Proteasomal Degradation of the Papillomavirus E2 Protein Is Inhibited by Overexpression of Bromodomain-Containing Protein 4

The papillomavirus (PV) E2 protein is an important regulator of the viral life cycle. It has diverse roles in viral transcription, DNA replication, and genome maintenance. Our laboratory has previously identified the cellular bromodomain protein Brd4 as a key interacting partner of E2. Brd4 mediates the transcriptional activation function of E2 and plays an important role in viral genome maintenance in dividing cells. E2 interacts with the C-terminal domain (CTD) of Brd4, and the CTD functions in a dominant-negative manner through binding E2 and interfering with E2's interaction with the full-length Brd4 protein. Previous studies have shown that PV E2 proteins are short lived; however, the mechanisms regulating their stability and degradation have not yet been well established. In this study, we explored the role of Brd4 in the regulation of bovine PV 1 (BPV1) and human PV 16 (HPV16) E2 stability. Expression of the Brd4 CTD dramatically increases E2 levels. Both BPV1 E2 and HPV16 E2 are regulated by ubiquitylation, and Brd4 CTD expression blocks this ubiquitylation, thus stabilizing the E2 protein. Furthermore, we have identified the cullin-based E3 ligases and specifically cullin-3 as potential components of the ubiquitylation machinery that targets both BPV1 and HPV16 E2 for ubiquitylation. Expression of the Brd4 CTD blocks the interaction between E2 and the cullin-3 complex. In addition to Brd4's role in mediating E2 transcription and genome tethering activities, these data suggest a potential role for Brd4 in regulating E2 stability and protein levels within PV-infected cells.Papillomaviruses (PVs) are small, double-stranded DNA tumor viruses that infect squamous epithelial cells. Their life cycle is closely linked to the differentiation program of these cells (15). The full-length PV E2 protein has several roles in the viral life cycle (15). Together with the viral E1 helicase, E2 initiates origin-specific viral DNA replication. Through its ability to link viral genomes to host chromosomes during mitosis, E2 is also required for viral genome maintenance. In addition, E2 is an important regulator of viral transcription. The structure of the E2 protein resembles that of a prototypical transcription factor, with an amino-terminal transcriptional activation domain and a carboxy-terminal DNA binding and dimerization domain separated by a less well-conserved hinge region. Depending upon the location of its cognate DNA binding sites within the promoter region, E2 can act either as a transcriptional activator or as a repressor. For the high-risk human PVs (HPVs), including HPV16 and HPV18, E2 represses expression from the LCR promoter that controls expression of the E6 and E7 viral oncogenes (33). During progression to cervical cancer, the HPV DNA is frequently integrated into the host cellular chromosomes in a manner that abolishes E2 expression, thus leading to increased E6 and E7 expression. The deregulated expression of the E6 and E7 oncogenes is thought to be a critical step in HPV-mediated transformation and ...

Section: Discussionmentioning

confidence: 99%

Brd4 Regulation of Papillomavirus Protein E2 Stability

Zheng

Schweiger

Martínez-Noël

et al. 2009

“…Cellular coactivators such as Brd4, hBrm, Gps2 (also known as AMF-1), and Tax1BP1 interact with E2 and enhance E2-dependent transcriptional activation (19-24). Brd4 and Tax1BP1 stabilize E2 protein, which partially explains how these factors increase E2 transcriptional activity (20,(24)(25)(26). …”

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confidence: 99%

“…Cellular coactivators such as Brd4, hBrm, Gps2 (also known as AMF-1), and Tax1BP1 interact with E2 and enhance E2-dependent transcriptional activation (19)(20)(21)(22)(23)(24). Brd4 and Tax1BP1 stabilize E2 protein, which partially explains how these factors increase E2 transcriptional activity (20,(24)(25)(26). Association with the Brm ATP-dependent chromatin remodeling complex enhances E2-dependent transcriptional activity specifically on episomally maintained templates (19).…”

