Involvement of Brd4 in different steps of the papillomavirus life cycle

Abstract

Bromodomain-containing protein 4 (Brd4) is a cellular chromatin-binding factor and transcriptional regulator that recruits sequence-specific transcription factors and chromatin modulators to control target gene transcription. Papillomaviruses (PVs) have evolved to hijack Brd4's activity in order to create a facilitating environment for the viral life cycle. Brd4, in association with the major viral regulatory protein E2, is involved in multiple steps of the PV life cycle including replication initiation, viral gene transcription, and viral genome segregation and maintenance. Phosphorylation of Brd4, regulated by casein kinase II (CK2) and protein phosphatase 2A (PP2A), is critical for viral gene transcription as well as E1- and E2-dependent origin replication. Thus, pharmacological agents regulating Brd4 phosphorylation and inhibitors blocking phospho-Brd4 functions are promising candidates for therapeutic intervention in treating human papillomavirus (HPV) infections as well as associated disease.

Keywords: Brd4; E2 protein; Papillomavirus life cycle; Papillomavirus replication; Papillomavirus transcription.

Fig. 1

Human papillomavirus genome and its transcription in relation to high-risk or low-risk HPV types. The papillomavirus genome contains approximately 8000 bp and encodes 7–10 open reading frames (ORFs). The viral E2 protein functions as a transcriptional activator or a repressor depending on the location and sequence context of the E2 binding sites in relation to the TATA box. URR: upstream regulatory region; E2BS: E2-binding site.

Fig. 2

Papillomavirus E2 protein domains and functions. The crystal structures have been obtained from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/; Berman et al., 2000). The crystal structure of the transactivation domain is a representation of HPV16 E2 in complex with Brd4 (PDB 2NNU; Abbate et al., 2006). The C-terminal domain of Brd4, which interacts with the E2 N-terminal domain, is highlighted in purple. The crystal structure of the DNA-binding/dimerization domain shows a dimer of the C-terminus of HPV18 E2 bound to double-stranded DNA, which is shown in green (PDB 1JJ4; Kim et al., 2000). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Domain contact and model for Brd4 interaction with high-risk and low-risk E2. (A) Domain interactions between Brd4 and E2. Bromodomain I (BD1), bromodomain II (BD2), N-terminal phosphorylation sites (NPS), basic residue-enriched interaction domain (BID), extra-terminal domain (ET), and C-terminal motif (CTM) in Brd4 are shown from the N-terminus (N) on the left to the C-terminus (C) on the right, whereas DNA-binding domain (DBD), hinge region, and the transactivation domain (TAD) of high-risk (HR) or low-risk (LR) E2 are depicted from C to N. (B) Model for BRD4 domain interaction with low-risk E2 regulated by CK2 and PP2A. (C) Model for BRD4 domain interaction with high-risk E2 regulated by CK2 and PP2A.

Fig. 4

Model for MMP-9 gene transcription in proliferating and differentiating keratinocytes regulated by E2, NFκB and AP-1 family members. Binding of JunB and JunD to three AP-1 sites in MMP-9 in proliferating keratinocytes is replaced by c-Jun binding to two promoter-proximal AP-1 sites upon differentiation, which is coupled with differentiation-induced translocation of NFκB from the cytoplasm to the nucleus. DC-1 is a phospho-Brd4-targeting peptoid (Cai et al., 2011) that effectively blocks phosphorylation-dependent Brd4 function in stimulating high-risk (HR) E2 binding to its cognate sequence in proliferating keratinocytes or in enhancing NFκB binding to the NFκB site upon differentiation.

Fig. 5

Phospho-Brd4 is critical for HPV origin replication and viral early promoter-driven transcription. Phosphorylation of Brd4 plays a positive role in enhancing E1- and E2-dependent HPV origin replication but is also required for E2-mediated inhibition of HPV early promoter activity (upper panel). In the presence of phospho-PDID (pPDID)-targeting compounds such as DC-1 peptoid, Brd4 interaction with E2 is inhibited, leading to suppressed origin replication but enhanced early promoter transcription (lower panel).