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1.  Localization of HPV-18 E2 at Mitochondrial Membranes Induces ROS Release and Modulates Host Cell Metabolism 
PLoS ONE  2013;8(9):e75625.
Papillomavirus E2 proteins are predominantly retained in the nuclei of infected cells, but oncogenic (high-risk) HPV-18 and 16 E2 can shuttle between the host nucleus and cytoplasm. We show here that cytoplasmic HPV-18 E2 localizes to mitochondrial membranes, and independent mass spectrometry analyses of the E2 interactome revealed association to the inner mitochondrial membrane including components of the respiratory chain. Mitochondrial E2 association modifies the cristae morphology when analyzed by electron microscopy and increases production of mitochondrial ROS. This ROS release does not induce apoptosis, but instead correlates with stabilization of HIF-1α and increased glycolysis. These mitochondrial functions are not shared by the non-oncogenic (low-risk) HPV-6 E2 protein, suggesting that modification of cellular metabolism by high-risk HPV E2 proteins could play a role in carcinogenesis by inducing the Warburg effect.
PMCID: PMC3782431  PMID: 24086592
2.  Loss of HPV16 E2 Protein Expression Without Disruption of the E2 ORF Correlates with Carcinogenic Progression 
The Open Virology Journal  2012;6:163-172.
Integration of the viral DNA in the cellular genome has been suggested to be critical in carcinogenic progression of HPV-associated cervical neoplasia. This event can be accompanied by disruption of the open reading frame (ORF) encoding the E2 repressor, thus leading to transcriptional up-regulation of the E6 and E7 viral oncogenes. At this stage, it is unclear whether disruption of the E2 ORF is mandatory for carcinogenic progression. We measured E2 RNA and protein expression in clinical samples of various grades of HPV16-associated cervical neoplasia and compared it with the status of the viral genome. RNA extracted from paraffin embedded tissues was hybridized to specific probes and quantified by the NanoString technology. Protein expression was appreciated by immunohistochemistry and the status of viral DNA was determined by in situ hybridization, all performed on serial sections of the same samples. E2 protein was found highly expressed in CIN1, CIN2 lesions where the HPV DNA was highly replicative, while it was decreased in more advanced grade lesions where replication is decreased or lost (CIN3 and SCC). In contrast, E2 transcripts could be elevated even in conditions of no or low expression of the protein, as found in the Caski cell line. Our data demonstrate that integration of the viral DNA in the cellular genome does not always lead to disruption of the E2 ORF and drastic reduction of E2 transcripts, while in contrast, expression of the E2 protein is always drastically reduced.
PMCID: PMC3547325  PMID: 23341852
HPV; transcription; IHC staining; E2; E6; E7; cyclin B1; viral genome status.
3.  The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation 
PLoS Pathogens  2011;7(9):e1002256.
Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.
Author Summary
High-risk human papillomavirus infection can cause cancer of the uterine cervix. The viral proteins leading to transformation of the infected keratinocytes are the E6 and E7 oncogenes which interact with and induce degradation of the cell cycle regulators p53 and pRB. In cervical carcinoma cells, repression of E6/E7 stabilizes the p53 transcription factor leading to activation of a large group of cellular p53 target genes. Here we show that repression of E6/E7 also induces transcriptional activation of an additional large set of genes involved in cell adhesion including previously described p63 target genes. Indeed, we further demonstrated that these p63 target genes are activated by TAp63β and not by p53 or by the ΔNp63α or β isoforms, even though these transcription factors are also expressed in these cells. In cervical carcinoma cells, E6 expression therefore leads to TAp63β degradation thereby allowing anchorage independent growth. Our work describes a new E6-dependent transformation pathway in HPV-associated carcinogenesis. TAp63β inhibition may also represent a common pathway to activate anchorage independent growth in cancers.
PMCID: PMC3182928  PMID: 21980285
4.  Tumor suppressor or oncogene? A critical role of the human papillomavirus (HPV) E2 protein in cervical cancer progression 
The papillomavirus (PV) E2 proteins have been shown to exert many functions in the viral cycle including pivotal roles in transcriptional regulation and in viral DNA replication. Besides these historical roles, which rely on their aptitude to bind to specific DNA sequences, E2 has also been shown to modulate the host cells through direct protein interactions mainly through its amino terminal transactivation domain. We will describe here some of these new functions of E2 and their potential implication in the HPV-induced carcinogenesis. More particularly we will focus on E2-mediated modulation of the host cell cycle and consequences to cell transformation. In all, the HPV E2 proteins exhibit complex functions independent of transcription that can modulate the host cells in concert with the viral vegetative cycle and which could be involved in early carcinogenesis.
