The Experts below are selected from a list of 6171 Experts worldwide ranked by ideXlab platform
Kenneth M Kaye - One of the best experts on this subject based on the ideXlab platform.
-
Kaposi's Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance.
Journal of Virology, 2019Co-Authors: Franceline Juillard, Erika De Leon Vazquez, Shijun Li, Kenneth M KayeAbstract:Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates Episome persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic-chromosome binding. Although amino- and carboxy-terminal LANA are essential for Episome persistence, they are not sufficient, since deletion of all internal LANA sequence renders LANA highly deficient for Episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to Episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within the region compromised segregation of TR DNA to daughter cells, as assessed by retention of green fluorescent protein (GFP) expression from a replication-deficient TR plasmid. However, deletion of this upstream sequence did not reduce Episome maintenance. In contrast, deletions that included an 80-amino-acid sequence immediately downstream resulted in highly deficient Episome persistence. LANA with this downstream sequence deleted maintained the ability to replicate and segregate TR DNA, suggesting a unique role for the residues. Therefore, this work identifies adjacent LANA regions with distinct roles in Episome segregation and persistence.IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds the KSHV terminal-repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to Episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within the region reduced segregation of TR DNA to daughter cells, but not Episome maintenance. In contrast, mutants with deletions of 80 amino acids immediately downstream were highly deficient for Episome persistence yet maintained the ability to replicate and segregate TR DNA, the two principal components of Episome persistence, suggesting another role for the residues. In summary, this work identifies adjacent LANA sequence with distinct roles in Episome segregation and persistence.
-
Silencing NDRG1results in highly deficient viral Episome persistence.
2019Co-Authors: Fang Zhang, Kenneth M Kaye, Deguang Liang, Xiaoxi Lin, Zhe Zou, Rui Sun, Xing Wang, Xiaozhen Liang, Ke LanAbstract:(A) The knockdown efficiency of NDRG1 in KMM-shNDRG1 cells were determined by western blotting. (B) The viral genomes in KMM-shNDRG1 and KMM-shcon cells were extracted and measured by qPCR and normalized to the copy number of the GAPDH gene. The relative viral genomic copy number in the vector group was set as 1.0. Data were shown as mean ± SD, n = 3, *p
-
cross species conservation of Episome maintenance provides a basis for in vivo investigation of kaposi s sarcoma herpesvirus lana
PLOS Pathogens, 2017Co-Authors: Aline C Habison, Bruno Correia, Marta Pires De Miranda, Pedro J Simas, S. Arismar Cerqueira, Edward J. Usherwood, Chantal Beauchemin, Colin E Mcvey, Rajesh Ponnusamy, Kenneth M KayeAbstract:Many pathogens, including Kaposi’s sarcoma herpesvirus (KSHV), lack tractable small animal models. KSHV persists as a multi-copy, nuclear Episome in latently infected cells. KSHV latency-associated nuclear antigen (kLANA) binds viral terminal repeat (kTR) DNA to mediate Episome persistence. Model pathogen murine gammaherpesvirus 68 (MHV68) mLANA acts analogously on mTR DNA. kLANA and mLANA differ substantially in size and kTR and mTR show little sequence conservation. Here, we find kLANA and mLANA act reciprocally to mediate Episome persistence of TR DNA. Further, kLANA rescued mLANA deficient MHV68, enabling a chimeric virus to establish latent infection in vivo in germinal center B cells. The level of chimeric virus in vivo latency was moderately reduced compared to WT infection, but WT or chimeric MHV68 infected cells had similar viral genome copy numbers as assessed by immunofluorescence of LANA intranuclear dots or qPCR. Thus, despite more than 60 Ma of evolutionary divergence, mLANA and kLANA act reciprocally on TR DNA, and kLANA functionally substitutes for mLANA, allowing kLANA investigation in vivo. Analogous chimeras may allow in vivo investigation of genes of other human pathogens.
-
The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.
The Journal of biological chemistry, 2015Co-Authors: Min Tan, Bruno Correia, Franceline Juillard, Colin E Mcvey, Rajesh Ponnusamy, J. Pedro Simas, Maria Arménia Carrondo, Kenneth M KayeAbstract:Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral Episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates Episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated Episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and Episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing Episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of Episome maintenance function. Overall, the reductions in Episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced Episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and Episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.
