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 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 Identification of High-Risk Human Papillomavirus DNA, p16 and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1008
Author(s):  
Andrejs Lifsics ◽  
Valerija Groma ◽  
Maksims Cistjakovs ◽  
Sandra Skuja ◽  
Renars Deksnis ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Squamous Cell ◽  
Surrogate Marker ◽  
Hpv Infection ◽  
Hpv16 E6 ◽  
Hpv Dna ◽  
Molecular Virology ◽  
Hpv E6 ◽  
E6 And E7

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.

scholarly journals p21cip1 Degradation in Differentiated Keratinocytes Is Abrogated by Costabilization with Cyclin E Induced by Human Papillomavirus E7

2001 ◽  
Vol 75 (13) ◽  
pp. 6121-6134 ◽  
Author(s):  
Francisco Noya ◽  
Wei-Ming Chien ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Cyclin E ◽  
Human Keratinocytes ◽  
S Phase ◽  
Brdu Incorporation ◽  
Primary Human Keratinocytes ◽  
Differentiated Cells ◽  
Hpv E7 ◽  
Cyclin E2

ABSTRACT The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.

scholarly journals Human Papillomavirus Oncoproteins E6 and E7 Independently Abrogate the Mitotic Spindle Checkpoint

1998 ◽  
Vol 72 (2) ◽  
pp. 1131-1137 ◽  
Author(s):  
Jennifer T. Thomas ◽  
Laimonis A. Laimins
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Human Keratinocytes ◽  
Mitotic Checkpoint ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Control ◽  
Mitotic Spindle Checkpoint ◽  
E6 And E7

ABSTRACT The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.

scholarly journals Sp100 Provides Intrinsic Immunity against Human Papillomavirus Infection

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Wesley H. Stepp ◽  
Jordan M. Meyers ◽  
Alison A. McBride
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Human Keratinocytes ◽  
Hpv Infection ◽  
Nuclear Bodies ◽  
Host Cells ◽  
Viral Transcription ◽  
Primary Human Keratinocytes ◽  
Dna Viruses ◽  
Hpv Dna

ABSTRACTMost DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA.IMPORTANCEThe intrinsic immune system provides a first-line defense against invading pathogens. Host cells contain nuclear bodies (ND10) that are important for antiviral defense, yet many DNA viruses localize here upon cell entry. However, viruses also disrupt, reorganize, and modify individual components of the bodies. In this study, we show that one of the ND10 components, Sp100, limits the infection of human skin cells by human papillomavirus (HPV). HPVs are important pathogens that cause many types of infection of the cutaneous and mucosal epithelium and are the causative agents of several human cancers. Understanding how host cells counteract HPV infection could provide insight into antimicrobial therapies that could limit initial infection.

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 Conditionally Activated E7 Proteins of High-Risk and Low-Risk Human Papillomaviruses Induce S Phase in Postmitotic, Differentiated Human Keratinocytes

2006 ◽  
Vol 80 (13) ◽  
pp. 6517-6524 ◽  
Author(s):  
N. Sanjib Banerjee ◽  
Nicholas J. Genovese ◽  
Francisco Noya ◽  
Wei-Ming Chien ◽  
Thomas R. Broker ◽  
...  
Keyword(s):  
High Risk ◽  
Human Keratinocytes ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Low Risk ◽  
Binding Motif ◽  
Primary Human Keratinocytes ◽  
Hpv Genotypes ◽  
17Β Estradiol ◽  
E7 Proteins

ABSTRACT The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17β-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17β-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17β-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.

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.

Sign in / Sign up

Export Citation Format

Share Document