CRT). Specific 38 anti-viral reagents are desperately required, particularly for HPV+HNSCC whose 39 incidence is increasing and for which there are no diagnostic tools available for 40 combating this disease. Using data from The Cancer Genome Atlas (TCGA) ourselves 41 and others determined that the estrogen receptor α (ERα) is overexpressed in 42 HPV+HNSCC, and that elevated levels are associated with an improved disease 43 outcome. This has led to the proposal that estrogen treatment could be a novel 44 therapeutic approach for combating HPV+cancers. Here we demonstrate that estrogen 45 attenuates the growth of HPV+epithelial cells using multiple mechanisms, supporting 46 the idea that estrogen has potential as a therapeutic agent for the treatment of 47 65 correlates with increased survival in HPV+HNSCC(20, 21). These reports suggest ERα 66 as a diagnostic marker but also raise the possibility of using estrogen as a therapeutic 67 for the treatment of HPV+HNSCC. In...

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“…E1 has also been shown to induce DNA damage, arrest cell growth, and recruit host cell machinery to increase viral replication [8]. Under stress, E1 replicates HPV DNA with low fidelity and the resulting double-stranded breaks, which could be a carcinogenic factor [9]. …”

Section: Introductionmentioning

confidence: 99%

Elevated HPV16 E1 Expression Is Associated with Cervical Cancer Progression

2017

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Objectives: The primary replication protein, HPV E1, has been shown to play a role in mitigating host defence and disrupting normal cell cycle processes, leading to the development of cancer. This study investigated the expression profile of HPV16 E1 in various stages of cervical cancer development and the factors that control E1 expression. Methods: One hundred and twenty-four HPV16-positive cervical samples ranging from normal to CIN 1, CIN 2/3, and SCC lesions were studied. E1 mRNA expression was determined by ddPCR. Methylation of promoters p97 and p670 was quantified by pyrosequencing, while PCR, qPCR, and sequencing were used to determine the physical state and variations of the HPV16 E1 genome. Results: Increased E1 mRNA expression related to disease progression (normal 0.18, CIN 1 0.41, CIN 2/3 0.65, and SCC 0.79) was demonstrated with a significant positive correlation (r = 0.661, p = 0.019). No association between physical state and E1 expression was found. Methylation of p97 and p670 promoters showed significant elevation in SCC compared to normal samples. Only 4.2% showed genomic variations of HPV16 E1 63-bp duplication. Conclusion: E1 may play a role in cancer development. The detection of E1 mRNA and promoter methylation may be useful as cancer prognostic markers.

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DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

Cited by 20 publications

References 49 publications

Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

Estrogen attenuates the growth of human papillomavirus positive epithelial cells

Elevated HPV16 E1 Expression Is Associated with Cervical Cancer Progression

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