Brd4 Regulation of Papillomavirus Protein E2 Stability

Self Cite

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 ...

“…Brd4 has been implicated in the regulation of full-length E2 protein stability (11,30,66). Also, it was reported previously that Brd4 knockdown could increase steady-state E2 levels (51).…”

Section: Discussionmentioning

confidence: 87%

NCoR1 Mediates Papillomavirus E8^E2C Transcriptional Repression

Powell

Smith

Sowa

et al. 2010

Self Cite

“…One of these binding partners, the chromatin modulator Brd4, has been shown in previous studies to interact via its extreme C-terminal domain (CTD) (also named the C-terminal motif [CTM]) ( Fig. 3A) with E2 proteins from multiple PVs (13,16,(35)(36)(37). BPV-1 E2 WT and Y102 mutant proteins were expressed in HEK293TT cells along with full-length FLAG-tagged Brd4, and protein complexes were immunoprecipitated with mouse monoclonal anti-FLAG (M2)-conjugated beads.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

Culleton

Kanginakudru

DeSmet

et al. 2017

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-toglutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.IMPORTANCE Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

“…Brd4 coding sequence was also inserted into BamHI/XhoI sites of pGEX-6P-1 (GE Healthcare) to obtain pGEX-6P-1-hBrd4 bearing a glutathione S-transferase (GST)-tagged hBrd4. pOZN-16E2, containing recoded HPV16 E2 fused with Flag and HA tags on its N terminus, has been described previously (31). HPV16 E2 was also amplified by PCR and inserted into BamHI/XhoI sites of pEGFPC1 (Clontech) to obtain green fluorescent protein (GFP)-16E2.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of Brd4 to the Human Papillomavirus Type 16 DNA Replication Complex Is Essential for Replication of Viral DNA

Wang

Helfer

Pancholi

et al. 2013

102

bReplication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase ␦. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(؉) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus. Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that replicate in differentiating cutaneous and mucosal epithelia (1). They are one of the most prevalent sexually transmitted pathogens in the world. High-risk HPVs are known etiological agents of cervical, anogenital, and head and neck cancers (2), with HPV16 being responsible for over 50% of cervical cancer cases worldwide (3-5).HPVs specifically infect basal epithelial cells. HPV genome replication occurs during two different stages of the viral life cycle. In the infected basal epithelial cells, the viral genomes replicate an average of once per cell cycle during S phase, in synchrony with the host DNA replication (6). This allows the viral genome to be maintained as stable episomes at 50 to 100 copies per cell. This stage of DNA replication ensures a persistent infection in the basal layer of the epidermis. Terminal differentiation of infected cells triggers vegetative viral DNA replication, producing viral genomes, which can then be assembled into virions and be released from the surface of differentiated epithelium (7).Replication of the HPV genome is carried out by viral E1 and E2 proteins in combination with various components of the cellular DNA replication machinery (7). E2 binds to several consensus E2 binding sites near the HPV origin of replication (Ori) and recruits E1 to the...