Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle

Evans, Michael R.; James, Claire D.; Bristol, Molly L.; Nulton, Tara J.; Wang, Xu; Kaur, Namsimar; White, Elizabeth; Windle, Brad; Morgan, Iain M.

doi:10.1128/jvi.01941-18

Cited by 33 publications

(34 citation statements)

References 60 publications

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“…Figure 1B summarizes our previous data from our RNA-seq analysis in N/Tert-1 cells. TWIST1 was found to be significantly downregulated in N/Tert-1 cells expressing HPV16 E2 or the entire viral episome compared to parental control cells (33,34). Figure 2A validates the downregulation of TWIST1 RNA expression in N/Tert-1 cells expressing E2 or the entire HPV16 genome (compare lanes 2 and 3, respectively, with lane 1).…”

Section: Resultssupporting

confidence: 66%

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes

Fontan

Das

Bristol

et al. 2020

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Section: Resultssupporting

confidence: 66%

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes

Fontan

Das

Bristol

et al. 2020

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“…It is of note that overexpression of IFIT1 in HN-22 only reduces replication by about 50% (Figure 5 C). There are likely other factors that negatively regulate HPV replication, as numerous innate immune factors like IFIT1, have been shown to be downregulated by HPV16 in cells of the head and neck region and in HPV+HNSCC clinical samples [54,62]. While further characterization of the HPV16 IFIT1-E1…”

Section: Discussionmentioning

confidence: 99%

A Head and Neck Squamous Cell Carcinoma Model to Study and Target HPV Replication and Transcription

Evans¹,

Fontan²,

James³

et al. 2019

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The incidence of human papillomavirus-related head and neck squamous cell carcinoma (HPV+HNSCC) has reached epidemic levels in the last decade. While prophylactic vaccines will prevent future HPV infections, there are currently no HPV-specific antiviral drugs to treat current HPV infections or HPV+HNSCC. HPV replication and transcription are promising targets for anti-HPV therapeutics, as modulation of these processes can alter expression levels of HPV E6 and E7, which are required for maintenance of the transformed phenotype. This is a particularly attractive target in in HPV+HNSCC where the majority of tumors have episomal genomes replicating in an E1-E2 dependent manner. Here, we describe a model system to study HPV16 E1-E2 mediated DNA replication and HPV16 E2-mediated transcriptional activation and repression in multiple HNSCC cell lines. Our results demonstrate that low levels of IFIT1 are required for HPV16 replication in HNSCC cell lines and HPV16 E1 interacts with IFIT1. Restoration of IFIT1 expression in HNSCC cell lines partially inhibits HPV16 E1-E2 mediated replication. This system can be used to study replication and transcription by HPV16 E1 and E2 in HNSCC as well as be utilized to screen potential anti-HPV therapeutics that target HPV16 replication and transcription.

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“…Recently we demonstrated one potential unique use of this system. We expressed the HPV16 E2 protein in NOKs and determined host gene regulation by E2 in these cells that had a highly significant overlap with the genes regulated by the entire HPV16 genome (44). Therefore, this system has allowed us to identify and characterize a new function for the E2 protein; regulation of host gene transcription during infection.…”

Section: Discussionmentioning

confidence: 99%

SAMHD1 regulates human papillomavirus 16 induced cell proliferation and viral replication during differentiation of oral keratinocytes

Morgan

James

Prabhakar

et al. 2019

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4Human papillomaviruses induce a host of anogenital cancers, and also oropharyngeal cancer 5 (HPV+OPC); HPV16 is causative in around 90% of HPV+OPC. Using TERT immortalized 6 "normal" oral keratinocytes (NOKs) we have identified significant host gene reprogramming by 7 HPV16 (NOKs+HPV16), and demonstrated that NOKs+HPV16 support late stages of the viral life 8 cycle. Expression of the cellular dNTPase and homologous recombination factor SAMHD1 is 9 transcriptionally regulated by HPV16 in NOKs, and here we demonstrate that E6 and E7 regulate 10 expression of SAMHD1 at the transcriptional and post-transcriptional levels. CRISPR/Cas9 11 removal of SAMHD1 from NOKs and NOKs+HPV16 demonstrate that SAMHD1 controls cell 12 proliferation of NOKs only in the presence of HPV16; deletion of SAMHD1 promotes hyper-13 proliferation of NOKs+HPV16 cells in organotypic raft cultures but has no effect on NOKs. Viral 14 replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify 15 a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral 16 replication; such studies are problematic in non-immortalized primary oral keratinocytes due to 17 their limited lifespan. To confirm the relevance of our results we repeated the analysis with human 18 tonsil keratinocytes immortalized by HPV16 (HTK16) and observe the same hyper-proliferative 19 phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three 20 independent CRISPR/Cas9 guide RNAs. The isogenic pairing of NOKs with NOKs+HPV16, 21 combined with HTK16, presents a unique system to identify host genes whose products 22 functionally interact with HPV16 to regulate host cellular growth in oral keratinocytes. 23 Importance 24Head and neck cancer is the sixth most common cancer worldwide. The incidence of HPV+OPC 25 has been rising steadily since the 1970s and has recently reached epidemic proportions, 26 according to the WHO. Upwards of 70% of the 600,000 new OPC cases per year are HPV 27 positive, with high-risk type 16 present in 90% of those incidences. A better understanding of the 28 viral life cycle will facilitate the development of novel therapeutics to combat this ongoing 29 3 epidemic, as well as other HPV positive cancers. Here we present a unique oral keratinocyte 30 model to identify host proteins that specifically interact with HPV16. Using this system, we report 31 that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein level in oral 32 keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes 33 enhanced cellular proliferation by HPV16 in oral keratinocytes and elevated viral replication, but 34 not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay 35 between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system 36 to assist exploring host factors critical for HPV pathogenesis. 37 38

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Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle

Cited by 33 publications

References 60 publications

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes

A Head and Neck Squamous Cell Carcinoma Model to Study and Target HPV Replication and Transcription

SAMHD1 regulates human papillomavirus 16 induced cell proliferation and viral replication during differentiation of oral keratinocytes

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