Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what other transcripts are dependent on or augmented by the association of NFX1-123 with 16E6. Microarray analysis revealed enhanced expression of Notch1 mRNA in 16E6-expressing keratinocytes when NFX1-123 was overexpressed. A moderate increase in Notch1 mRNA was seen with overexpression of NFX1-123 alone, but with 16E6 coexpression the increase in Notch1 was enhanced. The PAM2 motif and R3H protein domains in NFX1-123, which were important for increased hTERT expression, were also important in the augmentation of Notch1 expression by 16E6. These findings identify a second gene coregulated by 16E6 and NFX1-123 and the protein motifs in NFX1-123 that are important for this effect. Human papillomaviruses (HPVs) are nonenveloped doublestranded DNA viruses. Of the more than 150 HPVs that have been identified to date, over 30 types are capable of infecting the genital and oral mucosa. HPVs that infect mucosa are divided into low-risk (LR) and high-risk (HR) categories based on their associated risk for anogenital and, increasingly, head and neck cancers (1-11). As these HR HPV infections are linked to malignancies, much focus has been placed on studying the cellular changes these viruses trigger that lead to cancer and on studying the functions of two oncogenes expressed by HR HPV, E6 and E7.HR E6 induces oncogenic changes primarily through its interactions with host cell proteins, and those interactions block apoptotic signaling and drive cellular immortalization (for a review, see reference 12). E6-associated protein (E6AP) is an E3 ubiquitin ligase that is the most well-studied protein partner of HR E6 (13-19). However, HR E6 interacts with other cellular proteins, such as NFX1 splice variants, and these are important in oncogenesis as well (20,21).In human epithelial cells, NFX1 is a gene expressed as two splice variants, . NFX1-91 has been shown to transcriptionally repress telomerase expression in epithelial cells, and HR HPV 16E6 with E6AP targets NFX1-91 for degradation (22,23). Our work has focused on NFX1-123, the longer splice variant of NFX1, and on its role with 16E6 in oncogenesis. Previously we had shown that 16E6 bound NFX1-123 in its central domain, but unlike NFX1-91 it was not degraded; together, 16E6 and NFX1-123 increased hTERT expression and telomerase activity in human foreskin keratinocytes (HFKs) through a posttranscriptional mechanism (20). 16E6 and NFX1-123 increased hTERT by utilizing both the PAM2 motif and R3H domain of the NFX1-123 protein. The N-terminal PAM2 motif is required to interact with cytopl...

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

NFX1-123 and Human Papillomavirus 16E6 Increase Notch Expression in Keratinocytes

Vliet-Gregg¹,

Hamilton²,

Katzenellenbogen

2013

“…pRB1 inactivation also triggers cell cycle control checkpoints, and one mechanism by which high-risk HPV oncoproteins inactivate these checkpoints is the targeting of the tumor suppressor p53 (10) for proteasome-mediated degradation (11). High-risk HPV E6 recruits the cellular ubiquitin ligase E6AP to ubiquitinate p53 in this reaction (10,11), and critically, it is only the high-risk genus alpha HPV E6 that can bind both p53 and E6AP (12,13). In cervical cancer cells, p53 levels are quite low due to E6-mediated proteolysis.…”

mentioning

confidence: 99%

Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes

White

Walther

Javanbakht

et al. 2014

Self Cite

The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCEThis study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer.H uman papillomaviruses (HPVs) are DNA viruses with small double-stranded DNA genomes and a tropism for squamous epithelial cells. Different HPVs have been associated with various neoplastic diseases: they are the established cause of cervical cancer and have been associated with other anogenital cancers, they are responsible for an increasing proportion of head and neck oropharyngeal cancers, and they cause hyperproliferative lesions in patients with the genetic disorder epidermodysplasia verruciformis (EV) (1). Most HPV infections are benign, and the vast majority of HPV infections resolve without progression to cancer. This diversity of pathology is related to and reflected by the phylogenetic relationships between HPV types.The more than 178 HPVs now identified are grouped on the basis of the sequence of their L1 gene into five genera (2, 3). Genus alpha human papillomaviruses (alpha HPVs) infect the mucosal epithelium, and a subset of these are associated with cancer. The "high-ris...

“…Thus, the N-terminal regions of the E6 proteins of beta-HPVs appear to be functionally similar to the C-terminal region of E1A. It is unclear why a recent unbiased proteomic study (28) and two hybrid interaction studies (27) did not identify DYRK1/HAN11 interaction with the beta-HPV E6 proteins. A major difference might that we used transient overexpression of different E6 proteins.…”

mentioning

confidence: 93%

“…The beta-HPV E6 proteins have been reported to suppress UV-induced apoptosis by interacting with the proapoptotic molecule BAK and targeting it for degradation (23,24). Recent protein-protein interaction studies using affinity purification and mass-spectrometric analyses and yeast two-hybrid interaction approaches identified interactions with cellular proteins such as MAML1, p300, and Ccr4-Not (25)(26)(27)(28). These studies have provided novel insights into the functions of beta-HPV E6 proteins.…”

mentioning

confidence: 99%

Functional Similarity between E6 Proteins of Cutaneous Human Papillomaviruses and the Adenovirus E1A Tumor-Restraining Module

Kuppuswamy

Subramanian

Kostas-Polston

et al. 2013

bThe adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins. Functional commonality is a prominent theme by which the oncoproteins of small DNA tumor viruses such as the polyomaviruses, papillomaviruses, and adenoviruses subvert cell growth-regulatory activities. It is well known that the transforming proteins of these viruses target the tumor suppressor protein p53 to inactivate its cell growth-inhibitory activities (reviewed in reference 1). The second example is the targeting of the retinoblastoma protein (pRb) by which the viral proteins such as adenovirus E1A, simian virus 40 (SV40) T antigen, and human papillomavirus (HPV) E7 disable the cell cycle restriction imposed by retinoblastoma family proteins (reviewed in references 2 and 3). Since these viruses infect quiescent target cells, they induce a cellular proliferative state to facilitate their DNA replication by inactivating the cell cycle regulatory checkpoints conferred by antiproliferative molecules such as p53 and pRb. The transforming protein E4-ORF1 of HAdv9 and E6 proteins of high-risk HPVs have been shown to target the epithelial cell tight junction and polarity proteins, such as hDgl-1, Scribble, and PATJ, through conserved PDZ-binding motifs to disrupt epithelial differentiation and promote oncogenesis (4).Studies on the transforming activities of HAdv5/2 E1A proteins have revealed that the N-terminal region of E1A promotes cell proliferation and inhibits cell differentiation to mediate oncogenic cell transformation (5) by interacting with retinoblastoma family proteins, p300/CBP (6, 7), and p400/TRRAP protein complexes (8-10). In contrast to the N-terminal region, the C-terminal region antagonizes cell proliferation (11) and differentiation activities (12, 13). Deletion of the C-terminal region in HAdv5/2 E1A confers a hypertransforming function to E1A (12,(14)(15)(16)(17). The transformed cells that express the E1A C-terminal mutants and the activated Ras oncogene are also highly tumorigenic and metastatic (14,17). The E1A C-terminal region interacts with three different protein complexes, FOXK1/K2, DYRK1A/1B/ HAN11 (referred to here as DYRK1/HAN11), and CtBP1/2 to collectively confer the tumor-restraining activity to the E1A C-terminal region (11).Of the more than 120 known human papillomaviruses (HPVs), a predominant number belong to the genus beta. Unlike the alpha-HPVs, which are linked to anogenital and head and neck cancers, the beta-HPVs are less extensively studied. Certain beta-HPV species...