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).

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.

The papillomavirus (PV) E2 protein is a DNA binding, protein interaction platform that recruits viral and host factors necessary for transcription and replication. We recently discovered phosphorylation of a tyrosine (Y102) in bovine PV (BPV) E2. To identify the responsible factor, we tested several candidate tyrosine kinases that are highly expressed in keratinocytes for binding to BPV-1 E2. Fibroblast growth factor receptor 3 (FGFR3) coimmunoprecipitated with the BPV-1 E2 protein, as did human papillomavirus 31 (HPV-31) E2, which also colocalized with FGFR3 within the nucleus. A constitutively active mutant form of FGFR3 decreased BPV-1 and HPV-31 transient replication although this result also occurred in a BPV-1 E2 mutant lacking a previously identified phosphorylation site of interest (Y102). Furthermore, FGFR3 depletion in cell lines that maintain HPV-31 episomes increased viral copy number. These results suggest that FGFR3 kinase activity may regulate the PV reproductive program through phosphorylation of the E2 protein although this is unlikely to occur through the Y102 residue of HPV E2. The papillomavirus (PV) is a double-stranded DNA tumor virus infecting cervix, mouth, and throat tissues. The viral protein E2 is responsible for the replication of the virus. Understanding the mechanisms of the replicative life cycle of the virus may bring to light direct targets and treatments against viral infection. We recently found that the fibroblast growth factor receptor 3 (FGFR3) interacts with and mediates PV E2 function through phosphorylation of the E2 protein. Our study suggests that the function of the E2 protein may be regulated through a direct FGFR3 target during the maintenance stage of the PV life cycle.

Several serine and threonine residues of the papilloma virus early E2 protein have been found to be phosphorylated. By contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this paper we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the non-phosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither bind the C-terminus of Brd4, but in all other aspects, the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in-vitro assay, while HPV-31 Y102E binds E1 and replicated albeit at lower levels than wild type. To examine effect of E2 mutations under more native-like infection conditions, a neomycin selectable marker was inserted into L1/L2 of HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days post-transfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines. IMPORTANCE Post-translational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

In this special issue, work is presented linking metacognition among persons with schizophrenia with a range of psychosocial outcomes including vocational functioning, empathy, motivation, self-evaluation, and other cognitive functions. This overview will highlight how these works allow for the quantitative study of processes which underpin alterations in self-experience in schizophrenia, which in turn allows self-experience to be studied as part of a larger set of brain-based and social phenomena whose interaction influences the trajectory of one's life and illness. We explore the hypothesis that metacognitive capacity, as a node in a larger biopsychosocial network, may be accessible by psychosocial treatment and, if successfully targeted, may disrupt the processes which perpetuate disability. Limitations and directions for future research are also discussed.