RESUMEN
Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.
Asunto(s)
Herencia Extracromosómica , Genoma Viral/efectos de los fármacos , Papillomavirus Humano 16/genética , Interferón gamma/farmacología , Queratinocitos/efectos de los fármacos , Infecciones por Papillomavirus/genética , Plásmidos/genética , Integración Viral/genética , Antivirales/farmacología , Transformación Celular Viral/efectos de los fármacos , Células Cultivadas , ADN Viral/genética , Humanos , Queratinocitos/virología , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.
Asunto(s)
Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 16/genética , Queratinocitos/virología , ARN Mensajero/metabolismo , Recombinación Genética , Integración Viral , Transformación Celular Viral , Células Cultivadas , Células Clonales/virología , Femenino , Genoma Viral , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos/metabolismo , Masculino , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Neoplasias del Cuello Uterino/virologíaRESUMEN
Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.
Asunto(s)
Carcinoma/virología , Transformación Celular Viral , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Secuencias Reguladoras de Ácidos Nucleicos , Origen de Réplica , Transcripción Genética , Neoplasias del Cuello Uterino/virología , Secuencia de Bases , Línea Celular , Femenino , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/fisiología , Humanos , Queratinocitos/virología , Datos de Secuencia MolecularRESUMEN
Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in approximately 25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.
Asunto(s)
Papillomavirus Humano 18/fisiología , Queratinocitos/virología , Southern Blotting , Células Cultivadas , Cuello del Útero/citología , Cuello del Útero/virología , Femenino , Prepucio/citología , Prepucio/virología , Humanos , Masculino , Tonsila Palatina/citología , Tonsila Palatina/virología , Papillomaviridae/fisiología , Transcripción Genética/fisiología , Replicación Viral/fisiologíaRESUMEN
Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-beta synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-beta as a potential boost in infection. Treatment with low levels of human IFN-beta induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1(-/-), IRF-2(-/-) and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.
Asunto(s)
Genoma Viral , Papillomavirus Humano 11/genética , Papillomavirus Humano 16/genética , Factor 1 Regulador del Interferón/genética , Interferón beta/farmacología , Replicación Viral , Animales , Inmunoprecipitación de Cromatina , Cartilla de ADN/química , Ensayo de Cambio de Movilidad Electroforética , Femenino , Regulación Viral de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/metabolismo , Factor 1 Regulador del Interferón/fisiología , Factor 2 Regulador del Interferón/fisiología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/virología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Elementos de Respuesta , Transcripción Genética/efectos de los fármacosRESUMEN
Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3' of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the 'initiator' YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3' enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the 'initiator' YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-'initiator' factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.
Asunto(s)
Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras/metabolismo , Sitio de Iniciación de la Transcripción/fisiología , Factor de Transcripción YY1/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Viral de la Expresión Génica/fisiología , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Represoras/genéticaRESUMEN
Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1- mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5' mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1- genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.
Asunto(s)
Genes , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Secuencia de Bases , Línea Celular , Replicación del ADN , ADN Viral/biosíntesis , Prueba de Complementación Genética , Papillomavirus Humano 16/fisiología , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Sitios de Empalme de ARN , TATA Box , Sitio de Iniciación de la Transcripción , Replicación ViralRESUMEN
A conserved E8(wedge)E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8--E2 (16-E8--E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8--E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8--E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8--E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8--E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8--E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8(wedge)E2 limits the viral copy number but is not required for plasmid persistence and maintenance.
Asunto(s)
ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/fisiología , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Replicación del ADN , Células HeLa , Humanos , Transcripción Genética , Replicación ViralRESUMEN
Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection.
Asunto(s)
Queratinocitos/virología , Papillomaviridae/fisiología , Cultivo de Virus/métodos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Nutrientes , Fibroblastos , Genoma Viral , Humanos , Control de Calidad , Transfección , Replicación ViralRESUMEN
The E2 open reading frame of bovine papillomavirus (BPV)-1 encodes a 410 amino acid (aa) transcriptional activator, E2-TA, and collinear polypeptides--E2-TR (243 aa) and E8^E2 (196 aa). E8^E2 and E2-TR share the DNA-binding domain of E2-TA, and both have been defined as transcriptional repressors. Although purified E2-TR and E8^E2 proteins specifically bound E2 sites with similar affinities, only the E2-TR stimulated transcription. Here we show that E2-TR trans-activates E2-dependent promoters 5 to 10-fold in cooperation with cellular factors and in a dose-dependent fashion in epithelial cells and fibroblasts of animal or human origin while E2-TA activated >100-fold and the E8^E2 had no effect. However, in contrast to E2-TA, E2-TR activated transcription from a promoter-proximal position. E2-TR also partially inhibited the BPV-1 P89 or heterologous promoters whereas E8^E2 led to complete repression. Thus, the BPV-1 E2-TR modulates viral gene expression in a manner distinct from other E2 proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Eliminación de Secuencia , Transactivadores/genética , Proteínas Virales/genéticaRESUMEN
Interferon regulatory factors (IRFs) are critical mediators of gene expression, cell growth and immune responses. We previously demonstrated that interferon (IFN) induction of early viral transcription and replication in several mucosal HPVs requires IRF-1 binding to a conserved interferon response element (IRE). Here we show that the IRF-2 protein serves as a baseline transactivator of the HPV-16 major early promoter, P97. Cotransfections in IRF knockout cells confirmed that basal HPV-16 promoter activity was supported by both IRF-1 and IRF-2 complexes interacting with the promoter-proximal IRE in a dose-dependent manner. Furthermore, HPV-16 E7 expression downregulates the IRF-2 promoter, thus linking IRF-2 levels to viral transforming gene expression through a negative feedback mechanism. Taken together, these observations reveal a complex viral strategy utilizing multiple signal transduction pathways during the establishment and maintenance of HPV persistence.
Asunto(s)
Papillomavirus Humano 16/genética , Factor 2 Regulador del Interferón/metabolismo , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Transcripción Genética , Línea Celular , Regulación Viral de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/metabolismo , Queratinocitos/virología , Proteínas E7 de Papillomavirus , Transducción de SeñalRESUMEN
Transcription from the major upstream early gene promoter, P89, of bovine papillomavirus (BPV)-1 is detectable in transfected cells lacking viral gene products yet also responds to viral E2 proteins. In contrast to human papillomaviruses (HPVs), the BPV upstream regulatory region (URR) functions as a transcriptional enhancer in epithelial cells and fibroblasts of bovine, murine or human origin. Mutations of Sp1 and/or two novel transcriptional enhancer factor (TEF)-1 sites within the 5' URR of the intact BPV-1 genome dramatically reduced P89-initiated mRNA levels, leading to decreased BPV-1 plasmid amplification and inefficient formation of transformed cell foci. However, cell lines transformed with wt or mutant BPV-1 genomes contained similar levels of unintegrated BPV-1 DNA, P89 mRNA and E2-dependent transactivation. We conclude that cellular factors necessary for activating viral early gene transcription, establishment of viral plasmid replication and cell immortalization are not required during the maintenance phase of BPV-1 infection.