-
kaposi s sarcoma associated herpesvirus lana recruits the dna polymerase clamp loader to mediate efficient replication and virus persistence
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Toshiki Tsurimoto, Franceline Juillard, Erika De Leon Vazquez, Shijun Li, Lin Li, She Chen, Kenneth M KayeAbstract:Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular Episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and Episome persistence. RFC depletion had a negative impact on LANA’s ability to replicate and maintain viral DNA in cells containing artificial KSHV Episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.
Arnold J. Berk - One of the best experts on this subject based on the ideXlab platform.
-
robust in vivo transduction of a genetically stable epstein barr virus Episome to hepatocytes in mice by a hybrid viral vector
Journal of Virology, 2009Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:To make a safe, long-lasting gene delivery vehicle, we developed a hybrid vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd vector is recombined to form a circular plasmid. This Episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the Episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this hybrid approach is highly efficient at delivering EBV Episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of vector-transduced mice demonstrated that luciferase transgene expression from the hybrid was robust and compared well to a traditional HDAd vector. Quantitative PCR analysis confirmed that the EBV Episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV hybrid to a variety of disease targets and the potential benefits of this approach are discussed.
-
874. Long-Term Transgene Expression In Vivo from a Helper Dependent Adenovirus|[mdash]|Epstein-Barr Virus Hybrid Vector
Molecular Therapy, 2005Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal Episome in replicating B cells. Maintenance is mediated by the virally-expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the Episome and tethers it to metaphase chromosomes for segregation into daughter cells during mitosis. In our hybrid binary system, an HDA vector (HDA-EBV) is used to deliver a linearized, EBV-based Episome to target cells. Co-infection with a second HDA vector expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked Episome sequence, and places a promoter upstream of the transgene. The Episome also contains a human origin and the EBNA-1 binding site for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA-EBV vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular Episomes in vitro and that the EBV elements significantly prolong transgene expression1.
-
development of a novel helper dependent adenovirus epstein barr virus hybrid system for the stable transformation of mammalian cells
Journal of Virology, 2004Co-Authors: Oliver Dorigo, Sean D. Gallaher, Michele P Calos, Pedro R Lowenstein, Maria G Castro, Arnold J. BerkAbstract:Epstein-Barr virus (EBV) Episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV Episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV Episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV Episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV Episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV Episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV Episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.
-
773. Non-Invasive In Vivo Detection of Episomes Delivered to Mouse Hepatocytes by a Helper Dependent Adenovirus- Epstein-Barr Virus Hybrid Vector System
Molecular Therapy, 2004Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal Episome in replicating B cells. Maintenance is mediated by the virally expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the Family of Repeats (FR) region of the EBV Episome, and tethers it to metaphase chromosomes for segregation during telophase. In our hybrid binary system, an HDA vector (HDA.EBV) is used to deliver a linear EBV-based Episome to target cells. Co-infection with a second HDA expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked Episome sequence, and places a CMV promoter upstream of the transgene. The Episome also contains a human origin and the FR for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA.EBV vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular Episomes in vitro and that EBNA-1 and the FR significantly prolong transgene expression (in publication).
-
an adenovirus epstein barr virus hybrid vector that stably transforms cultured cells with high efficiency
Journal of Virology, 1999Co-Authors: Lily Wu, Arnold J. BerkAbstract:EBV Episomes are nuclear plasmids that are stably maintained through multiple cell divisions in primate and canine cells (J. L. Yates, N. Warren, and B. Sugden, Nature 313:812–815, 1985). In this report, we describe the construction and characterization of an E1-deleted recombinant adenovirus vector system that delivers an EBV Episome to infected cells. This adenovirus-EBV hybrid vector system utilizes Cre-mediated, site-specific recombination to excise an EBV Episome from a target recombinant adenovirus genome. We demonstrate that this vector system efficiently delivers the EBV Episome and stably transforms a large fraction of infected canine D-17 cells. Using a colony-forming assay, we demonstrate stable transformation of 37% of cells that survive the infection. However, maximal transformation efficiency is achieved at doses of the E1-deleted recombinant adenoviruses that are toxic to the infected cells. Consequently, E1-deleted vector toxicity imposes a limitation on our current vector system.
Paul M. Lieberman - One of the best experts on this subject based on the ideXlab platform.
-
Control of Viral Latency by Episome Maintenance Proteins.
Trends in microbiology, 2019Co-Authors: Alessandra De Leo, Abram Calderon, Paul M. LiebermanAbstract:The human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) share the common property of persisting as multicopy Episomes in the nuclei of rapidly dividing host cells. These Episomes form the molecular basis for viral latency and are etiologically linked to virus-associated cancers. Episome maintenance requires epigenetic programming to ensure the proper control of viral gene expression, DNA replication, and genome copy number. For these viruses, Episome maintenance requires a dedicated virus-encoded Episome maintenance protein (EMP), namely LANA (KSHV), EBNA1 (EBV), and E2 (HPV). Here, we review common features of these viral EMPs and discuss recent advances in understanding how they contribute to the epigenetic control of viral Episome maintenance during latency.
