Cellular Steady-State Levels of “High Risk” but Not “Low Risk” Human Papillomavirus (HPV) E6 Proteins Are Increased by Inhibition of Proteasome-Dependent Degradation Independent of Their p53- and E6AP-Binding Capabilities

Kehmeier, Eva; Rühl, Heiko; Voland, Britta; Stöppler, Melissa Conrad; Androphy, Elliot J.; Stöppler, Hubert

doi:10.1006/viro.2002.1502

Cited by 39 publications

(41 citation statements)

References 62 publications

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“…Conversely, if the silencing of E6AP led to a concomitant decrease in E6 protein levels, we would have expected our microarray results to be similar to those observed, and while our overall conclusionthat all E6 functions are dependent on E6AP-would have been the same, the underlying mechanism would have been quite distinct. Importantly, however, there are no reports in the literature to suggest that E6AP affects E6 protein expression levels, and a previous study has shown that steady-state levels of functional, epitope-tagged E6 proteins are not influenced, either positively or negatively, by their ability to interact with E6AP (28). It therefore seems unlikely that the silencing of E6AP would have affected E6 protein levels in our experiments.…”

Section: Vol 79 2005 Effects Of E6 and E6ap On Gene Expression Profmentioning

confidence: 66%

The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

Kelley

Keiger

Lee

et al. 2005

J Virol

100

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The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of ϳ31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.Human papillomaviruses (HPVs) are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells. Approximately 30 HPV types are sexually transmitted and can be further categorized according to their associations with malignancies (65). Low-risk HPV types (e.g., types 6 and 11) are associated with benign lesions, while DNAs of high-risk HPV types (e.g., types 16, 18, 33, and 39) are found in Ͼ95% of all cervical cancers, strongly suggesting that HPV is a causative agent of this disease (65). The only two viral proteins that are generally expressed in HPV-positive cervical cancers, E6 and E7, have been shown to be necessary and sufficient to immortalize human foreskin keratinocytes, further suggesting that they are cooperating oncoproteins (21, 41). HPV E7 interacts with and inactivates the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130 (40). E7 inactivation of pRb activates E2F transcription factors, which results in increased transcription of E2F-responsive genes and S-phase cell cycle progression. While several other E7-interacting proteins have been identified (including histone deacetylase [38], p21, TATA-binding protein, and TAF110 [40]), results from animal studies indicate that the major biologic activities of E7 are related ...

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Section: Vol 79 2005 Effects Of E6 and E6ap On Gene Expression Profmentioning

confidence: 66%

The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

Kelley

Keiger

Lee

et al. 2005

J Virol

100

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“…Interestingly, the levels are restored by proteasome inhibitor treatment 2 hr before harvesting. Both E6 and E6*I are stabilized by MG132 suggesting that not only full-length E6 23 but also E6*I is degraded along the proteasome pathway. As a control we also carried out the same fractionation experiment on the wt HPV-18 E6 construct and on a HPV-16 E7 expressing construct, to show that the localization is consistent with that of the 2 proteins taken singularly (Fig.…”

Section: Hpv-18 E6*i Regulates the Levels Of Hpv-18 Full-length E6mentioning

confidence: 99%

HPV‐18 E6*I modulates HPV‐18 full‐length E6 functions in a cell cycle dependent manner

Guccione

Pim

Banks

2004

Intl Journal of Cancer

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The E6 ORFs of the high-risk Human Papillomavirus (HPV) Types 16 and 18 have been shown to encode (besides the full-length product) several truncated forms, termed E6*. We have reported previously that the HPV-18 E6*I protein interacts with the full-length E6 protein as well as with the ubiquitin ligase E6-AP and, as a result of this, E6* can inhibit E6-mediated degradation of p53. Moreover, ectopic expression of the HPV-18 E6*I protein has an antiproliferative effect in cervical cancer-derived cell lines. These results led us to investigate further the modulatory functions of E6*I on E6. Using epitope tagged versions of the 2 proteins we have analyzed the sub-cellular distribution of the full-length HPV18 E6 and HPV18 E6*I, as well as their respective cellular abundance during the cell cycle, and show specific upregulation of E6*I during G2/M. We also investigated the effect of E6*I overexpression in cell lines derived from cervical tumors, with respect to the expression levels of E6 target proteins, such as p53, hDlg and Scribble and find a corresponding increase in p53 expression also during G2/M. In addition we show that the overexpression of E6*I reduces the amount of E6 in the insoluble nuclear and membrane fractions of the cell. E6 levels can, however, be restored by the addition of a specific proteasome inhibitor, suggesting that the interaction between E6 and E6*I leads to the destabilization of a subset of the E6 protein. These results suggest that the E6*I protein can function as a fine regulator of the fulllength E6 protein by direct interaction that leads both to changes in its cellular abundance as well as its distribution during particular phases of the cell cycle. © 2004 Wiley-Liss, Inc. Key words: E6*I; E6; cell cycle; HPV; proteasome degradationHuman papillomaviruses (HPVs) are linked to the development of a number of human malignancies. 1 These include squamous cell carcinomas (SCCs) in immunosuppressed individuals and in patients with epidermodysplasia verruciformis (EV), head and neck cancers, anogenital cancers and, most notably, carcinoma of the uterine cervix. 2 Although more than 130 HPV types have been described so far, infection by only a small subset of virus types (high-risk HPVs) is associated with the aforementioned malignancies, whereas others give rise to benign condylomas, and cutaneous plantar and palmar warts (low-risk HPVs). 3 High-risk mucosal HPVs encode 2 major oncoproteins, E6 and E7, both of which have oncogenic activity in a variety of cell transformation assays. 4 -7 E7 has the major role of deregulating the cell cycle, uncoupling it from differentiation, 8 and it plays a critical role in tumor promotion. In contrast E6 contributes more strongly during tumor progression, accelerating the malignant conversion of benign tumors. 9 High-risk, but not low-risk HPVs transcribe both E6 and E7 as bicistronic and polycistronic mRNAs. 10 -13 The resulting transcripts encode the 2 full-length proteins and 4 shorter peptides termed E6*. 14 These alternatively spliced transcripts ...

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“…It has also been suggested that the subcellular localiza-tion of E6 is essential for its p53 degradation activity, based on the observation that high-risk HPV16, -18, and -31 E6 proteins can localize to the nucleus after transfection whereas low-risk HPV6 and -11 E6 proteins are predominantly cytoplasmic (35,37,41,57,59,60,63), although HPV11 E6 has also been detected in the nucleus (24). In another study, the expression levels of E6 have been shown to vary between high-and low-risk types (31). Specifically, high-risk HPV16 E6 was shown to possess a lower steadystate expression level than E6 from the low-risk HPV6 and -11 or the cutaneous HPV5 and -8.…”

mentioning

confidence: 99%

p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

Mésplède

Gagnon

Bergeron-Labrecque

et al. 2012

J Virol

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Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high-and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC 50 ]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC 50 s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some highrisk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants.

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Cellular Steady-State Levels of “High Risk” but Not “Low Risk” Human Papillomavirus (HPV) E6 Proteins Are Increased by Inhibition of Proteasome-Dependent Degradation Independent of Their p53- and E6AP-Binding Capabilities

Cited by 39 publications

References 62 publications

The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

HPV‐18 E6*I modulates HPV‐18 full‐length E6 functions in a cell cycle dependent manner

p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

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