Cell Biology & Molecular Genetics

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

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 beta-papillomavirus, with mitotic chromosomes. The E2 protein consists of a conserved N-terminal 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 S-phase, 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 C-terminal 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 protein-protein or protein-RNA interactions, respectively. Collectively, these studies have added to our current understanding of the interaction of HPV8 E2 protein with host chromosomes.

Cell Biology & Molecular Genetics

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