PMCID: PMC3180061  PMID: 21968515
HPV; E2; transformation; transcription; chromatin; replication; cell cycle; apoptosis; cervical carcinoma
5.  The Human Papillomavirus Type 18 E2 Protein Is a Cell Cycle-Dependent Target of the SCFSkp2 Ubiquitin Ligase▿  
Journal of Virology  2009;84(1):437-444.
The human papillomavirus type 18 (HPV-18) E2 gene is inactivated in cervical carcinoma after integration of the viral DNA into the host cellular genome. Since E2 represses the transcription of the two viral oncogenes E6 and E7, integration which allows their strong expression is considered a major step in transformation by HPV. We show here that E2 is specifically degraded at the end of the G1 phase in a Brd4-independent manner, implying that its regulatory functions are cell cycle dependent. Degradation of E2 occurs via the Skp1/Cullin1/F-box Skp2 (SCFSkp2) ubiquitin ligase, since silencing of Skp2 induces stabilization of E2. In addition, the amino-terminal domain of E2 can interact with Skp2 as shown by coimmunoprecipitation experiments. We previously showed that E2 inhibits the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to accumulation of several of its substrates. We demonstrate here that Skp2, which is a known APC/C substrate in G1, is also stabilized by E2. Therefore, by negative feedback, SCFSkp2 activation could lead to E2 degradation and E6/E7 expression specifically in the late G1 phase. Moreover, since the SCFSkp2 can trigger S-phase entry and Skp2 itself is a known oncogene, we believe that E2-mediated accumulation of Skp2, together with E2 degradation leading to putative release of E6 and E7 inhibition, could induce premature S-phase entry in HPV-infected cells, pointing to a direct role of E2 in the early steps of HPV-mediated transformation.
PMCID: PMC2798402  PMID: 19828607
6.  A New E6/P63 Pathway, Together with a Strong E7/E2F Mitotic Pathway, Modulates the Transcriptome in Cervical Cancer Cells▿  
Journal of Virology  2007;81(17):9368-9376.
Cervical carcinoma is associated with certain types of human papillomaviruses expressing the E6 and E7 oncogenes, which are involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. A critical event on the path to malignant transformation is often manifested by the loss of expression of the viral E2 transcription factor due to the integration into the host genome of the viral DNA. Using microarrays, we have previously shown that reintroduction of a functional E2 in the HeLa cervical carcinoma cell line activates a cluster of p53 target genes while at the same time severely repressing a group of E2F target genes. In the present study, using new high-density microarrays containing more than 22,000 human cDNA sequences, we identified a novel p63 pathway among E2-activated genes and 38 new mitotic genes repressed by E2. We then sought to determine the pathways through which these genes were modulated and used an approach that relies on small interfering RNA to demonstrate that the p63 target genes were activated through silencing of the E6/E6AP pathway while the mitotic genes were mainly repressed through E7 silencing. Importantly, a subset of the mitotic genes was shown to be significantly induced in biopsies of stage IV cervical cancers, which points to a prominent E7 pathway in cervical carcinoma.
PMCID: PMC1951466  PMID: 17582001
7.  HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo 
Journal of Clinical Investigation  2005;115(2):348-358.
Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein–1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-κB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.
PMCID: PMC546425  PMID: 15690084
8.  HMG-I(Y) and the CBP/p300 Coactivator Are Essential for Human Papillomavirus Type 18 Enhanceosome Transcriptional Activity 
Molecular and Cellular Biology  2003;23(7):2329-2340.
A strong epithelial specific enhancer drives transcription of the human papillomavirus type 18 (HPV18) oncogenes. Its activity depends on the formation of a higher-order nucleoprotein complex (enhanceosome) involving the sequence-specific JunB/Fra2 transcription factor and the HMG-I(Y) architectural protein. Here we show that proteins from HeLa cell nuclear extract cover almost all of the HPV18 enhancer sequences and that it contains seven binding sites for the purified HMG-I(Y) protein, providing evidence for a tight nucleoprotein structure. Binding of HMG-I(Y) and the AP1 heterodimer from HeLa nuclear extract to overlapping sites of the core enhanceosome is cooperative. The integrity of this specific HMG-I(Y) binding site is as essential as the AP1 binding site for the enhancer function, indicating the fundamental role played by this architectural protein. We demonstrate that the CBP/p300 coactivator is recruited by the HPV18 enhanceosome and that it is limiting for transcriptional activation, since it is sequestered by the adenovirus E1A protein and by the JunB/Fra2 positive factor in excess. We show the involvement of JunB and p300 in vivo in the HPV18 transcription by chromatin immunoprecipitation of HPV18 sequences in HeLa cells.
PMCID: PMC150719  PMID: 12640118
9.  Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain 
Journal of Virology  2001;75(16):7244-7251.
The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G1 growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.
PMCID: PMC114960  PMID: 11461997

Results 1-9 (9)