Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation

Centre for Cancer Research and Cell Biology, Queen's University, Belfast BT9 7BL, United Kingdom.
Journal of Virology (Impact Factor: 4.44). 03/2010; 84(10):5131-9. DOI: 10.1128/JVI.01965-09
Source: PubMed


The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to cause cellular transformation.
Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin
keratinocytes (HFKs) expressing E7 following methylcellulose-induced differentiation. Analysis of NPM expression in E7-expressing
cells and E6- and E7-expressing cells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel
analysis. The elevated expression of NPM was determined to be posttranscriptional and was attributed to increased v-akt murine
thymoma viral oncogene (AKT) activity in the E6- and E7-expressing cells. Depletion of NPM caused a reduction in the replicative
capacity of E7- and E6/E7-expressing HFKs and an increase in markers of differentiation. Also, the p53 and pRb tumor suppressor
levels are increased with the knockdown of NPM in E6/E7-expressing cells, and, interestingly, p14ARF is relocalized from the nucleolus to the nucleoplasm and cytoplasm in these cells. The results show for the first time that
NPM is required for the proliferation and inhibition of differentiation observed in HPV E6- and E7-expressing primary cells.

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Available from: Dennis J Mccance
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    • "ARF is considered to be a potential nucleolar integrator of growth signals, and Apicelli et al [17] recently proposed that basal ARF acts as a monitor of steady-state ribosome biogenesis and growth. ARF can be induced by protein E7 from the cottontail rabbit in E6/E7- or E7-immortalized rabbit keratinocytes [53] and by HPV16 E7 in E6/E7-expressing keratinocytes [52]. Our data show that E7 expression can induce p14ARF early after transduction of primary cervical keratinocytes, before their immortalization. "
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    ABSTRACT: High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.
    Full-text · Article · May 2014 · PLoS ONE
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    • "shER was provided by D. Picard (Geneva University, Geneva, Switzerland) siRNA for IKK or IKK was purchased from Ambion, and the sequence used was previously published (Accardi et al., 2011). siRNA for HPV16E6E7 or E7 was purchased from Dharmacon and previously published (Tang et al., 2006; McCloskey et al., 2010). The ISRE Luciferase minimal promoter was purchased from Stratagene. "
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    ABSTRACT: Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.
    Full-text · Article · Jun 2013 · Journal of Experimental Medicine
    • "Activates the p73 promoter + - Brooks et al. (2002) Upregulates the CDC25A promoter + ND Nguyen et al. (2002) Abrogates BRCA 1 inhibition of ER-α transcriptional activity + ND Zhang et al. (2005) Abrogates BRCA1 inhibition of E Box transcriptional activity + ND Zhang et al. (2005) Abrogates BRCA1 repression of hTERT + ND Zhang et al. (2005) Enhances c-myc dependent transcriptional transactivation + − Wang et al. (2007) Represses TLR9 promoter + ND Hasan et al. (2007) Abrogates E2F6 mediated transcriptional repression + + McLaughlin-Drubin et al. (2008) Induces the hTERT promoter + ND Liu et al. (2008) Upregulates Nucleophosmin + ND McCloskey et al. (2010) Activates HIF-1-mediated transcription + ND Bodily et al. (2011b) Co-activation, epigenetic control Abrogates BRG-1-mediated transcriptional repression of c-fos + ND Lee et al. (2002a) Abrogates p300 transcriptional co-activation function + o Bernat et al. (2003) Down-regulates steroid receptor coactivator 1 (SRC-1)- mediated transcription Relocalizes SRC-1 to the cytoplasm + + Baldwin et al. (2006) Abrogates FHL2 co-activation of beta-catenin and Fos/ Jun -dependent promoters + ND Campo-Fernandez et al. (2007) Stimulates DNMT1 activity + ND Burgers et al. (2007) Decreases the number of E2F6-PcG complexes + ND McLaughlin-Drubin et al. (2008) Induces EZH2 + + Holland et al. (2008) Upregulates SIRT1 + ND Allison et al. (2009) Represses E-cadherin promoter through a DNMT-1- dependent mechanism + ND Laurson et al. (2010) Upregulates KDM6A/B + ND McLaughlin-Drubin et al. (2011) Induces p16 INK4A expression through H3K27 demethylation + ND McLaughlin-Drubin et al. (2011) Modulation of cellular miR expression "
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    ABSTRACT: E7 is an accessory protein that is not encoded by all papillomaviruses. The E7 amino terminus contains two regions of similarity to conserved regions 1 and 2 of the adenovirus E1A protein, which are also conserved in the simian vacuolating virus 40 large tumor antigen. The E7 carboxyl terminus consists of a zinc-binding motif, which is related to similar motifs in E6 proteins. E7 proteins play a central role in the human papillomavirus life cycle, reprogramming the cellular environment to be conducive to viral replication. E7 proteins encoded by the cancer-associated alpha human papillomaviruses have potent transforming activities, which together with E6, are necessary but not sufficient to render their host squamous epithelial cell tumorigenic. This article strives to provide a comprehensive summary of the published research studies on human papillomavirus E7 proteins.
    No preview · Article · May 2013 · Virology
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