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

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.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Culleton

Kanginakudru

DeSmet

et al. 2017

“…For Southern blotting and flow cytometry analysis, C33A cells were transfected using the calcium phosphate method (32). Small interfering RNA (siRNA) transfection was performed using calcium phosphate, as previously described (33). To detect viral DNA replication, C33A cells were pulsed with 10 M BrdU at 44 hpt for 2 h and cultured with normal growth medium for another 2 h before acetone fixation.…”

Section: Methodsmentioning

confidence: 99%

Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

Tsang

Wang

et al. 2014

Self Cite

IMPORTANCEMCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM-and ATR-mediated DDR pathways accumulate in MCPyV LT-positive nuclear foci. Such a phenotype was recapitulated using our previously established system for visualizing MCPyV replication complexes in cells. By combining immunofluorescent staining, fluorescence in situ hybridization, and BrdU incorporation analysis, we demonstrate that DDR proteins are important for maintaining robust MCPyV DNA replication. This study not only provides the first look into the microscopic details of DDR factor/LT replication complexes at the MCPyV origin but also provides a platform for further studying the mechanistic role of host DDR factors in the MCPyV life cycle and virus-associated oncogenesis.

“…Brd4 is known to bind to the E2 N-terminal transcriptional activation domain, and localizes to replication foci during the initiation of viral replication before being displaced from actively replicating origins (Sakakibara et al, 2013;Wang et al, 2013). There are three was investigated by co-immunoprecipitation experiments with each of the Brd4 binding regions (Figure 3.11).…”

Section: Hpv31 E2 Interactions With Host Factors Are Not Perturbed Bymentioning

confidence: 99%

“…Brd4 binding deficient mutant R37A/I73A is also defective in viral replication (Wang, Helfer, Pancholi, Bradner, & You, 2013). Brd4 is known to bind to the E2 N-terminal transcriptional activation domain, and localizes to replication foci during the initiation of viral replication before being displaced from actively replicating origins (Sakakibara et al, 2013;Wang et al, 2013).…”

Section: Hpv31 E2 Interactions With Host Factors Are Not Perturbed Bymentioning

confidence: 99%

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

Thomas

Androphy

2018

iii ACKNOWLEDGEMENTS I would like to thank my supervisor Elliot J. Androphy for his guidance, and encouragement over these many years. I truly appreciate your faith and continuous support.I also want to thank all members of the Androphy lab, for your guidance, critiques, and support.Thank you to my thesis committee, without whom none of this would be possible.Your guidance has been invaluable. Michael Klemsz, Suk-Hee Lee, Lindsey Mayo, and Andy Yu. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. v When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111. Human Papillomaviruses and DiseaseThere are over 200 HPVs that have been described and they manifest in a variety of ways; they can be asymptomatic, or they can result in benign lesions (papillomas) or progress to malignancy (de Villiers, Fauquet, Broker, Bernard, & zur Hausen, 2004). In the United States, HPV is the most common sexually transmitted infection and nearly all sexually active adults contract it at least once. E1The PV E1 protein is an essential viral replication factor (Lusky & Botchan, 1985;Ustav, Ustav, Szymanski, & Stenlund, 1991). E1 is the viral helicase and initiates replication by binding to specific sequences on the viral genome with the assistance of the E2 protein (Frattini & Laimins, 1994).There is sequence and functional homology between PV E1 and SV (simian virus) 40 Large T Antigen which allowed for many of the predictions concerning E2 shows differential affinity for the E2BS and it is proposed that at low levels, E2b...