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

Abstract

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...

Acetylation of E2 by P300 Mediates Topoisomerase Entry at the Papillomavirus Replicon

“…HPV replication is dependent on acetylation at K111 by P300. Previous experiments suggested the regulatory role of E2 on PV replication by mutating lysine 111 to arginine, glutamine, and alanine (12). After confirming that Topo1 recruitment is P300 specific (Fig.…”

“…We previously reported that the K111 E2 mutations did not alter E2 DNA binding or E1 recruitment to the replication origin. However, the K111R mutant was unable to induce origin unwinding and RPA accumulation (12). We suspected the requirement for Topo1 during replication because of the relevant functions and made two key discoveries.…”

“…The cells were then washed with PBS and harvested. HPV31 E2 mutant plasmids were constructed by site-directed mutagenesis (QuikChange II; Agilent) and verified by sequencing of the entire E2 gene (12). HA-tagged HPV31 E2 was obtained from P. Howley (30), codon-optimized FLAG HPV31 E2 was supplied by A. McBride (31), and the HPV31 E1, pCI-RLuc, and PFLORI31 constructs were obtained from J. Archambault (32).…”

“…Importantly, both E2 mutant proteins coimmunoprecipitated with relevant factors, including E1, Brd4, TopBP1, and GPS2. While E2 K111R recruited E1 to the origin, replication protein A (RPA) was not detected, indicating the inability to form stable single-stranded DNA at the replication forks where detectable levels of RPA were deposited (12). To uncover the mechanism by which this acetylation regulates viral replication, we focused on topoisomerase 1 (Topo1), which is necessary for resolving the torsional stress resulting from unwinding double-stranded DNA as it is duplicated by polymerases (13).…”

Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

“…RNA (1 g) was used for cDNA synthesis using a SuperScript III first-strand synthesis system (Thermo Fisher Scientific). cDNA was analyzed by quantitative PCR for HPV18 E6 and actin using previously published primers (59).…”