RESUMEN
Human 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.IMPORTANCE Human 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.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Interacciones Huésped-Patógeno , Papillomavirus Humano 11/fisiología , Proteínas Nucleares/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Línea Celular , Humanos , Unión ProteicaRESUMEN
The Long Control Region (LCR) of the human papillomavirus (HPV) genome encompasses the early promoter (EP) that drives expression of the viral oncogenes in infected cells and HPV-associated cancers. Here, we report on a natural variant of HPV33 that displays higher EP activity than the prototype in transfected C33A and HeLa cervical carcinoma cells, and in the osteosarcoma U2OS cell line which supports replication of HPV episomes. This increased promoter activity was ascribed to a single nucleotide variation in the LCR, T7791C, in a putative binding site for the transcription factor C/EBPß. T7791C abrogated binding of recombinant C/EBPß to this site in vitro and stimulated the EP in vivo, suggesting that it abrogates a negatively-acting regulatory element. A second C/EBPß binding site was identified in vitro that activated the EP in vivo and whose function and location in the epithelial-specific enhancer is shown to be conserved in the highly prevalent HPV18. These results suggest that C/EBPß is both an activator and a repressor of the HPV33 EP, acting via two distinct binding sites. Prediction of C/EBPß sites in the LCR of 186 HPV types suggests that C/EBPß regulation of the EP is common among high-risk viruses from the α genus.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Papillomaviridae/metabolismo , Papillomaviridae/patogenicidad , Sitios de Unión/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Femenino , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Papillomaviridae/genética , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
The human papillomavirus (HPV) Long Control Region (LCR) encompasses the early promoter (EP) that drives transcription of the E6 and E7 oncogenes in keratinocytes and HPV-associated cancers. In this study, the transcriptional activities of the HPV33 EP from the prototype LCR and from eight variants representative of the worldwide diversity of the virus were examined in primary human keratinocytes (PHK) and in the HeLa cervical carcinoma cell line by luciferase reporter-gene assays. Remarkably, the two variations with the greatest effect on the EP in PHK were C7732G and a 79-bp deletion that were associated with high-grade cervical lesions and persistent infections, respectively, in epidemiological studies. In contrast, the three variations most active in HeLa cells were C7537A, A7874C and A7879G. A7874C, which lies within an E2-binding sequence, is also shown to increase the activity and binding of E2 at this site. Collectively, these results indicate that naturally-occurring variations affect the HPV33 EP differentially in PHK than in cancer cells and, furthermore, that they can also alter its regulation by E2. These findings provide a molecular basis for rationalizing the results of previous epidemiological studies and for understanding the contribution of LCR polymorphisms to the oncogenicity and persistence of HPV33 infections.
Asunto(s)
Papillomaviridae/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética , Proteínas del Envoltorio Viral/genética , Células HeLa , Humanos , Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Papillomaviridae/patogenicidad , Cultivo Primario de CélulasRESUMEN
We have mapped a global network of virus-host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV- but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers.Significance: In this study, we created a protein-protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474-89. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinogénesis/patología , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Interacciones Huésped-Patógeno , Papillomaviridae/fisiología , Infecciones por Papillomavirus/complicaciones , Biomarcadores de Tumor/genética , Carcinogénesis/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Humanos , Mutación , Infecciones por Papillomavirus/virología , Mapas de Interacción de ProteínasRESUMEN
Within immunocompromised populations, the JC polyomavirus is the cause of the often-fatal disease Progressive Multifocal Leukoencephalopathy (PML). JC virus encodes a protein, termed T-antigen (T-ag), which is essential for its replication and pathogenicity. Previous studies of JCV T-ag have, in general, used antibodies raised against SV40 T-ag. Unfortunately, SV40 T-ag is also detected in humans and therefore there have been concerns about cross-reactivity. To address this issue, we have isolated a monoclonal antibody that binds to the JCV, but not the SV40, T-ag origin-binding domain (OBD). Furthermore, the region on the surface of the JCV T-ag OBD that is recognized by the "anti-JCV OBD mAb" has been mapped. We also demonstrate that the "anti-JCV OBD mAb" will be a useful reagent for standard techniques (e.g., Westerns blots and ELISAs). Finally, we note that additional monoclonal Abs that are specific for the T-ags encoded by the other human polyomaviruses could be generated by adopting the approach described herein.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Antígenos Virales de Tumores/química , Antígenos Virales de Tumores/inmunología , Virus JC/inmunología , Dominios y Motivos de Interacción de Proteínas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Antígenos Virales de Tumores/metabolismo , Sitios de Unión , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Ratones , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
UNLABELLED: The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE: While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 11/fisiología , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Papillomavirus Humano 11/genética , Humanos , Estructura Terciaria de Proteína , Eliminación de Secuencia , Proteínas Virales/genéticaRESUMEN
The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Replicación del ADN/fisiología , ADN Viral/metabolismo , Virus JC/fisiología , Replicación Viral/fisiología , Secuencia de Aminoácidos , Antígenos Virales de Tumores/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , ADN Viral/química , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
