scholarly journals Centrosome Abnormalities and Genomic Instability by Episomal Expression of Human Papillomavirus Type 16 in Raft Cultures of Human Keratinocytes

2001 ◽  
Vol 75 (16) ◽  
pp. 7712-7716 ◽  
Author(s):  
Stefan Duensing ◽  
Anette Duensing ◽  
Elsa R. Flores ◽  
Anh Do ◽  
Paul F. Lambert ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Genomic Instability ◽  
Transcriptional Control ◽  
Ectopic Expression ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Hpv 16 ◽  
Hpv E6 ◽  
Copy Numbers ◽  
E6 And E7

ABSTRACT Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.

scholarly journals Human Papillomavirus E6 Proteins Mediate Resistance to Interferon-Induced Growth Arrest through Inhibition of p53 Acetylation

2007 ◽  
Vol 81 (23) ◽  
pp. 12740-12747 ◽  
Author(s):  
Christy Hebner ◽  
Melanie Beglin ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
Human Fibroblasts ◽  
Antiviral Agents ◽  
Growth Arrest ◽  
Physiological Role ◽  
Human Keratinocytes ◽  
Hpv E6 ◽  
E6 And E7 ◽  
E7 Proteins ◽  
Mutant Forms

ABSTRACT The high-risk human papillomavirus (HPV) E6 and E7 proteins act cooperatively to mediate multiple activities in viral pathogenesis. For instance, E7 acts to increase p53 levels while E6 accelerates its rate of turnover through the binding of the cellular ubiquitin ligase E6AP. Interferons are important antiviral agents that modulate both the initial and persistent phases of viral infection. The expression of HPV type 16 E7 was found to sensitize keratinocytes to the growth-inhibitory effects of interferon, while coexpression of E6 abrogates this inhibition. Treatment of E7-expressing cells with interferon ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by p300/CBP at lysine 382. Cells expressing mutant forms of E6 that are unable to bind p300/CBP or bind p53 failed to block acetylation of p53 at lysine 382 and were sensitive to growth arrest by interferon. In contrast, mutant forms of E6 that are unable to bind E6AP remain resistant to the effects of interferon, demonstrating that the absolute levels of p53 are not the major determinants of this activity. Finally, p53 acetylation at lysine 382 was found not to be an essential determinant of other types of senescence such as that induced by overexpression of Ras in human fibroblasts. This study identifies an important physiological role for E6 binding to p300/CBP in blocking growth arrest of human keratinocytes in the presence of interferon and so contributes to the persistence of HPV-infected cells.

scholarly journals Degradation of p53, Not Telomerase Activation, by E6 Is Required for Bypass of Crisis and Immortalization by Human Papillomavirus Type 16 E6/E7

2004 ◽  
Vol 78 (11) ◽  
pp. 5698-5706 ◽  
Author(s):  
H. R. McMurray ◽  
D. J. McCance
Keyword(s):  
Human Papillomavirus ◽  
Human Keratinocytes ◽  
Telomerase Activity ◽  
Transformation Process ◽  
Dominant Negative ◽  
Primary Human Keratinocytes ◽  
Telomere Shortening ◽  
Human Papillomavirus Type 16 ◽  
Tert Gene ◽  
E6 And E7

ABSTRACT Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.

scholarly journals Roles of the E6 and E7 Proteins in the Life Cycle of Low-Risk Human Papillomavirus Type 11

2004 ◽  
Vol 78 (5) ◽  
pp. 2620-2626 ◽  
Author(s):  
Stephen T. Oh ◽  
Michelle S. Longworth ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
High Risk ◽  
Translation Termination ◽  
Human Keratinocytes ◽  
Low Risk ◽  
Hpv E6 ◽  
E6 And E7 ◽  
Substitution Mutations ◽  
E7 Proteins

ABSTRACT Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.

scholarly journals Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

2001 ◽  
Vol 75 (16) ◽  
pp. 7602-7611 ◽  
Author(s):  
Cristina Balagué ◽  
Francisco Noya ◽  
Ramon Alemany ◽  
Louise T. Chow ◽  
David T. Curiel
Keyword(s):  
Human Papillomavirus ◽  
Anticancer Agents ◽  
Human Keratinocytes ◽  
Cell Killing ◽  
Oncolytic Adenoviruses ◽  
Hpv E6 ◽  
Stratified Squamous Epithelium ◽  
Infected Cells ◽  
E6 And E7 ◽  
Adenovirus Replication

ABSTRACT Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

scholarly journals Genetic Analysis of High-Risk E6 in Episomal Maintenance of Human Papillomavirus Genomes in Primary Human Keratinocytes

2002 ◽  
Vol 76 (22) ◽  
pp. 11359-11364 ◽  
Author(s):  
Regina B. Park ◽  
Elliot J. Androphy
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
High Risk ◽  
Viral Replication ◽  
Human Keratinocytes ◽  
Binding Motif ◽  
Primary Human Keratinocytes ◽  
Hpv16 E6 ◽  
E6 And E7 ◽  
Episomal Maintenance

ABSTRACT Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.

scholarly journals Global Effects of Human Papillomavirus Type 18 E6/E7 in an Organotypic Keratinocyte Culture System

2004 ◽  
Vol 78 (17) ◽  
pp. 9041-9050 ◽  
Author(s):  
Peggy A. Garner-Hamrick ◽  
J. M. Fostel ◽  
Wei-Ming Chien ◽  
N. Sanjib Banerjee ◽  
Louise T. Chow ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Culture System ◽  
Human Keratinocytes ◽  
Protein Translation ◽  
Primary Human Keratinocytes ◽  
Raft Culture ◽  
Dna And Rna ◽  
E6 And E7 ◽  
E7 Proteins ◽  
Hpv 18

ABSTRACT The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in ≥12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.

scholarly journals Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

2010 ◽  
Vol 84 (16) ◽  
pp. 8219-8230 ◽  
Author(s):  
Monika Somberg ◽  
Stefan Schwartz
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Binding Sites ◽  
Mrna Levels ◽  
Coding Region ◽  
Cervical Lesions ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

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