-
HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus
Journal of virology, 2016Co-Authors: Jayaraju Dheekollu, Andreas Wiedmer, Daniel Sentana-lledo, Joel Cassel, Troy E. Messick, Paul M. LiebermanAbstract:ABSTRACT Epstein-Barr virus (EBV) establishes latent infections as multicopy Episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and Episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and Episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV Episomes from Burkitt9s lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and Episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation. IMPORTANCE EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control.
-
Chromatin Structure of Epstein–Barr Virus Latent Episomes
Current topics in microbiology and immunology, 2015Co-Authors: Paul M. LiebermanAbstract:EBV latent infection is characterized by a highly restricted pattern of viral gene expression. EBV can establish latent infections in multiple different tissue types with remarkable variation and plasticity in viral transcription and replication. During latency, the viral genome persists as a multi-copy Episome, a non-integrated-closed circular DNA with nucleosome structure similar to cellular chromosomes. Chromatin assembly and histone modifications contribute to the regulation of viral gene expression, DNA replication, and Episome persistence during latency. This review focuses on how EBV latency is regulated by chromatin and its associated processes.
-
molecular basis for oligomeric dna binding and Episome maintenance by kshv lana
PLOS Pathogens, 2013Co-Authors: John Domsic, Horng-shen Chen, Fang Lu, Ronen Marmorstein, Paul M. LiebermanAbstract:LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and Episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD) to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and Episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to Episome maintenance function. The structural features of LANADBD suggest a novel mechanism of Episome maintenance through DNA-binding induced oligomeric assembly.
-
Timeless-Dependent DNA Replication-Coupled Recombination Promotes Kaposi's Sarcoma-Associated Herpesvirus Episome Maintenance and Terminal Repeat Stability
Journal of virology, 2013Co-Authors: Jayaraju Dheekollu, Kenneth M Kaye, Horng-shen Chen, Paul M. LiebermanAbstract:Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable Episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV Episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV Episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.
Nicholas Coleman - One of the best experts on this subject based on the ideXlab platform.
-
STAT3 is important for cell cycle progression and HPV18 genome amplification in differentiated keratinocytes.
2018Co-Authors: Ethan L. Morgan, Nicholas Coleman, Christopher W. Wasson, Lucy Hanson, David Kealy, Ieisha Pentland, Victoria Mcguire, Cinzia Scarpini, Simon J. C. Arthur, Joanna L. ParishAbstract:Representative western blots of phosphorylated and total STAT3, HPV18 E6 and E7, cyclin D1, p21, involucrin and filaggrin in A) calcium or B) methylcellulose-differentiated HPV18 containing keratinocytes in the presence or absence of 10 μM cryptotanshione. Data shown represent at least three biological repeats. C) Southern blot analysis of HPV18 Episomes in keratinocytes treated with 10 μM cryptotanshinone and differentiated in methylcellulose for 120 hours. DNA was linearized with EcoRI, producing a single band running at approximately 8 kbp, demonstrating a differentiation-dependent increase in viral Episome copy number in untreated control cells and a reduction in Episome copy number in cells treated with cryptotanshinone. Digestion with BglII shows a lack of detectable multimeric/integrated HPV genomes in all experimental conditions. Data shown are representative of two donor cell lines. D) Signal intensity was quantified using ImageJ software. Bars represent means ± standard deviation of two biological repeats from two donor cell lines.
-
In vitro Progression of Human Papillomavirus 16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis
Cancer research, 2010Co-Authors: Elizabeth Gray, Mark R. Pett, Ian Roberts, Margaret Stanley, Dawn Ward, David M. Winder, Cinzia G Scarpini, Nicholas ColemanAbstract:An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ∼15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 Episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the Episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in Episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated Episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive Episome-retaining cells showed high levels of HPV16 E2/E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state. Cancer Res; 70(10); 4081–91. ©2010 AACR.
-
an increase in dna double strand breaks induced by ku70 depletion is associated with human papillomavirus 16 Episome loss and de novo viral integration events
The Journal of Pathology, 2007Co-Authors: Margaret Stanley, Nicholas Coleman, David M. Winder, N. Foster, Mkk Shivji, Mt Herdman, Ar VenkitaramanAbstract:Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 Episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 Episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 Episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.