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confidence: 99%

Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300

Quinlan

Culleton

et al. 2013

fThe p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an in vitro acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression.T he papillomavirus E2 protein controls transcription of viral early gene products by binding to specific DNA motifs (ACCN6GGT) critically placed within the viral genome (1). While viral gene expression is controlled by a variety of cellular transcription factors, including TFIID, Sp1, Oct1, and AP1 (2-6), expression of E2 results in increased transcriptional activation from the bovine papillomavirus (BPV) promoter and enhancer elements containing E2 binding sites (7,8). DNA binding activity is conferred by the C-terminal DNA binding domain (DBD); however, the N-terminal transactivation domain (TAD) of E2 is also necessary for specific activity (9-12). The ability of the TAD to activate transcription in the absence of the DBD (13) indicates that its function is at least partly mediated through complex formation with cellular factors (10, 12).Prior studies have identified E2-interacting proteins that facilitate E2-dependent transcription; however, the precise mechanisms through which these factors contribute to E2 activation of the viral early promoter remain unclear. Several reports have demonstrated that general transcription factors TFIIB and TFIID, including the TATA binding protein (TBP) and several TBP-associated factors, interact with E2 (14-18) and that TFIIB and TBP can enhance transcription by E2 (3,16,18). Cellular coactivators such as Brd4, hBrm, Gps2 (also known as AMF-1), and Tax1BP1 interact with E2 and enhance E2-dependent transcriptional activation (19-24). Brd4 and Tax1BP1 stabilize E2 protein, which partially explains how these factors increase E2 transcriptional activity (20,(24)(25)(26).

“…This protein contains 12 amino acids from the E8 ORF fused to the C-terminal DNA binding/dimerization domain of E2 (E2C). To date, E8 E2C transcripts have been detected for bovine papillomavirus type 1 (BPV-1) and human papillomavirus types 11,16,31,; the short segment from E8 exhibits high conservation among the different HPVs for which it has been described and shows partial conservation with the BPV-1 E8 domain (5,8,44,54).Early studies demonstrated that the N-terminal domain of E2 is necessary for transcriptional repression. Evidence for this came from comparative studies with full-length BPV-1 E2 (E2TA) and a second, truncated BPV-1-specific E2 repressor (E2TR), which is translated from an alternative initiating methionine residue and therefore lacks most of the N-terminal transactivation domain (TAD) (29).…”

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confidence: 99%

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confidence: 99%

NCoR1 Mediates Papillomavirus E8^E2C Transcriptional Repression

Powell

Smith

Sowa

et al. 2010

The papillomavirus E2 open reading frame encodes the full-length E2 protein as well as an alternatively spliced product called E8 E2C. E8 E2C has been best studied for the high-risk human papillomaviruses, where it has been shown to regulate viral genome levels and, like the full-length E2 protein, to repress transcription from the viral promoter that directs the expression of the viral E6 and E7 oncogenes. The repression function of E8 E2C is dependent on the 12-amino-acid N-terminal sequence from the E8 open reading frame (ORF). In order to understand the mechanism by which E8 E2C mediates transcriptional repression, we performed an unbiased proteomic analysis from which we identified six high-confidence candidate interacting proteins (HCIPs) for E8 E2C; the top two are NCoR1 and TBLR1. We established an interaction of E8 E2C with an NCoR1/HDAC3 complex and demonstrated that this interaction requires the wild-type E8 open reading frame. Small interfering RNA (siRNA) knockdown studies demonstrated the involvement of NCoR1/HDAC3 in the E8 E2C-dependent repression of the viral long control region (LCR) promoter. Additional genetic work confirmed that the papillomavirus E2 and E8 E2C proteins repress transcription through distinct mechanisms.Papillomaviruses (PVs) infect the squamous epithelia, inducing proliferative lesions. The PV life cycle is tightly linked with the squamous cell differentiation program. In particular, late viral gene synthesis, genome amplification, and virion production occur with the progression of epithelial cell differentiation.The PVs are small DNA viruses that contain approximately 10 open reading frames (ORFs). Like those of many viruses, PV regulatory proteins are multifunctional. The E2 ORF encodes several viral products (21). The full-length E2 protein is the most studied; it is a prototypic transcription factor, with a C-terminal DNA binding and dimerization domain, an internal hinge region, and an N-terminal "transactivation" domain (34). Initially, E2 was characterized as a transcriptional activator, and subsequently, it was shown to be a potent repressor of the long control region (LCR), which is the viral promoter that controls E6 and E7 expression (21). In addition to its transcriptional activities, E2 functions in concert with the viral E1 helicase to initiate replication of the viral DNA (21). Also, the E2 protein is involved in tethering viral genomes to the host chromatin for maintenance during mitosis (23, 50). Both the C-terminal portion of E2 (E2C), which mediates dimerization and recognition of consensus E2-binding sequences (ACCN 6 GGT) within the viral LCR, and the N terminus of E2 are required for its various transcription, replication, and genome maintenance activities (9,13,21,33,35,38,45,64).In addition to full-length E2, PVs encode an alternatively spliced product of E2 called E8 E2C. This protein contains 12 amino acids from the E8 ORF fused to the C-terminal DNA binding/dimerization domain of E2 (E2C). To date, E8 E2C transcripts have been detected for bovine ...