DNA damage response is hijacked by human papillomaviruses to complete their life cycle

ABSTRACTHuman 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.IMPORTANCEHPV16 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.

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Modulation of the DNA damage response during the life cycle of human papillomaviruses

Virus Research ◽

10.1016/j.virusres.2016.11.006 ◽

2017 ◽

Vol 231 ◽

pp. 41-49 ◽

Cited By ~ 35

Author(s):

Daniel C. Anacker ◽

Cary A. Moody

Keyword(s):

Dna Damage ◽

Life Cycle ◽

Dna Damage Response ◽

Human Papillomaviruses ◽

Damage Response

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

10.1101/450601 ◽

2018 ◽

Author(s):

Dipon Das ◽

Molly L Bristol ◽

Nathan W Smith ◽

Xu Wang ◽

Pietro Pichierri ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Life Cycle ◽

Dna Replication ◽

Dna Damage Response ◽

Mammalian Cells ◽

Human Papillomaviruses ◽

Repair Protein ◽

Damage Response ◽

Dna Repair Protein

AbstractHuman 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 replication (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.ImportanceHPV16 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 where SIRT1 deacetylation of WRN promotes its interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication.

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

Pathogens ◽

10.3390/pathogens9060467 ◽

2020 ◽

Vol 9 (6) ◽

pp. 467 ◽

Cited By ~ 1

Author(s):

Claire D. James ◽

Dipon Das ◽

Molly L. Bristol ◽

Iain M. Morgan

Keyword(s):

Immune Response ◽

Dna Damage ◽

Life Cycle ◽

Dna Damage Response ◽

Innate Immune Response ◽

Adaptive Immune Response ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Innate Immune ◽

Damage Response

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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Regulation of the life cycle of HPVs by differentiation and the DNA damage response

Future Microbiology ◽

10.2217/fmb.13.127 ◽

2013 ◽

Vol 8 (12) ◽

pp. 1547-1557 ◽

Cited By ~ 34

Author(s):

Shiyuan Hong ◽

Laimonis A Laimins

Keyword(s):

Dna Damage ◽

Life Cycle ◽

Dna Damage Response ◽

Damage Response

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Interaction of the Human Papillomavirus E1 Helicase with UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

mBio ◽

10.1128/mbio.00152-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 1

Author(s):

Marit Orav ◽

David Gagnon ◽

Jacques Archambault

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Human Papillomaviruses ◽

Viral Genomes ◽

Damage Response ◽

Theta Replication ◽

Infected Cells ◽

Cellular Dna ◽

Medical Burden

ABSTRACTHuman papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCEHuman papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

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The Role of the DNA Damage Response throughout the Papillomavirus Life Cycle

Viruses ◽

10.3390/v7052450 ◽

2015 ◽

Vol 7 (5) ◽

pp. 2450-2469 ◽

Cited By ~ 46

Author(s):

Caleb McKinney ◽

Katherine Hussmann ◽

Alison McBride

Keyword(s):

Dna Damage ◽

Life Cycle ◽

Dna Damage Response ◽

Damage Response

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FANCD2 Binds Human Papillomavirus Genomes and Associates with a Distinct Set of DNA Repair Proteins to Regulate Viral Replication

mBio ◽

10.1128/mbio.02340-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 20

Author(s):

Chelsey C. Spriggs ◽

Laimonis A. Laimins

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Human Papillomavirus ◽

Life Cycle ◽

High Risk ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Repair Pathway ◽

Damage Response ◽

Dna Repair Proteins

ABSTRACTThe life cycle of human papillomavirus (HPV) is dependent on the differentiation state of its host cell. HPV genomes are maintained as low-copy episomes in basal epithelial cells and amplified to thousands of copies per cell in differentiated layers. Replication of high-risk HPVs requires the activation of the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) DNA repair pathways. The Fanconi anemia (FA) pathway is a part of the DNA damage response and mediates cross talk between the ATM and ATR pathways. Our studies show that HPV activates the FA pathway, leading to the accumulation of a key regulatory protein, FANCD2, in large nuclear foci. These HPV-dependent foci colocalize with a distinct population of DNA repair proteins, including ATM components γH2AX and BRCA1, but infrequently with p-SMC1, which is required for viral genome amplification in differentiated cells. Furthermore, FANCD2 is found at viral replication foci, where it is preferentially recruited to viral genomes compared to cellular chromosomes and is required for maintenance of HPV episomes in undifferentiated cells. These findings identify FANCD2 as an important regulator of HPV replication and provide insight into the role of the DNA damage response in the differentiation-dependent life cycle of HPV.IMPORTANCEHigh-risk human papillomaviruses (HPVs) are the etiological agents of cervical cancer and are linked to the development of many other anogenital and oropharyngeal cancers. Identification of host cellular pathways involved in regulating the viral life cycle may be helpful in identifying treatments for HPV lesions. Mutations in genes of the Fanconi anemia (FA) DNA repair pathway lead to genomic instability in patients and a predisposition to HPV-associated malignancies. Our studies demonstrate that FA pathway component FANCD2 is recruited to HPV DNA, associates with members of the ATM DNA repair pathway, and is essential for the maintenance of viral episomes in basal epithelial cells. Disruption of the FA pathway may result in increased integration events and a higher incidence of HPV-related cancer. Our study identifies new links between HPV and the FA pathway that may help to identify new therapeutic targets for the treatment of existing HPV infections and cancers.

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

10.1101/2020.05.18.103309 ◽

2020 ◽

Author(s):

Claire D. James ◽

Dipon Das ◽

Ethan L. Morgan ◽

Raymonde Otoa ◽

Andrew Macdonald ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Damage ◽

Life Cycle ◽

Dna Replication ◽

Viral Replication ◽

Dna Damage Response ◽

Basal Cell ◽

Viral Life Cycle ◽

Cervical Lesions ◽

Damage Response

AbstractHuman 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 when compared with 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.ImportanceHPV16 is the major human viral carcinogen, responsible for around 3-4% 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 and 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 as 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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Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

mBio ◽

10.1128/mbio.01433-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 23

Author(s):

Oliver I. Fregoso ◽

Michael Emerman

Keyword(s):

Dna Damage ◽

Life Cycle ◽

Dna Damage Response ◽

Viral Life Cycle ◽

Primate Species ◽

Accessory Gene ◽

Defense Proteins ◽

Knock Out ◽

Damage Response ◽

Hiv 1

ABSTRACTThere has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.IMPORTANCEHIV-1 and HIV-2 belong to a family of viruses called lentiviruses that infect at least 40 primate species, including humans. Lentiviruses have been circulating in primates for at least 5 million years. In order to better fight HIV, we must understand the viral and host factors necessary for infection, adaptation, and transmission of these viruses. Using the natural variation of HIV-1, HIV-2, and related lentiviruses, we have investigated the role of the DNA damage response in the viral life cycle. We have found that the ability of lentiviruses to activate the DNA damage response is largely conserved. However, we also found that the SLX4 host factor is not required for this activation, as was previously proposed. This indicates that the DNA damage response is an important player in the viral life cycle, and yet the mechanism(s) by which HIV-1, HIV-2, and other primate lentiviruses engage the DNA damage response is still unknown.

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