-
An increase in DNA double‐strand breaks, induced by Ku70 depletion, is associated with human papillomavirus 16 Episome loss and de novo viral integration events
The Journal of pathology, 2007Co-Authors: David M. Winder, Margaret Stanley, Pett, N. Foster, Mkk Shivji, Mt Herdman, Ar Venkitaraman, Nicholas ColemanAbstract:Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 Episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 Episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 Episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.
-
Interferon-β treatment of cervical keratinocytes naturally infected with human papillomavirus 16 Episomes promotes rapid reduction in Episome numbers and emergence of latent integrants
Carcinogenesis, 2006Co-Authors: M. Trent Herdman, Mark R. Pett, Ian Roberts, William Alazawi, Andrew E. Teschendorff, Xiao-yin Zhang, Margaret Stanley, Nicholas ColemanAbstract:Following integration of human papillomavirus (HPV) into the host genome, overexpression of the viral oncogenes E6 and E7 requires loss of the transcriptional repressor functions of E2. A key step in HPV-related carcinogenesis is therefore clearance of residual viral Episomes, which encode E2. As spontaneous loss of HPV-16 Episomes in vitro is associated with increased expression of antiviral genes inducible by type I interferon (IFN), we used the W12 model to examine the effects of exogenous IFN-beta on cervical keratinocytes containing HPV-16 Episomes as a result of 'natural' infection in vivo. In contrast to studies of cells transfected with HPV-31 or bovine papillomavirus, IFN-beta caused rapid reduction in numbers of HPV-16 Episomes. This was associated with the emergence of cells bearing previously latent integrants, in which there was increased expression of E6 and E7. Our data indicate that integrated HPV-16 can exist in a minority of cells in a mixed population without exerting a selective advantage until Episome numbers are reduced. The kinetics of cell death and changes in viral transcription and translation that we observed support a model where integrants are initially present in cells also containing Episomes, with generalized Episome clearance by IFN-beta resulting in integrant de-repression. We conclude that IFN-beta can hasten the transition from episomal to integrated HPV-16 in naturally infected cervical keratinocytes. Greater emphasis should be placed on Episome loss in models of HPV-related carcinogenesis. We provide the strongest evidence to date that treating HPV-16 lesions by inducing an IFN response may cause clinical progression.
Lily Wu - One of the best experts on this subject based on the ideXlab platform.
-
an adenovirus epstein barr virus hybrid vector that stably transforms cultured cells with high efficiency
Journal of Virology, 1999Co-Authors: Lily Wu, Arnold J. BerkAbstract:EBV Episomes are nuclear plasmids that are stably maintained through multiple cell divisions in primate and canine cells (J. L. Yates, N. Warren, and B. Sugden, Nature 313:812–815, 1985). In this report, we describe the construction and characterization of an E1-deleted recombinant adenovirus vector system that delivers an EBV Episome to infected cells. This adenovirus-EBV hybrid vector system utilizes Cre-mediated, site-specific recombination to excise an EBV Episome from a target recombinant adenovirus genome. We demonstrate that this vector system efficiently delivers the EBV Episome and stably transforms a large fraction of infected canine D-17 cells. Using a colony-forming assay, we demonstrate stable transformation of 37% of cells that survive the infection. However, maximal transformation efficiency is achieved at doses of the E1-deleted recombinant adenoviruses that are toxic to the infected cells. Consequently, E1-deleted vector toxicity imposes a limitation on our current vector system.
-
An Adenovirus–Epstein-Barr Virus Hybrid Vector That Stably Transforms Cultured Cells with High Efficiency
Journal of Virology, 1999Co-Authors: Lily Wu, Arnold J. BerkAbstract:EBV Episomes are nuclear plasmids that are stably maintained through multiple cell divisions in primate and canine cells (J. L. Yates, N. Warren, and B. Sugden, Nature 313:812–815, 1985). In this report, we describe the construction and characterization of an E1-deleted recombinant adenovirus vector system that delivers an EBV Episome to infected cells. This adenovirus-EBV hybrid vector system utilizes Cre-mediated, site-specific recombination to excise an EBV Episome from a target recombinant adenovirus genome. We demonstrate that this vector system efficiently delivers the EBV Episome and stably transforms a large fraction of infected canine D-17 cells. Using a colony-forming assay, we demonstrate stable transformation of 37% of cells that survive the infection. However, maximal transformation efficiency is achieved at doses of the E1-deleted recombinant adenoviruses that are toxic to the infected cells. Consequently, E1-deleted vector toxicity imposes a limitation on our current vector system.