BACKGROUND: Genome-wide expression profiling is increasingly being used to identify transcriptional changes induced by drugs and environmental stressors. In this context, the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) project generated transcriptional profiles from rat liver samples and human/rat cultured primary hepatocytes exposed to more than 100 different chemicals. OBJECTIVES: To assess the capacity of the cell culture models to recapitulate pathways induced by chemicals in vivo, we leveraged the TG-GATEs data set to compare the early transcriptional responses observed in the liver of rats treated with a large set of chemicals with those of cultured rat and human primary hepatocytes challenged with the same compounds in vitro. METHODS: We developed a new pathway-based computational pipeline that efficiently combines gene set enrichment analysis (GSEA) using pathways from the Reactome database with biclustering to identify common modules of pathways that are modulated by several chemicals in vivo and in vitro across species. RESULTS: We found that some chemicals induced conserved patterns of early transcriptional responses in in vitro and in vivo settings, and across human and rat genomes. These responses involved pathways of cell survival, inflammation, xenobiotic metabolism, oxidative stress, and apoptosis. Moreover, our results support the transforming growth factor beta receptor (TGF-ßR) signaling pathway as a candidate biomarker associated with exposure to environmental toxicants in primary human hepatocytes. CONCLUSIONS: Our integrative analysis of toxicogenomics data provides a comprehensive overview of biochemical perturbations affected by a large panel of chemicals. Furthermore, we show that the early toxicological response occurring in animals is recapitulated in human and rat primary hepatocyte cultures at the molecular level, indicating that these models reproduce key pathways in response to chemical stress. These findings expand our understanding and interpretation of toxicogenomics data from human hepatocytes exposed to environmental toxicants.
Asunto(s)
Contaminantes Ambientales/toxicidad , Hepatocitos/efectos de los fármacos , Animales , Células Cultivadas , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Expresión Génica , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratas , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/genética , Especificidad de la Especie , Transcripción GenéticaRESUMEN
UNLABELLED: The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE: The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication.
Asunto(s)
Replicación del ADN , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Papillomaviridae/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Virales/metabolismo , Línea Celular , Humanos , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virales/genéticaRESUMEN
Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches.
Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN , ADN Helicasas/metabolismo , Biología Molecular/métodos , Papillomaviridae/enzimología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bromodesoxiuridina/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN Helicasas/química , Histonas/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Transporte de Proteínas , Fracciones Subcelulares/enzimologíaRESUMEN
A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Oncogénicas Virales/metabolismo , Proteolisis , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Vectores Genéticos/metabolismo , Humanos , Luciferasas/metabolismo , Plásmidos/metabolismo , TransfecciónRESUMEN
Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.
Asunto(s)
Replicación del ADN , Ensayos Analíticos de Alto Rendimiento/métodos , Papillomavirus Humano 31/fisiología , Replicación Viral/fisiología , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Papillomavirus Humano 31/efectos de los fármacos , Humanos , Hidroxiurea/farmacología , Luciferasas/metabolismo , Modelos Biológicos , Plásmidos/metabolismo , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.
Asunto(s)
Replicación del ADN/fisiología , ADN Viral/fisiología , Virus JC/fisiología , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica/fisiología , Humanos , Virus JC/genética , Modelos Moleculares , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
UNLABELLED: The human papillomavirus (HPV) E1 helicase promotes viral DNA replication through its DNA unwinding activity and association with host factors. The E1 proteins from anogenital HPV types interact with the cellular WD repeat-containing factor UAF1 (formerly known as p80). Specific amino acid substitutions in E1 that impair this interaction inhibit maintenance of the viral episome in immortalized keratinocytes and reduce viral DNA replication by up to 70% in transient assays. In this study, we determined by affinity purification of UAF1 that it interacts with three deubiquitinating enzymes in C33A cervical carcinoma cells: USP1, a nuclear protein, and the two cytoplasmic enzymes USP12 and USP46. Coimmunoprecipitation experiments indicated that E1 assembles into a ternary complex with UAF1 and any one of these three USPs. Moreover, expression of E1 leads to a redistribution of USP12 and USP46 from the cytoplasm to the nucleus. Chromatin immunoprecipitation studies further revealed that E1 recruits these threes USPs to the viral origin in association with UAF1. The function of USP1, USP12, and USP46 in viral DNA replication was investigated by overproduction of catalytically inactive versions of these enzymes in transient assays. All three dominant negative USPs reduced HPV31 DNA replication by up to 60%, an effect that was specific, as it was not observed in assays performed with a truncated E1 lacking the UAF1-binding domain or with bovine papillomavirus 1 E1, which does not bind UAF1. These results highlight the importance of the USP1, USP12, and USP46 deubiquitinating enzymes in anogenital HPV DNA replication. IMPORTANCE: Human papillomaviruses are small DNA tumor viruses that induce benign and malignant lesions of the skin and mucosa. HPV types that infect the anogenital tract are the etiological agents of cervical cancer, the majority of anal cancers, and a growing proportion of head-and-neck cancers. Replication of the HPV genome requires the viral protein E1, a DNA helicase that also interacts with host factors to promote viral DNA synthesis. We previously reported that the E1 helicase from anogenital HPV types associates with the WD40 repeat-containing protein UAF1. Here, we show that UAF1 bridges the interaction of E1 with three deubiquitinating enzymes, USP1, USP12, and USP46. We further show that these deubiquitinases are recruited by E1/UAF1 to the viral origin of DNA replication and that overexpression of catalytically inactive versions of these enzymes reduces viral DNA replication. These results highlight the need for an E1-associated deubiquitinase activity in anogenital HPV genome replication.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Papillomaviridae/fisiología , Ubiquitina Tiolesterasa/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Línea Celular Tumoral , Cromatografía de Afinidad , Replicación del ADN , Interacciones Huésped-Patógeno , Humanos , Inmunoprecipitación , Unión ProteicaRESUMEN
Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2448â471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2448â471 complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2448â471 complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.
