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Browsing by Author "Klemsz, Michael"
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Item HPV replication regulation by acetylation of a conserved lysine in the E2 protein(2017-06-26) Thomas, Yanique Serge Gillana; Androphy, Elliot J.; Klemsz, Michael; Yu, Andy; Mayo, Lindsey; Lee, Suk-HeePapillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. 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. 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.Item Regulation of papillomavirus E2 protein by posttranslational modification(2015-04-24) Culleton, Sara Poirier; Androphy, Elliot J.; Klemsz, Michael; Mayo, Lindsey; Nakshatri, Harikrishna; Sullivan, William J., Jr.Papillomaviruses (PVs) are small, double-stranded DNA viruses. Hundreds of species have evolved to replicate in mammals, birds, and reptiles. Approximately two hundred species are estimated to infect humans alone, and these human papillomaviruses (HPVs) cause diseases ranging from benign warts to anogenital and oropharyngeal cancers. While vaccination is effective at preventing the majority of these infections and their disease outcomes, there are no successful treatments for existing infections; thus, exploration of novel therapeutic targets is warranted. PVs control expression and function of their gene products through alternative splicing, alternate start codons, and post-translational modification (PTM). The viral E2 protein regulates transcription, replication, and genome maintenance in infected cells, and PTMs have been demonstrated for E2 proteins from multiple papillomavirus types. Serine phosphorylation events were reported to influence E2 stability, and our laboratory was the first to describe in vitro acetylation events with implications for E2 transcription function. Here we report confirmation of these acetylation events in vivo and additional data elucidating the role of these PTMs in viral transcription. Moreover, we present a novel phosphorylation site for bovine papillomavirus type 1 (BPV-1) E2 at tyrosine 102 (Y102). Using phospho-deficient and phospho-mimetic point mutants, we found that this site influences E2-mediated transcription and replication, and we hypothesize that phosphorylation at Y102 regulates these activities by interrupting the association of E2 with its binding partners. We also report interaction of BPV-1 E2 and HPV-31 E2 with different receptor tyrosine kinases (TKs), most notably members of the fibroblast growth factor receptor family. We hypothesize that Y102 phosphorylation by these receptors occurs early in infection to limit viral replication and gene expression. Further studies will cement the role of RTKs in PV biology and could reveal novel therapeutic strategies.