Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300

Abstract

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).

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

“…We ensured that the abrogation of replication from the K111R mutant HPV31 E2 protein was not due to reduced cell viability (Figure 3.4). These experiments also demonstrated that while replication is affected by K112 in the context of BPV1 (Quinlan et al, 2012), mutations at this position in HPV31 E2 did not exhibit a similar dependence (Figure 3.3). Results were normalized to renilla luciferase and E2 transcription and presented as fold over E1 only control.…”

“…for BPV1 E2 transcriptional activation (Quinlan et al, 2012). While E2 is known to bind and localize the E1 helicase to the replication origin, which is flanked by high affinity E2 binding sites, additional role(s) for E2 in DNA replication have not been elucidated.…”

“…Sumoylation has been shown to increase the stability of E2 from HPV11, 16, and 18 (Y. C. induced by an increase in expression of the acetyltransferase p300 (EP300) (Wong, Pickard, & McCance, 2010). Our lab has previously identified conserved lysine residues in Bovine PV (BPV1) E2 that were acetylated by p300 in vitro and regulate its transcriptional activation capability (Quinlan, Culleton, Wu, Chiang, & Androphy, 2012). Of the several conserved lysines that were identified, a dilysine motif located in the transactivation domain was of particular interest.…”

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

“…Similar sites have been found in human papillomavirus 8 (HPV-8) at S253 (30) and in HPV-16 at S243 (31). We previously reported acetylation at lysine 111 (K111) and K112 of BPV-1 E2 and that mutation to arginine (R) at the former site inhibited viral transcription (32).…”

Papillomavirus Replication

“…Controlled expression of these truncation forms may allow PVs to alternate between different E2 functions. Posttranslational modifications such as acetylation (Quinlan et al 2012) and phosphorylation may also play a role in controlling E2 activity (see below).…”