Asunto(s)
Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Interacciones Huésped-Patógeno/fisiología , Factor de Transcripción TFIIH/metabolismo , Proteínas Virales/metabolismo , Animales , Antígenos Nucleares del Virus de Epstein-Barr/química , Humanos , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Factor de Transcripción TFIIH/química , Activación Transcripcional , Proteínas Virales/químicaRESUMEN
JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel "pocket"; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.
Asunto(s)
Antígenos Virales de Tumores/química , Replicación del ADN/genética , Virus JC/química , Virus JC/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Cristalización , Cristalografía por Rayos X , Virus JC/fisiología , Datos de Secuencia Molecular , Estructura Cuaternaria de ProteínaRESUMEN
BACKGROUND: The genomic diversity of high-risk human papillomaviruses (HPV) has been associated with viral persistence and HPV-induced lesions. Studies on HPV56 persistence are still pending. OBJECTIVE: To assess the association between HPV56 polymorphism and HPV56 persistence and presence of high-grade cervical intraepithelial neoplasia (CIN2,3) or cancer. STUDY DESIGN: HPV56-positive cervical specimens from 204 women selected from a total of 4669 participants recruited in 5 epidemiological studies (parent studies) were further analyzed by PCR-sequencing of the long control region (LCR). RESULTS: Of the 81 women followed prospectively in cohort studies who could be classified, 34 had persistent and 47 had transient HPV56 infections. Variant HPV56-LCR-MTL-21 was detected more frequently in persistent infections (52.9%, 95% CI: 36.7-68.6%) than in transient infections (25.5%, 95% CI: 15.1-39.4). Considering only women recruited in a cohort of women infected or at high risk for HIV infection, infection with variant HPV56-LCR-MTL-21 (OR=4.4, 95% CI: 1.3-14.5) was significantly associated with HPV56 persistence controlling in multivariate analysis for high risk HPV detection and HIV infection. A variation at nucleotide 7800 in HPV56-LCR-MTL-21 resulted in the loss of a binding site for Elf-1 embedded in one of the E2 binding sites, a potential activator or repressor of expression of the HPV genome. HPV56 polymorphism was not associated with CIN2,3 or cancer in women enrolled in cross-sectional and case-control studies. CONCLUSION: Polymorphism in HPV56 may influence the risk that infections with this type will persist.
Asunto(s)
Papillomaviridae/genética , Neoplasias del Cuello Uterino/virología , Adulto , Anciano , Canadá/epidemiología , Femenino , Humanos , Persona de Mediana Edad , Polimorfismo Genético , Estudios Prospectivos , Neoplasias del Cuello Uterino/epidemiología , Adulto Joven , Displasia del Cuello del Útero/virologíaRESUMEN
Like other acidic transactivation domains (TAD), the minimal TAD from the erythroid-specific transcription factor EKLF (EKLFTAD) has been shown to contribute both to its transcriptional activity as well as to its ubiquitin(UBI)-mediated degradation. In this article, we examine the activation-degradation role of the acidic TAD of EKLF and demonstrate that the first 40 residues (EKLFTAD1) within this region form a noncovalent interaction with UBI. Nuclear magnetic resonance (NMR) structural studies of an EKLFTAD1-UBI complex show that EKLFTAD1 adopts a 14-residue α helix that forms the recognition interface with UBI in a similar manner as the UBI-interacting helix of Rabex5. We also identify a similar interaction between UBI and the activation-degradation region of SREBP1a, but not with the activation-degradation regions of p53, GAL4, and VP16. These results suggest that select activation-degradation regions like the ones found in EKLF and SREBP1a function in part through their ability to form noncovalent interactions with UBI.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/química , Ubiquitina/química , Sustitución de Aminoácidos , Sitios de Unión , Línea Celular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Factores de Transcripción de Tipo Kruppel/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ubiquitina/genéticaRESUMEN
E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner.