Dissertation Defense: Susannah Stephenson

Candidate: Susannah Stephenson

Major: Microbiology and Immunology

Advisor: John L. Casey, Ph.D.

Title: Investigating the Initiation Site of Hepatitis Delta Virus Genome Synthesis

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesised by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5’ end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. Circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected HDAg-Huh7 cells, thus enabling the detection of the 5’ end of the genome RNA. The 5’ end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA.