Human papillomavirus 16 E6 and E7 synergistically repress innate immune gene transcription

James, Claire D.; Fontan, Christian T.; Otoa, Raymonde; Das, Diptikanta; Prabhakar, Apurva T.; Wang, Xu; Bristol, Molly L.; Morgan, Iain M.

doi:10.1101/841007

Cited by 9 publications

(15 citation statements)

References 46 publications

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“…To investigate the role of WRN during the viral life cycle, we exploited our N/Tert-1 model. N/Tert-1 are hTERT immortalized human foreskin keratinocytes, and we generated cell lines containing HPV16, N/Tert-1+HPV16 ( 46 – 48 , 53 ). This system has the direct advantage of being able to manipulate control cells (N/Tert-1) without the viral genome but with the same genetic lesions as the N/Tert-1+HPV16 cells.…”

Section: Discussionmentioning

confidence: 99%

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

James

Das

Morgan

et al. 2020

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Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle. IMPORTANCE HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.

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Section: Discussionmentioning

confidence: 99%

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

James

Das

Morgan

et al. 2020

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“…We next investigated whether the transcriptional reprograming of N/Tert-1 cells carried out by HPV16 oncogenes alone could sensitize cells to estrogen and attenuate cellular growth. To do this we expressed E6 or E7 or E6+E7 in N/Tert-1 cells and further compared these cells to those expressing the full HPV16 genome (N/Tert-1+HPV16); these E6, E7, and E6+E7 cell lines were generated using retroviral delivery and have been described previously(26, 52). Figure 6A demonstrates again that in N/Tert-1 control cells, estrogen treatment does not attenuate cellular growth (Figure 6Ai) but the presence of the entire HPV16 genome promotes such attenuation (Figure 6Aii).…”

Section: Resultsmentioning

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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“…These studies employed overexpression of viral proteins, and monitored their effect on the cell and the DDR pathway. We recently reported that HPV16 activates the DDR in human keratinocytes irrespective of the expression of E6 and E7 ( Figure 4 ) [ 46 ]. This was done by introducing stop codons into the E6 and E7 genes (individually and combined) in the full HPV16 genome and generating N/Tert-1 (human foreskin keratinocytes immortalized by telomerase) cells containing wild type and mutant genomes.…”

Section: The Ddr During the Hpv Life Cyclementioning

confidence: 99%

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

et al. 2020

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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.

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Human papillomavirus 16 E6 and E7 synergistically repress innate immune gene transcription

Cited by 9 publications

References 46 publications

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Estrogen attenuates the growth of human papillomavirus positive epithelial cells

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

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