Characterization of the Interaction of the Hpv8 E2 Tethering Protein with Host Chromosomes

Title of Document: CHARACTERIZATION OF THE INTERACTION OF THE HPV8 E2 TETHERING PROTEIN WITH HOST CHROMOSOMES. Vandana Sekhar, Doctor of Philosophy, 2011 Directed By: Dr. Alison A McBride, DNA Tumor Virus Section, Laboratory of Viral Diseases, NIAID, NIH and Dr. Leslie Pick, MOCB, UMD One of the mechanisms by which papillomaviruses establish persistent infection of the host is by tethering their genomes to host chromosomes during mitosis. This ensures maintenance and partitioning of the viral genomes to daughter cells after each cell division. Although studies have shown that the viral E2 protein links the viral genome to host chromosomes in several papillomaviruses, the exact molecular mechanism of this interaction has yet to be elucidated for the beta-papillomaviruses. The studies described in this dissertation aimed to characterize the interaction of the E2 protein of the human papillomavirus type 8 (HPV8), a type of betapapillomavirus, with mitotic chromosomes. The E2 protein consists of a conserved Nterminal transactivation domain and a C-terminal DNA binding and dimerization domain that are linked by a flexible hinge. We have mapped a sixteen amino acid region in the hinge that, when linked to the DNA binding domain, is crucial and sufficient for chromosomal association. Further we have identified two residues in this region, arginine 250 (R250) and serine 253 (S253) within a highly conserved RXXS motif that are required for HPV8 E2 chromosome binding. Additionally, we have shown that the S253 residue is phosphorylated. To gain insight into the regulation of the E2 chromosome binding function, we investigated the role of phosphorylation of S253. We have shown that S253 is phosphorylated by PKA in Sphase, which increases the half-life of E2 protein and modulates its interaction with host chromatin. Since E2 is also involved in transcriptional regulation and viral genome replication, we examined if mutating residues R250 or S253 affected the transcriptional activation or replication functions of the HPV8 E2 protein. Furthermore using a domain swapping approach, we also explored the role of the Cterminal domain in the HPV8 E2 chromosome binding function. Finally to establish the mode of interaction responsible for mediating HPV8 E2 chromosome binding, we employed both a proteomics approach and ribonuclease treatment techniques, to examine whether HPV8 E2 chromosomal association is mediated through proteinprotein or protein-RNA interactions, respectively. Collectively, these studies have added to our current understanding of the interaction of HPV8 E2 protein with host chromosomes. CHARACTERIZATION OF THE INTERACTION OF THE HPV8 E2 TETHERING PROTEIN WITH HOST CHROMOSOMES