RESUMEN
Human papillomaviruses (HPVs) infect keratinocytes of stratified epithelia. Long-term persistence of infection is a critical risk factor for the development of HPV-induced malignancies. Through the actions of its oncogenes, HPV evades host immune responses to facilitate its productive life cycle. In this work, we discovered a previously unknown function of the HPV16 E5 oncoprotein in the suppression of interferon (IFN) responses. This suppression is focused on keratinocyte-specific IFN-κ and is mediated through E5-induced changes in growth factor signaling pathways, as identified through phosphoproteomics analysis. The loss of E5 in keratinocytes maintaining the complete HPV16 genome results in the derepression of IFNK transcription and subsequent JAK/STAT-dependent upregulation of several IFN-stimulated genes (ISGs) at both the mRNA and protein levels. We also established a link between the loss of E5 and the subsequent loss of genome maintenance and stability, resulting in increased genome integration.IMPORTANCE Persistent human papillomavirus infections can cause a variety of significant cancers. The ability of HPV to persist depends on evasion of the host immune system. In this study, we show that the HPV16 E5 protein can suppress an important aspect of the host immune response. In addition, we find that the E5 protein is important for helping the virus avoid integration into the host genome, which is a frequent step along the pathway to cancer development.
Asunto(s)
Genoma Viral , Papillomavirus Humano 16/metabolismo , Interferón Tipo I/metabolismo , Queratinocitos , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus , Plásmidos/metabolismo , Transducción de Señal , Línea Celular , Inestabilidad Genómica , Papillomavirus Humano 16/genética , Humanos , Interferón Tipo I/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Plásmidos/genéticaRESUMEN
Human papillomaviruses (HPVs) cause benign lesions that can lead to malignancy. How cellular changes induced by viral oncogenes contribute to the progeny virion production is not always clear. Stromally-derived growth factors and their receptors are critical for development of malignancy, but their impact on the pre-malignant HPV life cycle is unknown. We show that HPV16 increases levels of Met, a growth factor receptor critical for tumor cell invasion, motility, and cancer metastasis. The viral oncogene E5 is primarily responsible for Met upregulation, with E6 playing a minor role. Met induction by E5 requires the epidermal growth factor receptor, which is also increased by E5 at the mRNA level. E5-induced Met contributes motility of HPV-containing cells. Finally, Met signaling is necessary for viral gene expression, particularly in the differentiation-dependent phase of the viral life cycle. These studies show a new role for E5 in epithelial-stromal interactions, with implications for cancer development.
Asunto(s)
Papillomavirus Humano 16/metabolismo , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Regulación hacia Arriba , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Papillomavirus Humano 16/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
Persistent high-risk human papillomavirus (HPV) infection is the major causal factor in cervical and other anogenital cancers. Because there are currently no therapeutics capable of preventing neoplastic progression of HPV infections, understanding the mechanisms of HPV-mediated persistence, including immune evasion, is a major research priority. The multifunctional growth factor transforming growth factor beta (TGFß) has been shown to inhibit expression of early viral transcripts from cells harboring integrated HPV genomes or cells infected with retroviruses expressing HPV oncoproteins. However, the mechanism of TGFß-induced inhibition has not been fully defined. In this study, we have observed a previously uncharacterized ability of TGFß to repress the differentiation-induced upregulation of late HPV16 gene expression. In addition, interferon kappa (IFN-κ), a keratinocyte-specific, constitutively expressed cytokine suppressed by differentiation, can be transcriptionally induced by TGFß1. TGFß-mediated IFN-κ transcription only occurs in cells containing HPV16, and this is due to TGFß1-mediated reversal of HPV-induced methylation of the IFN-κ promoter through active DNA demethylation mediated by thymine DNA glycosylase (TDG). This novel interaction between growth factor and innate immune signaling may shed light on the mechanisms of HPV persistence and how the virus manipulates both immune and growth factor signaling to promote its life cycle.IMPORTANCE Persistent infection by high-risk HPVs is the primary risk factor for development of HPV-induced cancers. Persistence involves viral evasion of the immune response, including the IFN response. HPV is also known to suppress TGFß signaling, which inhibits viral gene expression. Here, we show that the TGFß and IFN pathways are interrelated in the context of HPV16 infection through the upregulation of IFN-κ by TGFß. The ability of TGFß to induce IFN-κ promoter demethylation and transcriptional activation provides a new explanation for why HPV has evolved mechanisms to inhibit TGFß in infected cells.
Asunto(s)
Metilación de ADN , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/metabolismo , Interferón Tipo I/metabolismo , Queratinocitos/metabolismo , Infecciones por Papillomavirus/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismo , Humanos , Queratinocitos/patología , Queratinocitos/virología , Infecciones por Papillomavirus/patologíaRESUMEN
Transcripts from the late promoter of human papillomavirus type 16 (HPV16) are upregulated upon host cell differentiation. Differentiation-dependent transcript regulation is thought to sequester viral antigens in the uppermost epithelial layers, facilitating immune evasion. The mechanisms regulating late promoter upregulation during differentiation are poorly characterized. We show that the late promoter is upregulated at the transcriptional level and that the viral enhancer stimulates promoter activity. Using kinase inhibition and chromatin immunoprecipitation analysis, we show evidence for differentiation-dependent enhancement of transcript elongation. Three factors that promote transcript elongation, cyclin dependent kinase 9 (CDK9), CDK8 (a subunit of the Mediator complex), and bromodomain containing protein 4 (Brd4) are recruited to viral genomes upon differentiation, and each plays a role in promoter activity. These results shed light on the transcriptional processes utilized by HPV16 for proper regulation of gene expression during the viral life cycle.
Asunto(s)
Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas de Ciclo Celular , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas de Transporte de Catión/genética , Invasividad Neoplásica/genética , Neoplasias de la Próstata/genética , Intercambiadores de Sodio-Hidrógeno/genética , Regulación hacia Arriba , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Invasividad Neoplásica/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Intercambiador 1 de Sodio-Hidrógeno , Activación Transcripcional , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
High-risk human papillomaviruses (HPVs) encode oncoproteins which manipulate gene expression patterns in the host keratinocytes to facilitate viral replication, regulate viral transcription, and promote immune evasion and persistence. In some cases, oncoprotein-induced changes in host cell behavior can cause progression to cancer, but a complete picture of the functions of the viral oncoproteins in the productive HPV life cycle remains elusive. E7 is the HPV-encoded factor most responsible for maintaining cell cycle competence in differentiating keratinocytes. Through interactions with dozens of host factors, E7 has an enormous impact on host gene expression patterns. In this review, we will examine the role of E7 specifically as a regulator of transcription. We will discuss mechanisms of regulation of cell cycle-related genes by E7 as well as genes involved in immune regulation, growth factor signaling, DNA damage responses, microRNAs, and others pathways. We will also discuss some unanswered questions about how transcriptional regulation by E7 impacts the biology of HPV in both benign and malignant conditions.